Trispecific antibodies

Abstract

The present application provides a trispecific antibody. The trispecific antibody comprises: a first binding region having a binding CD16 activity; a second binding region having a binding CD138 activity; and a third binding region having a binding IL-15 receptor activity. The trispecific antibody according to the embodiment of the present application simultaneously binds CD138 on tumor cells, CD16 and IL-15 receptor on NK cells, and the trispecific antibody promotes CD16+ lymphocytes to form an immune synapse with tumor cells by binding with CD138 molecules on the tumor cells, so as to activate the lymphocytes to kill CD138+ tumor cells, and the trispecific antibody can bind with IL-15 receptors on the surface of NK cells to provide a stimulation signal for the survival and proliferation of NK cells, so that the trispecific antibody can effectively treat and prevent cancer.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology. Specifically, this invention relates to a trispecific antibody, and more specifically, this invention relates to a trispecific antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition, and their uses. Background Technology

[0002] Natural killer cells (NK cells) are important members of the innate immune system. Unlike T cells, NK cells do not express antigen-specific receptors. NK cells possess a broad spectrum of tumor-killing capabilities and play a crucial role in enhancing antibody and T cell responses. Currently, NK cell-based tumor immunotherapy takes various forms and employs different methods. The CD16 molecule is an important marker on the surface of NK cells, which can activate the IgE NK cell receptor (FcεRIγ) and the immune receptor tyrosine activation motif (ITAM) of CD3ζ to induce ADCC. Therefore, the CD16 target is preferentially selected in NK cell-redirected bispecific antibodies. Although CD16-based bispecific antibodies can effectively promote NK cell killing of tumor cells, their effectiveness is limited due to the short lifespan of NK cells. Summary of the Invention

[0003] The present invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, the present invention provides a trispecific antibody that can simultaneously bind to CD138 on tumor cells, CD16 and IL-15 receptors on NK cells. By promoting the simultaneous binding of NK cells and tumor cells, it can induce the formation of synapses between the two cells, activate NK cells to kill CD138+ positive tumor cells, and stimulate NK cells to activate and proliferate.

[0004] This invention is based on the following discoveries of the inventors:

[0005] CD138, also known as Syndecan-1, is a 288-amino acid protein commonly expressed in epithelial cells and leukocytes. The Syndecan family comprises four members, all containing an intracellular C-terminus, a transmembrane region, and an extracellular N-terminus, with numerous extracellular polysaccharide branches. CD138 expression is aberrant in most tumors and other epithelial malignancies; for example, elevated CD138 expression is observed in over 60% of pathological samples from bladder cancer patients. Some studies have also shown that high CD138 expression in breast cancer patients is associated with poor prognosis. Furthermore, CD138 expression can be detected in the tumor epithelium and stromal cells of some ovarian cancer patients, but is not expressed in normal ovarian biopsy samples. CD138 expression is associated with functions such as invasive growth and metastatic tendency of tumor cells. These clinical findings suggest that CD138 could be a promising target for clinical treatment.

[0006] Human cytokine IL-15 is typically secreted by myeloid cells and some epithelial cells. IL-15 interacts with IL-15 receptors on the surface of dendritic cells, CD8+ T cells, and NK cells, thereby promoting the activation and proliferation of these cells and the secretion of corresponding cytokines. The IL-15 receptor has three chains: α, β, and γ. The IL-15 α chain is unique to IL-15, while the receptor β chain is identical to that of the IL-2 receptor. The IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 receptors all share the same γ chain. IL-15 typically binds to the IL-15Rα chain before binding to the β and γ chains to form aggregates.

[0007] Based on this, the present invention designs a novel trispecific antibody that can specifically bind to CD16, CD138, and the IL-15 receptor. This trispecific antibody, by binding to the CD138 molecule on tumor cells, promotes the formation of an immune synapse between CD16+ lymphocytes and tumor cells, thereby activating lymphocytes to kill CD138+ tumor cells. Simultaneously, this trispecific antibody can bind to the IL-15 receptor on the surface of NK cells, providing a stimulating signal for NK cell survival and proliferation, offering new inspiration and ideas for antibody therapy in tumor immunotherapy.

[0008] In one aspect of the present invention, a trispecific antibody is proposed. According to an embodiment of the present invention, the trispecific antibody comprises: a first binding region having CD16 binding activity; a second binding region having CD138 binding activity; and a third binding region having IL-15 receptor binding activity. The trispecific antibody according to the embodiment of the present invention simultaneously binds to CD138 on tumor cells, CD16 on NK cells, and the IL-15 receptor. By binding to CD138 molecules on tumor cells, the trispecific antibody promotes the formation of an immune synapse between CD16+ lymphocytes and tumor cells, thereby activating lymphocytes to kill CD138+ tumor cells. Simultaneously, the trispecific antibody can bind to the IL-15 receptor on the surface of NK cells, providing a stimulating signal for NK cell survival and proliferation, offering new inspiration and ideas for antibody therapy in tumor immunotherapy, and can effectively treat and prevent cancer.

[0009] In another aspect of the invention, a nucleic acid molecule is proposed. According to an embodiment of the invention, the nucleic acid molecule encodes the aforementioned trispecific antibody. The nucleic acid molecule according to an embodiment of the invention encodes the aforementioned trispecific antibody.

[0010] In another aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule. When the nucleic acid molecule is ligated to the vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself or can be exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule and the control elements can be operably linked. In this document, "operably linked" means ligating a foreign gene to the vector so that the control elements within the vector, such as transcription control sequences and translation control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. After the expression vector of some specific embodiments of the present invention is introduced into suitable recipient cells, the aforementioned trispecific antibody can be effectively expressed under the mediation of a regulatory system, thereby achieving the large-scale in vitro production of trispecific antibodies.

[0011] In another aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises: carrying the aforementioned nucleic acid molecule or the aforementioned expression vector; or expressing the nucleic acid molecule encoding the aforementioned trispecific antibody. Using this recombinant cell, under suitable conditions, the aforementioned trispecific antibody can be effectively expressed intracellularly.

[0012] In another aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: the aforementioned trispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. As is known before, the trispecific antibody simultaneously binds to CD138 on tumor cells, CD16 on NK cells, and IL-15 receptors. Therefore, a drug containing the aforementioned trispecific antibody can, by binding to CD138 molecules on tumor cells, induce CD16+ lymphocytes to form an immune synapse with tumor cells, thereby activating lymphocytes to kill CD138+ tumor cells. Simultaneously, the trispecific antibody can bind to IL-15 receptors on the surface of NK cells, providing a stimulating signal for NK cell survival and proliferation, and can be used for the prevention and / or treatment of cancer or tumors.

[0013] In another aspect of the invention, the invention provides for the use of the aforementioned trispecific antibody or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of cancer or tumors.

[0014] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0015] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0016] Figure 1 This is a schematic diagram of the configuration of the trispecific antibody designed in Example 1 of the present invention;

[0017] Figure 2 The results show the binding activity of the trispecific antibody with CD16 single antigen, CD138 single antigen, and CD16 and CD138 dual antigen in Example 3 of the present invention.

[0018] Figure 3 The results of the binding activity of the trispecific antibody with IL-15Rβ / IL-15Rγ and CD16 in Example 3 of this invention;

[0019] Figure 4 The results of the killing efficiency of the trispecific antibody against multiple myeloma MM.1S cells and NCI-H929 cells in Example 4 of this invention;

[0020] Figure 5 The results show the killing efficiency of the trispecific antibody against pancreatic cancer PANC1 cells in Example 4 of this invention. Detailed Implementation

[0021] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0022] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0023] In this document, the terms “comprising” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0024] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0025] In this article, the terms "triple antibody" and "trispecific antibody" are synonymous, both referring to antibodies that can recognize three antigenic epitopes. This is a broad interpretation, and the specific structure is not limited, as long as it can recognize three antigenic epitopes. For example, such as Figure 1 The double antibody shown.

[0026] In this paper, the term "knob into hole structure" refers to the formation of a button (hole) mutation in the CH3 region of the constant region of the antibody heavy chain, which facilitates heavy chain interlocking and the formation of a heterodimer. For example, this can be achieved by mutating the amino acids in the CH3 domain of the constant region of the human IgG1 heavy chain (T366S, L368A, Y407V, Y349C mutation in one chain, i.e., "hole"; and T366W, S354C mutation in the other chain, i.e., "knob").

[0027] In this paper, the term "fragment" refers to a target protein or polypeptide, and a target protein or polypeptide that has been truncated at an N-terminus (N-terminus) or C-terminus (C-terminus), and / or has an internal deletion.

[0028] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0029] In this paper, the term "at least 90% identity" means at least 90% identity with each reference sequence, which may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0030] In this document, the amino acid numbering of the IgG1 Fc portion is based on the EU numbering system. For example, position 366 refers to position 366 according to the EU numbering system; "T366W" means that threonine at position 366 according to the EU numbering system is replaced by tryptophan; and "L368A" means that leucine at position 368 according to the EU numbering system is replaced by alanine.

[0031] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the aforementioned functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0032] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this invention include immortalized hybridoma cells, NS / O myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0033] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0034] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0035] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The trispecific antibody or pharmaceutical composition of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0036] In this document, the term "treatment" refers to the administration of a drug or compound to an individual to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in an individual who is susceptible but has not yet been diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0037] This invention proposes a trispecific antibody, a nucleic acid molecule, an expression vector, a recombinant cell, a pharmaceutical composition, and their uses, which will be described in detail below.

[0038] Trispecific antibodies

[0039] In one aspect of the present invention, a trispecific antibody is provided. According to an embodiment of the present invention, the trispecific antibody comprises: a first binding region having CD16 binding activity; a second binding region having CD138 binding activity; and a third binding region having IL-15 receptor binding activity. The trispecific antibody according to the embodiment of the present invention simultaneously binds to CD138 on tumor cells, CD16 on NK cells, and the IL-15 receptor. By binding to CD138 molecules on tumor cells, the trispecific antibody promotes the formation of an immune synapse between CD16+ lymphocytes and tumor cells, thereby activating lymphocytes to kill CD138+ tumor cells. Simultaneously, the trispecific antibody can bind to the IL-15 receptor on the surface of NK cells, providing a stimulating signal for NK cell survival and proliferation, and can effectively treat or prevent cancer and tumors.

[0040] According to embodiments of the present invention, the above-mentioned trispecific antibody may further include at least one of the following technical features:

[0041] According to an embodiment of the present invention, the first bonding region includes a first heavy chain variable region and a first light chain variable region, and the first heavy chain variable region and the first light chain variable region are connected.

[0042] According to an embodiment of the present invention, the first heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 90% identity with it; and / or, the first light chain variable region has an amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence having at least 90% identity with it.

[0043] CD16 heavy chain variable region:

[0044] QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWMGYISYSGYTNYNPSLES RVTISRDTSKNQFSLKLSSVTAADTAVYYCAREVGLRLGWFAYWGQGTLVTVSS (SEQ ID NO: 1).

[0045] CD16 light chain variable region:

[0046] DIQMTQSPSSSLSASVGDRVTITCKASQSVNNDVAWYQQKPGKAPKLLIYYASNRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSSPWTFGGGTKLEIK (SEQ ID NO: 2).

[0047] According to an embodiment of the present invention, the second bonding region includes a second heavy chain variable region and a second light chain variable region, and the second heavy chain variable region and the second light chain variable region are connected.

[0048] According to an embodiment of the present invention, the second heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:3 or an amino acid sequence having at least 90% identity with it; and / or, the second light chain variable region has an amino acid sequence as shown in SEQ ID NO:4 or an amino acid sequence having at least 90% identity with it.

[0049] CD138 heavy chain variable region:

[0050] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYTIHWVRQAPGQGLEWIGWFYPGSDNIKYNEKF KDRATMTADRSISTAYMELSRLRSDDTAVYFCARHERGYSTPGDVWGQGTTVTVS (SEQ ID NO: 3).

[0051] CD138 light chain variable region:

[0052] DVVMTQSPLSLPVTLGQPASISCRSQQSLLHSNANTYLHWFQQRPGQSPRLLIYRVSNRFSGVPDR FSGSGSGTDFTLKISRVEAEDVGVYFCSQSSRIPWTTFGGGTKLEIK (SEQ ID NO: 4).

[0053] According to an embodiment of the present invention, the third binding region includes an IL-15 fragment and optionally an IL-15Rα fragment.

[0054] In a preferred embodiment of the present invention, the third binding region includes an IL-15 fragment and an IL-15Rα fragment. Thus, the dimer fragment composed of IL-15 and the IL-15 receptor α chain (IL-15Rα) in this trispecific antibody can better bind to the IL-15 receptor β and γ chains on the surface of NK cells, thereby providing a stimulatory signal for NK cell survival and proliferation.

[0055] According to an embodiment of the present invention, the IL-15 fragment is connected to the first heavy chain variable region or the first light chain variable region, and the IL-15Rα fragment is connected to the second heavy chain variable region or the second light chain variable region; or, the IL-15Rα fragment is connected to the first heavy chain variable region or the first light chain variable region, and the IL-15 fragment is connected to the second heavy chain variable region or the second light chain variable region.

[0056] According to embodiments of the present invention, the IL-15 fragment has an amino acid sequence as shown in SEQ ID NO:5 or an amino acid sequence having at least 90% identity with it; and / or, the IL-15Rα fragment has an amino acid sequence as shown in SEQ ID NO:6 or an amino acid sequence having at least 90% identity with it.

[0057] IL-15 fragment:

[0058] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 5).

[0059] IL-15Rα fragment:

[0060] ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDP (SEQ ID NO: 6).

[0061] According to an embodiment of the present invention, the N end of the first heavy chain variable region is connected to the C end of the first light chain variable region; or, the C end of the first heavy chain variable region is connected to the N end of the first light chain variable region.

[0062] According to an embodiment of the present invention, the first binding region further includes a first Fc fragment; wherein the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first Fc fragment, and the C-terminus of the first Fc fragment is connected to the N-terminus of the IL-15 fragment or the IL-15Rα fragment; or, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is connected to the N-terminus of the first Fc fragment, and the C-terminus of the first Fc fragment is connected to the N-terminus of the IL-15 fragment or the IL-15Rα fragment. The N-terminus of the L-15Rα fragment is connected; or, the C-terminus of the IL-15 fragment or IL-15Rα fragment is connected to the N-terminus of the first Fc fragment, the C-terminus of the first Fc fragment is connected to the N-terminus of the first light chain variable region, and the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first light chain variable region; or, the C-terminus of the IL-15 fragment or IL-15Rα fragment is connected to the N-terminus of the first Fc fragment, the C-terminus of the first Fc fragment is connected to the N-terminus of the first heavy chain variable region, and the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first light chain variable region.

[0063] According to an embodiment of the present invention, the first binding region further includes a first linker peptide; wherein the N-terminus of the first heavy chain variable region is connected to the C-terminus of the first linker peptide, and the N-terminus of the first linker peptide is connected to the C-terminus of the first light chain variable region; or, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first linker peptide, and the C-terminus of the first linker peptide is connected to the N-terminus of the first light chain variable region.

[0064] According to an embodiment of the present invention, the first binding region further includes a first CL fragment and / or a first CH1 fragment; wherein, the C-terminus of the first light chain variable region is connected to the N-terminus of the first CL fragment, the C-terminus of the first CL fragment is connected to the N-terminus of the first heavy chain variable region, and / or, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first CH1 fragment, the C-terminus of the first CH1 fragment is connected to the N-terminus of the first Fc fragment, and the C-terminus of the first Fc fragment is connected to the N-terminus of the IL-15 fragment or the IL-15Rα fragment; or, the C-terminus of the IL-15 fragment or the IL-15Rα fragment is connected to the N-terminus of the first Fc fragment, the C-terminus of the first Fc fragment is connected to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is connected to the N-terminus of the first CL fragment, the C-terminus of the first CL fragment is connected to the N-terminus of the first heavy chain variable region, and / or, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first CH1 fragment; or, the... The C-terminus of the first light chain variable region is connected to the N-terminus of the first CL fragment, the C-terminus of the first CL fragment is connected to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is connected to the N-terminus of the first heavy chain variable region, and / or, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first CH1 fragment, the C-terminus of the first CH1 fragment is connected to the N-terminus of the first Fc fragment, and the C-terminus of the first Fc fragment is connected to the N-terminus of the IL-15 fragment or the IL-15Rα fragment; or, the C-terminus of the IL-15 fragment or the IL-15Rα fragment is connected to the N-terminus of the first Fc fragment, the C-terminus of the first Fc fragment is connected to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is connected to the N-terminus of the first CL fragment, the C-terminus of the first CL fragment is connected to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is connected to the N-terminus of the first heavy chain variable region, and / or, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first CH1 fragment. The inventors unexpectedly discovered that antibodies containing CL and / or CH1 have higher affinity than single-chain antibodies (VH+VL antibodies).

[0065] According to an embodiment of the present invention, the first binding region further includes a second linker peptide; wherein the C-terminus of the first Fc fragment is connected to the N-terminus of the second linker peptide, and the C-terminus of the second linker peptide is connected to the N-terminus of the IL-15 fragment or the IL-15Rα fragment; or, the C-terminus of the IL-15 fragment or the IL-15Rα fragment is connected to the N-terminus of the second linker peptide, and the C-terminus of the second linker peptide is connected to the N-terminus of the first Fc fragment.

[0066] For example, the C-terminus of the first light chain variable region is connected to the N-terminus of the first CL fragment, the C-terminus of the first CL fragment is connected to the N-terminus of the first heavy chain variable region, and / or, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first CH1 fragment, the C-terminus of the first CH1 fragment is connected to the N-terminus of the first Fc fragment, the C-terminus of the first Fc fragment is connected to the N-terminus of the second linker peptide, and the C-terminus of the second linker peptide is connected to the N-terminus of the IL-15 fragment or the IL-15Rα fragment; or, the C-terminus of the IL-15 fragment or the IL-15Rα fragment is connected to the N-terminus of the second linker peptide, the C-terminus of the second linker peptide is connected to the N-terminus of the first Fc fragment, the C-terminus of the first Fc fragment is connected to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is connected to the N-terminus of the first CL fragment, the C-terminus of the first CL fragment is connected to the N-terminus of the first heavy chain variable region, and / or, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first CH1 fragment; or, the C-terminus of the first light chain variable region is connected to the N-terminus of the first CL fragment. The N-terminus of the first CL fragment is connected to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is connected to the N-terminus of the first heavy chain variable region, and / or, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first CH1 fragment, the C-terminus of the first CH1 fragment is connected to the N-terminus of the first Fc fragment, the C-terminus of the first Fc fragment is connected to the N-terminus of the second linker peptide, and the C-terminus of the second linker peptide is connected to the N-terminus of the IL-15 fragment or the IL-15Rα fragment; or, the IL-15 The C-terminus of the fragment or IL-15Rα fragment is connected to the N-terminus of the second linker peptide, the C-terminus of the second linker peptide is connected to the N-terminus of the first Fc fragment, the C-terminus of the first Fc fragment is connected to the N-terminus of the first light chain variable region, the C-terminus of the first light chain variable region is connected to the N-terminus of the first CL fragment, the C-terminus of the first CL fragment is connected to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is connected to the N-terminus of the first heavy chain variable region, and / or, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first CH1 fragment.

[0067] According to an embodiment of the present invention, the first Fc fragment has at least one of the following mutation sites relative to the amino acid sequence of the Fc fragment of human wild-type IgG1: L234A, L235A, S354C, and T366W.

[0068] The amino acid sequence (including hinge-CH2-CH3) of human wild-type IgG1 Fc is shown below in this paper:

[0069] PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:18).

[0070] According to an embodiment of the present invention, the first Fc fragment has an amino acid sequence as shown in SEQ ID NO:7.

[0071] CD16 heavy chain constant region (including hinge-CH2-CH3):

[0072] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:7).

[0073] According to an embodiment of the present invention, the amino acid sequence of the first linker peptide is (GGGGS)n, where n is an integer greater than or equal to 1, preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, more preferably 10, 11, 12, 13 or 14.

[0074] According to an embodiment of the present invention, the first linker peptide has an amino acid sequence as shown in SEQ ID NO:8.

[0075] GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 8).

[0076] According to an embodiment of the present invention, the first CL fragment is a wild-type CL fragment of human, primate, or mouse origin, preferably a wild-type human CL fragment.

[0077] According to an embodiment of the present invention, the first CL fragment has an amino acid sequence as shown in SEQ ID NO:9.

[0078] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9).

[0079] According to an embodiment of the present invention, the first CH1 fragment comprises a CH1 fragment of wild-type IgG1 selected from human, primate, or mouse sources.

[0080] According to an embodiment of the present invention, the first CH1 fragment has an amino acid sequence as shown in SEQ ID NO:10.

[0081] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE (SEQ ID NO: 10).

[0082] According to an embodiment of the present invention, the second linker peptide has an amino acid sequence as shown in SEQ ID NO:11.

[0083] PSGQAGAAASESLFVSNHAY (SEQ ID NO: 11).

[0084] In a preferred embodiment of the present invention, the C-terminus of the first light chain variable region is connected to the N-terminus of the first CL fragment, the C-terminus of the first CL fragment is connected to the N-terminus of the first linker peptide, the C-terminus of the first linker peptide is connected to the N-terminus of the first heavy chain variable region, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first CH1 fragment, the C-terminus of the first CH1 fragment is connected to the N-terminus of the first Fc fragment, the C-terminus of the first Fc fragment is connected to the N-terminus of the second linker peptide, and the C-terminus of the second linker peptide is connected to the N-terminus of the IL-15Rα fragment. This further improves the therapeutic effect on cancers expressing CD138.

[0085] Optionally, the first binding region has an amino acid sequence as shown in SEQ ID NO:19.

[0086] (SEQ ID NO:19).

[0087] According to an embodiment of the present invention, the second binding region includes a first polypeptide and a second polypeptide, the first polypeptide and the second polypeptide are linked by interchain disulfide bonds, the first polypeptide includes a second light chain variable region, and the second polypeptide includes a second heavy chain variable region.

[0088] According to an embodiment of the present invention, the first polypeptide further includes a second CL fragment, wherein the C-terminus of the second light chain variable region is connected to the N-terminus of the second CL fragment.

[0089] According to an embodiment of the present invention, the second polypeptide further includes a second CH1 fragment and a second Fc peptide, wherein the C-terminus of the second heavy chain variable region is connected to the N-terminus of the second CH1 fragment, the C-terminus of the second CH1 fragment is connected to the N-terminus of the second Fc peptide, and the C-terminus of the second Fc peptide is connected to the N-terminus of the IL-15 fragment or the IL-15Rα fragment.

[0090] According to an embodiment of the present invention, the second polypeptide further includes a third linker peptide, wherein the C-terminus of the second heavy chain variable region is linked to the N-terminus of the second CH1 fragment, the C-terminus of the second CH1 fragment is linked to the N-terminus of the second Fc peptide, the C-terminus of the second Fc peptide is linked to the N-terminus of the third linker peptide, and the C-terminus of the third linker peptide is linked to the N-terminus of the IL-15 fragment or the IL-15Rα fragment.

[0091] According to an embodiment of the present invention, the second Fc fragment has at least one of the following mutation sites relative to the amino acid sequence of the Fc fragment of human wild-type IgG1: L234A, L235A, Y349C, T366S, L368A, and Y407V.

[0092] According to an embodiment of the present invention, the second Fc peptide has an amino acid sequence as shown in SEQ ID NO:12.

[0093] PKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:12).

[0094] According to an embodiment of the present invention, the second CL fragment is a wild-type CL fragment of human, primate, or mouse origin, preferably a wild-type human CL fragment.

[0095] According to an embodiment of the present invention, the second CL fragment has an amino acid sequence as shown in SEQ ID NO:13.

[0096] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13).

[0097] According to an embodiment of the present invention, the second CH1 fragment includes a CH1 fragment of wild-type IgG1 selected from human, primate, or mouse sources.

[0098] According to an embodiment of the present invention, the second CH1 fragment has an amino acid sequence as shown in SEQ ID NO:14.

[0099] ASTKGPSVFPLAPSSKSGSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE (SEQ ID NO: 14).

[0100] According to an embodiment of the present invention, the third linker peptide has an amino acid sequence as shown in SEQ ID NO:15.

[0101] EASGGPE (SEQ ID NO:15).

[0102] According to an embodiment of the present invention, the first polypeptide has an amino acid sequence as shown in SEQ ID NO:16.

[0103] DVVMTQSPLSLPVTLGQPASISCRSQQSLLHSNANTYLHWFQQRPGQSPRLLIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCSQSSRIPWTTFGGGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:16).

[0104] According to an embodiment of the present invention, the second polypeptide has an amino acid sequence as shown in SEQ ID NO:17.

[0105] (SEQ ID NO:17).

[0106] According to an embodiment of the present invention, the first bonding region and the second bonding region are connected by a knot-into-hole structure.

[0107] Nucleic acid molecules, expression vectors and recombinant cells

[0108] In another aspect of the invention, a nucleic acid molecule is proposed. According to an embodiment of the invention, the nucleic acid molecule encodes the aforementioned trispecific antibody. The nucleic acid molecule according to an embodiment of the invention encodes the aforementioned trispecific antibody.

[0109] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0110] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0111] In another aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule. When the nucleic acid molecule is ligated to the vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself or can be exogenous, i.e., not derived from the vector itself. Of course, the nucleic acid molecule and the control elements can be operably linked. In this document, "operably linked" means ligating a foreign gene to the vector so that the control elements within the vector, such as transcription control sequences and translation control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. After the expression vector of some specific embodiments of the present invention is introduced into suitable recipient cells, the aforementioned trispecific antibody can be effectively expressed under the mediation of a regulatory system, thereby achieving the large-scale in vitro production of trispecific antibodies.

[0112] According to embodiments of the present invention, the expression vector includes those selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0113] According to an embodiment of the present invention, the expression vector is a plasmid expression vector.

[0114] In another aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises: carrying the aforementioned nucleic acid molecule or the aforementioned expression vector; or expressing the nucleic acid molecule encoding the aforementioned trispecific antibody. Using this recombinant cell, under suitable conditions, the aforementioned trispecific antibody can be effectively expressed intracellularly.

[0115] It should be noted that the "suitable conditions" mentioned in this application specification refer to conditions suitable for the expression of the trispecific antibody described in this invention. Those skilled in the art will readily understand that suitable conditions for the expression of the trispecific antibody include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the trispecific antibody based on the specific environment of their laboratory.

[0116] According to an embodiment of the present invention, the recombinant cells are obtained by introducing the aforementioned expression vector into host cells.

[0117] According to an embodiment of the present invention, the recombinant cell is a eukaryotic cell, preferably a mammalian cell.

[0118] Pharmaceutical Composition

[0119] In another aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: the aforementioned trispecific antibody, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell. As is known before, the trispecific antibody simultaneously binds to CD138 on tumor cells, CD16 on NK cells, and IL-15 receptors. Therefore, a drug containing the aforementioned trispecific antibody can, by binding to CD138 molecules on tumor cells, induce CD16+ lymphocytes to form an immune synapse with tumor cells, thereby activating lymphocytes to kill CD138+ tumor cells. Simultaneously, the trispecific antibody can bind to IL-15 receptors on the surface of NK cells, providing a stimulating signal for NK cell survival and proliferation, and can be used for the prevention and / or treatment of cancer or tumors.

[0120] According to embodiments of the present invention, pharmaceutically acceptable excipients are further included.

[0121] The pharmaceutical compositions of the present invention can be administered by any acceptable method of administration. The pharmaceutical compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms, such as injections or lyophilized powders, and current methods for preparing these dosage forms are known or obvious to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal routes. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of the present invention are formulated to allow the bioactive components contained therein to be bioavailable after administration to a patient.

[0122] use

[0123] In another aspect of the invention, the invention provides for the use of the aforementioned trispecific antibody or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of cancer or tumors.

[0124] In another aspect of the invention, the invention proposes the use of the aforementioned trispecific antibody or the aforementioned pharmaceutical composition in the prevention and / or treatment of cancer or tumors.

[0125] In another aspect of the invention, the invention provides the aforementioned trispecific antibody or the aforementioned pharmaceutical composition for the prevention and / or treatment of cancer or tumors.

[0126] According to embodiments of the present invention, the above-described uses may further include at least one of the following technical features:

[0127] The cancers include at least one of multiple myeloma, lung cancer, stomach cancer, colorectal cancer, pancreatic cancer, breast cancer, and bladder cancer.

[0128] Methods for treating or preventing cancer or tumors

[0129] In another aspect, the present invention provides a method for treating or preventing cancer or tumors. According to embodiments of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned trispecific antibody or the aforementioned pharmaceutical composition. The method of the present invention can effectively treat or prevent cancer or tumors.

[0130] The effective amount of the trispecific antibody described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0131] The trispecific antibodies of the present invention can be incorporated into drugs suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms, such as liquid, semi-solid, and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical drugs are in the form of injection solutions or infusion solutions. The aforementioned trispecific antibodies can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0132] According to embodiments of the present invention, the administration route of the method is subcutaneous injection or intravenous injection.

[0133] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0134] Example 1: Design and construction of Tri1613815 trispecific binding protein (also known as trispecific antibody)

[0135] Through screening of various configurations, the trispecific antibody configuration was determined, and this antibody was named Tri1613815. This antibody contains three units; see details below. Figure 1 As shown. The first unit is an IgG-like anti-CD16 form, having a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:2; the second unit is an anti-CD138 variable region sequence, having a heavy chain variable region as shown in SEQ ID NO:3 and a light chain variable region as shown in SEQ ID NO:4; the third unit is a human IL-15 / IL-15Rα fragment, having an IL-15 fragment as shown in SEQ ID NO:5 and an IL-15Rα fragment as shown in SEQ ID NO:6.

[0136] Using this design principle, the inventors designed three polypeptide chains. The first polypeptide chain includes the CD16 light chain variable region, light chain constant region, linker peptide 1, CD16 heavy chain variable region, heavy chain constant region, linker peptide 2, and human IL-15Rα, and its amino acid sequence is shown in SEQ ID NO:19. The second polypeptide chain includes the CD138 heavy chain variable region, heavy chain constant region, linker peptide 3, and human IL-15, and its amino acid sequence is shown in SEQ ID NO:17. The third polypeptide chain includes the CD138 light chain variable region and light chain constant region, and its amino acid sequence is shown in SEQ ID NO:16. The heavy chain constant regions of the first and second polypeptide chains are derived from the human antibody IgG1. Due to the molecule's unique asymmetric structure, to reduce the formation of homodimers, different amino acid mutations were introduced into the constant regions of the first polypeptide chain (containing S354C and T366W mutations) and the second polypeptide chain (containing Y349C, T366S, L368A, and Y407V mutations), forming a knock-in-hole structure. Simultaneously, to prevent cross-linking activation caused by the Fcγ receptor, (L234A / L235A) mutations were also introduced into the constant regions of the first and second polypeptide chains. For specific design configurations, refer to [reference needed]. Figure 1 .

[0137] The nucleotide sequence of the first polypeptide chain is shown in SEQ ID NO:20, and it encodes the amino acid sequence shown in SEQ ID NO:19:

[0138]

[0139] The nucleotide sequence of the second polypeptide chain is shown in SEQ ID NO:21, and it encodes the amino acid sequence shown in SEQ ID NO:17:

[0140]

[0141] The nucleotide sequence of the third polypeptide chain is shown in SEQ ID NO:22, and it encodes the amino acid sequence shown in SEQ ID NO:16:

[0142] (SEQ ID NO:22).

[0143] Example 2: Expression and purification of three specific binding proteins

[0144] The nucleotide sequences of the first, second, and third polypeptide chains encoding the humanized antibody from Example 1 (as shown in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22, respectively) were recombinantly cloned into the common expression vector pTT5 plasmid using molecular biology techniques. The specific preparation method is as follows:

[0145] Antibodies were prepared by transiently transfecting ExpiCHO-S cells (Gibco, catalog number A29127) with the pTT5 vector carrying the above-mentioned three polypeptide chain nucleotide sequences. One day before transfection, the ExpiCHO-S cells were adjusted to a cell density of (3–4) × 10⁻⁶ cells. 6 / mL, cultured overnight at 37℃, 8% CO2, and 120 rpm with shaking. On the day of transfection, cells grew to 7 × 10⁹ / mL. 6 ~1×10 7 When the cell viability is greater than 95%, prepare for transfection by diluting the cells to 6 × 10⁶ / mL using fresh, pre-warmed ExpiCHO medium (Gibco, catalog number A2910002). 6 Transfect pTT5 plasmid (plasmid ratio 1:1:2 or other ratio) carrying the first, second, and third polypeptide chains and ExpiFectamine CHO transfection reagent (Gibco, catalog number A29129) into ExpiCHO-S cells at 37°C, 8% CO2, and 120 rpm with shaking. 18-22 h post-transfection, immediately add ExpiFectamine CHO Enhancer and ExpiCHO Feed to the transfected cells, mix well, and incubate at 32°C, 5% CO2, and 120 rpm with shaking. On day 5 post-transfection, add another 8 mL of ExpiCHO Feed to the cells, mix well, and continue culturing. Observe cell count and cell viability daily. Harvest cells by centrifugation when cell viability drops below 80% or after 10-14 days of culture. The expression supernatant was filtered through a 0.22 μm filter membrane. A Mabselect Prism A affinity chromatography column (GE, catalog number 17549854) was used to capture the trispecific antibody containing the Fc domain from the expression supernatant. After equilibrating the column with phosphate buffer (pH 7.2), the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100 mM citrate, pH 2.7). Finally, the elution was concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE as having a purity of over 95%, indicating that a trispecific antibody (also known as Tri1613815 antibody) was finally obtained.

[0146] Example 3: Assay of the binding activity of trispecific antibodies to antigens (ELISA)

[0147] 3.1 Assay for the binding activity of trispecific antibodies to single antigens

[0148] Human CD16 antigen (ACRO biosystems, catalog number CDA-H5220) or CD138 antigen (ACRO biosystems, catalog number SY1-H5225) was diluted to 2 μg / mL with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), and 100 μL was added to each well of an ELISA plate and incubated overnight at 4°C. The plate was then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate and incubated at room temperature for 2 h. The plate was then washed three more times with PBST. Different concentrations of the trispecific antibody prepared in Example 2 were added to each well and incubated at room temperature for 1 h. The plate was then washed three more times with PBST. Add 100 μL of HRP-rabbit anti-human IgG secondary antibody (boster, catalog number BA1070) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash three times with PBST, add TMB to each well, and incubate at room temperature in the dark for 2-5 minutes. Stop the reaction with 2M sulfuric acid in each well. Finally, read the OD values ​​using a microplate reader. 450 Numerical values, results can be found in [link / reference]. Figure 2 A and Figure 2 B.

[0149] in, Figure 2 A demonstrates that the Tri1613815 trispecific binding protein can bind to the CD16 antigen; Figure 2 B demonstrates that the Tri1613815 trispecific binding protein can bind to the CD138 antigen.

[0150] 3.2 Assay of the binding activity of the trispecific antibody to CD16 and CD138 dual antigens

[0151] To test whether the trispecific antibody Tri1613815 could simultaneously bind to both CD16 and CD138 antigens to achieve bridging, human CD16 antigen (ACRO biosystems, catalog number CDA-H5220) was diluted to 2 μg / mL with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), and 100 μL was added to each well of an ELISA plate and incubated overnight at 4°C. The plate was then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate and incubated at room temperature for 2 hours. The plate was then washed three more times with PBST. Different concentrations of the trispecific antibody prepared in Example 2 were added to each well and incubated at room temperature for 1 hour. The plate was then washed three more times with PBST. Add 100 μL of biotinylated human CD138 antigen (ACRO biosystems, catalog number SY1-H82E2) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash three times with PBST. Add 100 μL of HRP-labeled streptavidin secondary antibody (SouthernBiotech, catalog number 7105-05) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash three times with PBST, add TMB to each well, and incubate at room temperature in the dark for 2-5 minutes. Stop the reaction with 2M sulfuric acid in each well. Finally, read the OD using a microplate reader. 450 Numerical values, specific results as follows Figure 2 As shown in C.

[0152] The results showed that as the concentration of trispecific antibody molecules increased, its OD... 450 The readings gradually increased, indicating that the trispecific antibody molecule can bind to both human CD16 and CD138 simultaneously.

[0153] 3.3 Assay of the binding activity of the trispecific antibody to IL-15Rβ / IL-15Rγ and CD16

[0154] To determine whether the trispecific antibody Tri1613815 can bind to the IL-15 receptor and simultaneously bind to CD16 to specifically target NK cells, human CD16 antigen (ACRO biosystems, catalog number CDA-H5220) was diluted to 2 μg / mL with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), and 100 μL was added to each well of an ELISA plate and incubated overnight at 4°C. The plate was then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate and incubated at room temperature for 2 h. The plate was then washed three more times with PBST. Different concentrations of the trispecific antibody prepared in Example 2 were added to each well and incubated at room temperature for 1 h. The plate was then washed three more times with PBST. Add 100 μL of biotinylated human IL-15Rβ / IL-15Rγ protein (ACRO biosystems, catalog number ILG-H82F3) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash three times with PBST. Add 100 μL of HRP-labeled streptavidin secondary antibody (SouthernBiotech, catalog number 7105-05) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash three times with PBST, add TMB to each well, and incubate at room temperature in the dark for 2-5 minutes. Stop the reaction with 2M sulfuric acid in each well. Finally, read the OD using a microplate reader. 450 Numerical values ​​were obtained. A CD16 monoclonal antibody (the heavy chain, from N-terminus to C-terminus, includes the heavy chain variable region as shown in SEQ ID NO:1, the CH1 fragment as shown in SEQ ID NO:10, and the wild-type IgG1 Fc fragment as shown in SEQ ID NO:18; the light chain, from N-terminus to C-terminus, includes the light chain variable region as shown in SEQ ID NO:2 and the CL fragment as shown in SEQ ID NO:9) was used as a control group. Specific results are as follows: Figure 3 As shown.

[0155] The results showed that as the concentration of trispecific antibody molecules increased, its OD... 450 The readings gradually increased, indicating that the trispecific antibody molecule can bind to human IL-15Rβ / IL-15Rγ and CD16 simultaneously.

[0156] Example 4: In vitro killing experiment of the three specific binding proteins

[0157] 1. Multiple myeloma cell killing assay

[0158] Multiple myeloma MM.1S cells or NCI-H929 cells were labeled with CellTrace Violet (thermo, C34557), and the cell density was adjusted to 1×10⁻⁶.5 / ml. PBMCs adjusted cell density 1×10⁻⁶ 6 PBMCs and labeled NCI-H929 cells were mixed at a 1:1 ratio and added to 96-well round-bottom plates. Then, gradient concentrations of the trispecific antibody prepared in Example 2 were added and mixed thoroughly. After incubation at 37°C for 24 h, the mixture was transferred to flow cytometry tubes through a 200-mesh nylon mesh. 7AAD (7-Aminoactinomycin D) was added, and the cell killing efficiency was measured after 10 min. The CTV+7AAD+ cell ratio was used to characterize the killing efficiency. Results are shown below. Figure 4 .

[0159] The results showed that as the concentration of the trispecific antibody Tri1613815 increased, PBMCs reduced their effectiveness against MM.1S cells (…). Figure 4 A) and NCI-H929 cells ( Figure 4 The killing efficiency of B) gradually increased, indicating that the trispecific antibody can promote the killing of multiple myeloma cells by PBMC.

[0160] 2. Pancreatic cancer cell killing experiment

[0161] Pancreatic cancer PANC1 cells were digested, counted, and the cell density was adjusted to 2 × 10⁻⁶. 5 / ml. Turn on the RTCA instrument (Agilent), select instrument type DP, select experimental mode, and fill in cell and drug information. Enter the schedule to set the experimental steps. Add 50μl of fresh culture medium (89% RPMI 1640 medium + 10% fetal bovine serum + 1% penicillin and streptomycin) to the plate and place it in the instrument. Close the instrument and click Start. After completion, remove the plate, add 100μl of cell suspension, incubate at room temperature for 15-30 minutes to prevent edge effects, place it in the instrument, and click Start. After growth reaches the logarithmic growth phase, pause and add 50μl of PBMC (4×10⁻⁶). 6 / ml), and added gradient concentrations of the trispecific antibody Tri1613815 prepared in Example 2, clicked start, and analyzed after a period of time. The results are shown in [link to relevant documentation]. Figure 5 .

[0162] The results showed that as the concentration of the trispecific antibody Tri1613815 increased, the killing efficiency of PBMC against pancreatic cancer PANC1 cells gradually increased, indicating that the trispecific antibody can promote the killing of pancreatic cancer cells by PBMC.

[0163] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0164] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A trispecific antibody, characterized in that, It consists of a first bonding region, a second bonding region, and a third bonding region: A third binding region, wherein the third binding region has IL-15 receptor binding activity, and the third binding region is composed of an IL-15 fragment and an IL-15Rα fragment; The first binding region, having CD16 binding activity, comprises a first light chain variable region, a first CL fragment, a first linker peptide, a first heavy chain variable region, a first CH1 fragment, a first Fc fragment, and a second linker peptide; wherein, the C-terminus of the first light chain variable region is connected to the N-terminus of the first CL fragment, the C-terminus of the first CL fragment is connected to the N-terminus of the first linker peptide, the C-terminus of the first heavy chain variable region is connected to the N-terminus of the first CH1 fragment, the C-terminus of the first CH1 fragment is connected to the N-terminus of the first Fc fragment, the C-terminus of the first Fc fragment is connected to the N-terminus of the second linker peptide, and the C-terminus of the second linker peptide is connected to the N-terminus of one of the IL-15 fragment and the IL-15Rα fragment in the third binding region; The second binding region, which has CD138 binding activity, is composed of a second light chain variable region, a second CL fragment, a second heavy chain variable region, a second CH1 fragment, a second Fc peptide, and a third linker peptide. The C-terminus of the second light chain variable region is connected to the N-terminus of the second CL fragment to form a first polypeptide. The C-terminus of the second heavy chain variable region is connected to the N-terminus of the second CH1 fragment, the C-terminus of the second CH1 fragment is connected to the N-terminus of the second Fc peptide, the C-terminus of the second Fc peptide is connected to the N-terminus of the third linker peptide, and the C-terminus of the third linker peptide is connected to the N-terminus of another fragment in the third binding region, namely the IL-15 fragment and the IL-15Rα fragment, to form a second polypeptide. in: The first bonding region and the second bonding region are connected by a knock-into-hole structure; The amino acid sequence of the IL-15 fragment is shown in SEQ ID NO:5; The amino acid sequence of the IL-15Rα fragment is shown in SEQ ID NO:6; The first light chain variable region has an amino acid sequence as shown in SEQ ID NO:2; The first CL fragment is a wild-type CL fragment of primate or rodent origin; The amino acid sequence of the first linker peptide is (GGGGS)n, where n is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; The first heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:1; The first CH1 fragment is selected from the CH1 fragment of wild-type IgG1 of primate or mouse origin; The first Fc fragment, relative to the amino acid sequence of the Fc fragment of human wild-type IgG1, has at least one of the following mutation sites: L234A, L235A, S354C, and T366W; The second linker peptide has the amino acid sequence shown in SEQ ID NO:11; The second light chain variable region has an amino acid sequence as shown in SEQ ID NO:4; The second CL fragment is a wild-type CL fragment of primate or rodent origin; The second heavy chain variable region has an amino acid sequence as shown in SEQ ID NO:3; The second CH1 fragment is selected from the CH1 fragment of wild-type IgG1 of primate or mouse origin; The second Fc fragment, relative to the amino acid sequence of the Fc fragment of human wild-type IgG1, has at least one of the following mutation sites: L234A, L235A, Y349C, T366S, L368A, and Y407V; The third linker peptide has the amino acid sequence shown in SEQ ID NO:

15.

2. The trispecific antibody according to claim 1, characterized in that, The first CL fragment is a human-derived wild-type CL fragment; Optionally, the second CL fragment is a wild-type CL fragment of human origin.

3. The trispecific antibody according to claim 1, characterized in that, The first CH1 fragment is the CH1 fragment of human wild-type IgG1; Optionally, the second CH1 fragment is a CH1 fragment of human wild-type IgG1.

4. The trispecific antibody according to claim 1, characterized in that, The first Fc fragment has the amino acid sequence shown in SEQ ID NO:7; Optionally, n can be 10, 11, 12, 13 or 14; Optionally, the first CL fragment is a human wild-type CL fragment; Optionally, the first CH1 fragment has an amino acid sequence as shown in SEQ ID NO:

10.

5. The trispecific antibody according to claim 1, characterized in that, The first linker peptide has the amino acid sequence shown in SEQ ID NO:

8.

6. The trispecific antibody according to claim 1, characterized in that, The first CL fragment has the amino acid sequence shown in SEQ ID NO:

9.

7. The trispecific antibody according to claim 1, characterized in that, The second Fc peptide has the amino acid sequence shown in SEQ ID NO:12; Optionally, the second CL fragment is a human wild-type CL fragment; Optionally, the second CH1 fragment has an amino acid sequence as shown in SEQ ID NO:

14.

8. The trispecific antibody according to claim 1, characterized in that, The first polypeptide has the amino acid sequence shown in SEQ ID NO:16; Optionally, the second polypeptide has an amino acid sequence as shown in SEQ ID NO:

17.

9. The trispecific antibody according to claim 1, characterized in that, The second CL fragment has the amino acid sequence shown in SEQ ID NO:

13.

10. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the trispecific antibody as described in any one of claims 1 to 9.

11. The nucleic acid molecule according to claim 10, characterized in that, The nucleic acid molecule in question is DNA.

12. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 10 or 11.

13. The expression vector according to claim 12, characterized in that, The expression vectors include those selected from eukaryotic expression vectors or prokaryotic expression vectors.

14. The expression vector according to claim 12, characterized in that, The expression vector is a plasmid expression vector.

15. A recombinant cell, characterized in that, include: Carrying the nucleic acid molecule according to any one of claims 10-11 or the expression vector according to any one of claims 12-14; or, Express the trispecific antibody as described in any one of claims 1 to 9.

16. The recombinant cell according to claim 15, characterized in that, The recombinant cells are obtained by introducing the expression vector according to any one of claims 12 to 14 into a host cell.

17. The recombinant cell according to claim 15, characterized in that, The recombinant cells are eukaryotic cells.

18. The recombinant cell according to claim 15, characterized in that, The recombinant cells are mammalian cells.

19. A pharmaceutical composition, characterized in that, include: The trispecific antibody according to any one of claims 1 to 9, the nucleic acid molecule according to any one of claims 10 to 11, the expression vector according to any one of claims 12 to 14, or the recombinant cell according to any one of claims 15 to 18.

20. The pharmaceutical composition according to claim 19, characterized in that, This further includes pharmaceutically acceptable excipients.

21. Use of the trispecific antibody according to any one of claims 1 to 9 or the pharmaceutical composition according to any one of claims 19 to 20 in the preparation of a medicament for the treatment of cancer; The cancer is selected from at least one of multiple myeloma, pancreatic cancer, breast cancer, and bladder cancer.

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