Trispecific antibodies and uses thereof

Abstract

The present invention relates to a trispecific antibody that specifically binds to CD19, CD3, and CD28, which comprises a fragment derived from the IgD hinge region as a linker. The present invention further relates to the use of the trispecific antibody for treating tumors, such as hematological malignancies.

Description

[Technical Field]

[0001] Related Applications This application claims priority from a Chinese patent application filed on July 11, 2022, with application number 202210822959.8, entitled "Trispecific Antibody and Use Thereof," the contents of which are incorporated herein by reference in their entirety.

[0002] [Technical field] The present invention is in the field of antibody drugs. The present invention provides a trispecific antibody that specifically binds to CD19, CD3, and CD28, and includes a fragment derived from the IgD hinge region as a linker sequence. The antibody is particularly suitable for use as a tumor therapeutic agent, especially for the treatment of relapsed or refractory B-cell malignancies. [Background technology]

[0003] Antibody immunotherapeutics are a hot topic in the field of tumor treatment. Tumor immunotherapy specifically eliminates microscopic residual tumor foci and suppresses tumor growth by activating immune cells and enhancing the body's anti-tumor immune response. Due to its minimal side effects and clear therapeutic efficacy, it is becoming a trend in future tumor treatment. Immune checkpoint blockade (ICB) can induce durable responses in a variety of solid tumor types and is currently considered the most successful immunotherapy. However, the majority of cancer patients still do not respond to treatment. These patients lack tumor immunogenicity and intrinsic MHC antigen presentation function. Therefore, other methods that can promote immune cell-mediated tumor cell killing are needed.

[0004] Bispecific T-cell engager (BiTE) molecules consist of two single-chain variable fragments (scFv), one of which targets the CD3ε chain of the T-cell receptor (TCR) on T cells and the other of which targets a tumor-associated antigen (TAA), allowing polyclonal T cells to induce cancer cell-directed lysis in an MHC-independent manner. Although the effective dose of bispecific T-cell engagers is more than 10,000-fold lower than that of typical antibodies, they have several limitations, including dose-limiting toxicity, poor pharmacokinetic properties, and limited therapeutic efficacy.

[0005] Naive T lymphocytes require the synergistic action of two activation signals to move from resting to full activation. When an antigen / MHC complex is recognized by the TCR, the first signal is transmitted via CD3. However, this signal is insufficient to fully activate T cells, and must be supplemented by signals generated by costimulatory molecules. A lack of costimulatory signals can easily lead to T cell incapacity or activation-induced T cell apoptosis (Non-Patent Document 1).

[0006] Among the currently discovered T cell co-activating molecules, CD28 and the B7 family are the most important co-stimulatory molecules. They bind to the ligands CD80 (B7-1) and CD86 (B7-2) to amplify the first signal of TCR / CD3 conduction, maintain T cell survival, and promote cytokine-induced T cell proliferation and differentiation. CD28 is a dimeric transmembrane glycoprotein and is expressed by approximately 80% of human CD4 + T cells and 50% CD8 + Because it is expressed on the surface of T cells, it is considered a good target for T cell activation (Non-patent Document 2; Non-patent Document 3).

[0007] CD28 costimulatory signals can extend T cell survival through two mechanisms: exogenous and endogenous. The exogenous mechanism primarily promotes the release of cytokines, particularly IL-2, which plays an important role in T cell development. The endogenous mechanism is manifested by enhancing the expression of the anti-apoptotic protein Bcl-XL (Non-Patent Document 4). When CD3 or CD28 signals are present alone, Bcl-XL expression is relatively weak, whereas when both signals are present, Bcl-XL expression is significantly enhanced (Non-Patent Document 5). Furthermore, CD28 costimulatory signals can promote the proliferation of effector T cells and memory T cells, among which memory T cells are rapidly activated when the body encounters similar tumor cells again, thereby effectively eliminating the tumor cells.

[0008] The importance of costimulatory signals for T cell activation has also been clearly demonstrated in the development of chimeric antigen receptor T cell (CAR-T)-based drugs (Non-Patent Document 6; Non-Patent Document 7). The structure of first-generation CAR molecules consists only of an extracellular scFv of a target tumor-associated antigen and a CD3ζ chain used for intracellular T cell activation. This limited proliferation of T cells bearing this type of CAR molecule and poor durability of pharmacological action prevented successful clinical validation. Second- and third-generation CAR molecules further incorporate costimulatory signal domains (e.g., CD28, 4-1BB, OX40, etc.), significantly enhancing the activation and durability of pharmacological action of CAR-T cells bearing this type of CAR molecule. Currently, cell therapies designed based on second-generation CARs have already been successfully applied to the treatment of several hematological cancers (Non-Patent Document 8; Non-Patent Document 9).

[0009] Based on BiTE, the present invention applies the principle of activating T cells with two signals to the design of multifunctional antibodies, developing a dual-targeting antibody that targets the scFv domains of CD3 and CD28 on T cells, respectively, and also adding the scFv domain of a target tumor cell-associated antigen (TAA) to form a trispecific antibody, thereby simultaneously achieving targeting of tumor cells and double-signal activation of T cells.

[0010] In our prior patent application, Patent Document 1, we disclosed a trispecific antibody that simultaneously binds to CD19, CD3, and CD28. The trispecific antibody in this application uses an 81-amino acid IgD hinge region sequence as a linker between the second and third domains, and a cysteine contained in the linker sequence allows the trispecific antibody to form a dimer. In this application, in vitro experiments were conducted to verify the expression of the trispecific antibody's monomer and dimer, its antigen-binding activity, cell binding assays (the antibody's ability to bind CD19-positive target cells and CD3 / CD28-positive effector cells to form a group), and cell-killing ability (the antibody was co-cultured with lymphoma cells and CIK cells and the target cell death was observed). However, no in vivo experiments were conducted to verify the efficacy of this type of antibody as a tumor therapeutic agent.

[0011] In fact, such trispecific antibody molecules can, in principle, achieve better efficacy; however, due to the complexity of their molecular structure, there is a need for continued creative work in this field to improve the structure and function of trispecific antibody molecules in order to provide new, safe and effective antitumor drugs, as it is difficult to ensure that each binding domain can exert its intended effect. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] Chinese Patent Application Publication No. 106589129 [Non-patent literature]

[0013] [Non-Patent Document 1] Schietinger A and Greenberg PD. Tolerance and exhaustion: defining mechanisms of T cell dysfunction. Trends in Immunology. 2014; 35: 51-60. [Non-patent document 2] Jeong S and Park SH. Co-stimulatory receptors in cancers and their implications for cancer immunotherapy. Immune Netw. 2020; 20(1): e3. [Non-patent document 3] Esensten JH, Helou YA, Chopra G, et al. CD28 costimulation: from mechanism to therapy. Immunity. 2017; 44: 973-988. [Non-patent document 4] Boise LH, Minn AJ, Noel PJ, June CH, Accavitti MA, Lindsten T, Thompson CB. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-xL. Immunity. 2010; 3: 87-98. [Non-patent document 5] Watts TH. Staying alive: T cell costimulation, CD28, and Bcl-xL. J Immunol. 2010; 185: 3785-3787. [Non-patent document 6] Pettitt D, Arshad Z, Smith J, et al. CAR-T cells: a systematic review and mixed methods analysis of the clinical trial landscape. Molecular Therapy. 2018; 26: 342-353. [Non-Patent Document 7] Ma S, Li XC, Wang XY, et al. Current Progress in CAR-T cell therapy for solid tumors. Int J Biol Sci. 2019; 15: 2548-2560. [Non-patent document 8] Savoldo B, Ramos CA, Liu E, et al. CD28 costimulation improves expansion and persistence of chimeric antigen receptor-modified T cells in lymphoma patients. Immunology. 2011; 121: 1822-1826. [Non-Patent Document 9] Guedan S, Posey Jr., AD, Shaw C, et al. Enhancing CAR T cell persistence through ICOS and 4-1BB costimulation. Immunology. 2018; 3(1): e96976. Summary of the Invention

[0014] Based on their previous research, the inventors have further developed a new T-cell engager antibody, which simultaneously binds to CD19, CD3, and CD28, and has designed a completely new linker sequence to connect the second and third domains, thereby completing the present invention.

[0015] In a first aspect, the present invention relates to a trispecific antibody, comprising: (1) a first binding domain that specifically binds to CD19; (2) a first linker sequence; (3) a second binding domain that specifically binds to CD3; (4) a second linker sequence, and (5) A third binding domain that specifically binds to CD28.

[0016] In a preferred embodiment, the first linker sequence is a flexible linker. In a specific embodiment, the first linker sequence is (G4S) n , (G4S) n G4, (SG4) n , G4(SG4) n , (G2S) n , (G2S) n G2, (SG2) n or G2 (SG2) n is the linker sequence of

[0017] In a preferred embodiment, the amino acid sequence of the second linker sequence does not contain a cysteine, so that the trispecific antibody cannot form a dimer. In a preferred embodiment, the amino acid sequence of the second linker sequence does not contain a glycosylation site. In a specific embodiment, the second linker sequence is an amino acid sequence derived from an IgD hinge region, or (EAAAK) n It is an amino acid sequence comprising:

[0018] In a second aspect, the invention provides an isolated nucleic acid molecule encoding the nucleotide sequence of the trispecific antibody of the first aspect.

[0019] In a third aspect, the invention provides an expression vector comprising the isolated nucleic acid molecule of the second aspect.

[0020] In a fourth aspect, the invention provides a host cell comprising an expression vector of the third aspect.

[0021] In a fifth aspect, the invention provides a method of preparing a multispecific antibody according to the first aspect.

[0022] In a sixth aspect, the present invention provides the use of a trispecific antibody according to the first aspect in the preparation of a medicament for the treatment of a B-cell-related disease, in particular a B-cell-related tumor, including B-cell leukemia and B-cell lymphoma. In particular, the B-cell-related tumor is a refractory or relapsed B-cell-related tumor. The B-cell lymphoma may be non-Hodgkin's lymphoma (NHL) or Hodgkin's lymphoma (HL). Examples of the B-cell-related diseases include classical Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma (FL), mucosa-associated lymphoid tissue lymphoma (MALT), small lymphocytic lymphoma (SLL), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), Burkitt lymphoma, acute lymphoblastic leukemia (ALL), chronic lymphatic leukemia (CLL), B cell prolymphocytic leukemia (B ... These include, but are not limited to, precursor B-cell lymphoblastic leukemia (B-PLL), precursor B-cell lymphoblastic leukemia, hairy cell leukemia (HCL), splenic B-cell lymphoma / leukemia, and splenic marginal zone lymphoma (SMZL).

[0023] In a seventh aspect, the invention relates to a method of treating a tumor, said method comprising the use of a trispecific antibody according to the first aspect.

[0024] In an eighth aspect, the present invention relates to a pharmaceutical composition comprising the trispecific antibody of the invention and a pharmaceutically acceptable vector.

[0025] In a ninth aspect, the invention relates to the combination of a trispecific antibody of the invention with another treatment or drug.

[0026] In a tenth aspect, the invention relates to the use of an amino acid fragment derived from the IgD hinge region as a linker sequence in a fusion protein.

[0027] The trispecific antibodies of the present invention have at least the following advantages:

[0028] (1) Long-lasting T cell activation provided by dual signaling mechanisms The trispecific antibodies of the present invention can simultaneously activate two T cell stimulatory signals, CD3 and CD28, and this "dual signal" activation mechanism more appropriately regulates TCR / CD3 signaling to generate long-lasting, effective immune responses in the body. That is, by inducing activated T cells rather than the drug itself to exert its effects in the body, the frequency of dosing can be reduced even when the drug has a relatively short half-life (compared to antibodies containing an Fc region), and there is no need to maintain long-term drug activation of T cells through frequent, continuous infusions.

[0029] Compared with the CD19 / CD3 bispecific antibody Blincyto® (blinatumomab), which also belongs to the T cell engager class, the trispecific antibody of the present invention offers significant convenience in terms of administration. Because blinatumomab lacks costimulatory signals, it is prone to T cell inactivation and activation-induced T cell apoptosis, necessitating frequent continuous infusions to maintain sustained drug-induced T cell activation.

[0030] Furthermore, compared to CAR-T cell therapy (e.g., CD19CAR-T), the trispecific antibody of the present invention can be industrially produced using production processes and routes similar to those of monoclonal antibodies, and can be widely applied to different individuals. In contrast, all approved CAR-T products and the vast majority of CAR-T clinical trials use autologous CAR-Ts, i.e., the patient's own T cells are used as the starting material. The production costs of such "privately customized" therapies are quite high, and the high price limits their market potential. Furthermore, the quality stability of CAR-T therapy is quite poor, with the patient's own T cells always lacking in quality and quantity, and there are significant quality differences between different product lots, making large-scale industrial production and standardized quality control difficult to achieve. Customized drug preparation and administration for various patients is required.

[0031] (2) Formation of immune synapses closer to the natural state Under physiological conditions, TCRs on T cells recognize antigenic peptides presented by the MHC of tumor cells, thereby forming an immune synapse (IS), a critical step in the T cell activation process. After immune synapse formation, the distance between T cells and tumor cells is typically approximately 15 nm. Some bispecific antibodies can simultaneously bind to both T cells and tumor cells, but due to limitations in their own structure and recognized antigen-binding epitopes, they are unable to draw tumor cells and T cells closer to the distance of approximately 15 nm. The present trispecific antibody, through structural optimization, achieves a synapse closer to the natural distance between T cells and tumor cells, demonstrating extremely high efficacy in polyclonal T cell-mediated tumor killing (Dickopf S, Georges GJ, Brinkmann U. Format and geometries matter: Structure-based design defines the functionality of bispecific antibodies. Computational and Structural Biotechnology Journal 18 (2020) 1221-1227).

[0032] (3) Convenience based on scFv structure Trispecific antibodies formed by scFvs also have the advantages of small molecular weight and strong tumor penetration. Compared to antibodies with Fc structures, antibodies with scFv structures have a shorter half-life. However, T cells activated by trispecific antibodies via the "dual signaling" activation mechanism are persistent and less prone to apoptosis after activation. Furthermore, trispecific antibodies utilizing the "dual signaling" activation mechanism can sufficiently activate T cells even at extremely low drug concentrations, enhancing T cell survival and inducing the differentiation of memory T cells, thereby achieving long-term killing of target cells.

[0033] (4) Realization of long-lasting immune responses by inducing memory T cells Existing preclinical research data have shown that the trispecific antibody of the present invention significantly enhances immune memory, induces the differentiation of memory T cells that have the ability to self-renew and replicate, have a long survival time in the body, and are capable of sustained tumor cell killing, primarily effector memory T cells (T EM ) and central memory T cells (T CM This is manifested in an increase in the number and proportion of tumors that can maintain a long-term effective immune response in vivo and prevent tumor recurrence.

[0034] (5) Advantages of the new linker sequence In the present invention, a new linker sequence is used to link the second and third binding domains. The linker sequence is a fragment derived from the hinge region of IgD and is obtained by modifying or removing glycosylation sites in the IgD hinge region, removing cysteines that may cause dimer formation, and / or shortening the overall length of the hinge region. The use of linker sequences obtained by these modifications in trispecific antibodies can result in more homogeneous antibody products, less generation of structural isomers or polymers, and reduced potential for off-target activation. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 is a schematic diagram of the structure of a trispecific antibody of the present invention. [Figure 2A] FIG. 2A shows the results of SDS-PAGE (silver staining) (A) analysis of LMOAME after purification in Example 2. [Figure 2B] FIG. 2B shows the SEC-HPLC (B) analysis results of purified LMOAME in Example 2. [Figure 3] FIG. 3 is a mass spectrum of LMOAME after purification in Example 2. [Figure 4] Figure 4, A and B, shows the results of SEC-HPLC analysis of LMOAME (A) and a control trispecific antibody (BLMOAM) (B) after affinity chromatography capture. [Figure 5]FIG. 5 shows the results of SDS-PAGE analysis of LMOAME and a control trispecific antibody (BLMOAM) after affinity chromatography capture (Coomassie Brilliant Blue staining). [Figure 6] FIG. 6 is a SEC-HPLC map of the purified MLMOF in Example 2. [Figure 7] FIG. 7 is a graph showing the binding curves of LMOAME with hCD19 and hCD28 antigens measured in Example 3. [Figure 8A] 8A shows the results of the T cell activation activity of LMOAME and blinatumomab measured in Example 5. CD4+ cell population. [Figure 8B] Figure 8B shows the results of the T cell activation activity of LMOAME and blinatumomab measured in Example 5. CD8+ cell population. [Figure 9] FIG. 9 shows the results of LMOAME and blinatumomab in mediating effector cell proliferation as measured in Example 6. [Figure 10] FIG. 10 shows the results of the two rods LMOAME and Blinatumomab measured in Example 7 in mediating effector cell differentiation. [Figure 11] FIG. 11 shows the results of the two rods measured in Example 8 in mediating the killing activity of LMOAME and Blinatumomab against target cells. [Figure 12] Figure 12, panels A and B, show the time course curves of body weight (mean ± SEM, g) (A) and tumor volume (mean ± SEM, mm) of mice after administration measured in the animal experiment in Example 10. Statistical analysis of tumor volume and G1 for each group was performed using Dunnett's multiple comparison test, with *: P<0.05 and **: P<0.01 indicating significant differences. [Figure 13] Figures 13A-B are photographs of mice and tumors at the end of administration (day 21) measured in the animal experiment in Example 10, where (A) is a photograph of a mouse at the end of administration; and (B) is a photograph of the excised tumor at the end of administration. [Figure 14]Figure 14 shows the average tumor weight of mice in each group at the end of dosing (day 28) in the animal experiment in Example 11. Tumor weights are shown as mean ± SEM. Statistical analysis of tumor weights between different groups was performed by examining independent samples. Compared to G1, * indicates a significant difference of P<0.05, ** indicates a significant difference of P<0.01, and *** indicates a significant difference of P<0.001. Compared to G2 and G6, and compared to G3 and G7, * indicates a significant difference of P<0.05. [Figure 15A] FIG. 15A shows the time course curve of body weight (mean±SEM, g) of mice after administration measured in the animal experiment of Example 12. [Figure 15B] Figure 15B shows the time course curve (mean ± SEM, p / sec / cm2 / sr) of tumor photon counts in mice after administration measured in the animal experiment of Example 12. Statistical analysis of tumor photon counts in each administration group and the G1 group was performed using Dunnett's multiple comparison test, with *: P<0.05, **: 0.001≦P<0.01; P<0.001 indicates a significant difference. [Figure 16] FIG. 16 shows the survival curves of mice in each group after administration measured in the animal experiment of Example 12. DETAILED DESCRIPTION OF THE INVENTION

[0036] [Definition] The term "antibody" is interpreted in a relatively broad sense in the context of the present invention, i.e., it includes not only conventional IgG class antibodies but also antibody mimetics and derivatives such as scFv. For example, IgG class antibodies are immunoglobulins with a Y-shaped structure that are produced by the immune system in response to foreign substances such as pathogens. Antibodies with a classical structure are homodimers, and each monomer has one heavy chain and one light chain connected by a disulfide bond. The light chain contains one variable region (V L ) and one constant region (C L ), and the heavy chain consists of one variable region (V H ) and three constant regions (C H 1. C H 2, and C H3). Each variable region contains three "complementarity-determining regions (CDRs)" that constitute the antigen-binding site responsible for complementarity with the antigen. L , C L , V H and C H The antigen-binding fragment consisting of 1 is called "Fab". The remaining part, i.e., the "stem" part below the Y-shaped structure, is called the "Fc" region, which is the constant region of the antibody heavy chain, C H 2 and C H The "hinge region" is the C domain in IgG, IgA, and IgG immunoglobulin molecules. H 1 and C H 2, connecting the Fab and Fc regions.

[0037] "scFv" refers to a single-chain fragment variable (VH) and is sometimes abbreviated as single-chain antibody. It is a type of fusion protein consisting of an antibody heavy chain variable region (VH) and light chain variable region (VL). In scFv, the order of VH and VL can be reversed; VH can be located at the N-terminus, or VL can be located at the N-terminus. VH and VL can be connected by a "linker" sequence. A single-chain variable fragment can also contain a heavy chain variable region fragment (single variable domain on a heavy chain (VHH)), and is abbreviated as nanobody. Because it lacks a light chain, nanobody only has the three antigen-recognition regions (CDR regions) belonging to the heavy chain. Its molecular weight is approximately one-tenth that of a conventional antibody and approximately half that of an antigen-binding fragment (scFv, VH-VL).

[0038] "IgD" is one of the immunoglobulin isotypes and has a Y-shaped structure similar to that of IgG. In the present invention, a fragment of the hinge region of IgD is used as a linker sequence.

[0039] A "multispecific antibody" refers to an antibody or antibody mimetic that can bind simultaneously and specifically to multiple targets.

[0040] A "triabody" refers to an antibody or antibody mimetic capable of simultaneously and specifically binding to three targets. In the context of the present invention, a triabody specifically refers to a single-chain fusion protein in which three domains with binding specificities for different antigen targets are formed in tandem, for example, three different scFvs are formed in tandem.

[0041] "Specifically binds" or "specifically binds to" means that an antibody exhibits preferential binding to a particular target compared to other proteins, although this type of specificity need not be absolute binding specificity. An antibody is considered to have "specificity" for its expected target if its binding can be used to determine the presence of the target protein in a sample and does not result in undesirable results, such as false positives. Compared to its affinity for non-target proteins, antibodies or antigen-binding fragments thereof of the present invention bind to the target protein with an affinity that is at least 2-fold, preferably at least 10-fold, more preferably at least 20-fold, and optimally at least 100-fold higher. Alternatively, or based on this, antibodies or antigen-binding fragments thereof of the present invention may have a binding affinity for their target protein, e.g., 1 x 10 -7 M, 1 x 10 -8 Lower than M, 1×10 -9 M (1 nM) lower than 1 × 10 -10 Lower than M, 1×10 -11 Lower than M or even 1×10 -12 K lower than M (1 pM) D When an antibody of the present application binds to a polypeptide containing a given amino acid sequence (e.g., human CD19, human CD3, human CD28) but does not bind to a protein lacking the sequence, the antibody is said to specifically bind to a polypeptide containing the given amino acid sequence.

[0042] "Binding domain" or "antigen-binding domain", as used herein in the context of multispecific antibodies, refers to a polypeptide structure capable of specifically recognizing and binding to a target, and is sometimes abbreviated to "domain".

[0043] In the context of the present invention, the term "linker sequence" refers to a peptide fragment for linking two binding domains, and also refers to a peptide fragment for connecting VH and VL in one binding domain. The term "first linker sequence" specifically refers to a linker for connecting the first binding domain and the second binding domain, and the term "second linker sequence" specifically refers to a linker for connecting the second binding domain and the third binding domain. The first and second linker sequences are both linkers for connecting different binding domains, and are therefore also collectively referred to as "inter-domain linkers." When a linker is also present between the VH and VL contained in one domain, it is called an "intra-domain linker."

[0044] "T cell engager antibodies" refer to antibodies that specifically bind to two types of cells, bringing them into close proximity and "gluing" them together in a predictable manner.

[0045] The term "CD19xCD3xCD28 antibody" as used herein refers to a trispecific antibody that comprises a binding domain that specifically binds to CD19, a binding domain that specifically binds to CD3, and a binding domain that specifically binds to CD28.

[0046] "G4S" refers to Gly-Gly-Gly-Gly-Ser, i.e., a pentapeptide unit consisting of four glycines and one serine, and one or more such units are commonly used linker sequences in recombinant fusion proteins. For the same reason, "G2S" refers to Gly-Gly-Ser.

[0047] The terms "rigid" and "flexible" are used herein to describe the conformational rigidity or flexibility of a molecule. Specifically, when describing a peptide linker sequence, a "flexible linker" refers to a peptide linker that allows the two connected polypeptides or functional domains to change their relative positions or interact to some extent, whereas a "rigid linker" typically does not have a similar effect and provides a more rigid conformation.

[0048] In the context of the present invention, "percent homology" or "% homology" is used to describe the degree of similarity between two nucleotide sequences or two amino acid sequences, and has the same meaning as "percent identity." The percent homology of two sequences is calculated by aligning the two sequences, dividing the number of positions with identical residues by the total length of the aligned sequences, and multiplying the result by 100%. Methods and tools for comparing two amino acid or nucleotide sequences are known in the art, such as the BLAST kit (Altschul, SF et al. (1990) J. Mol. Biol. 215: 403-410) available on the NCBI website.

[0049] "Conservative substitution" of amino acids is well known in the art and generally refers to changing one amino acid residue for another amino acid residue having a similar side chain in terms of domain function. For example, the table below provides a list of exemplary conservative substitutions. TIFF2025525535000001.tif88142

[0050] "Cross-reactivity" refers to the ability of an antibody that specifically binds to one antigen to bind to another antigen. When the two antigens are from the same species, this type of cross-reactivity can be referred to as "intra-species cross-reactivity" or "intra-target cross-reactivity." When the two antigens are from different species, this type of cross-reactivity is referred to as "inter-species cross-reactivity." Unless otherwise specified, the "cross-reactivity" of an antibody in the context of the present invention refers to inter-species cross-reactivity, i.e., the ability to react with homologous or orthologous proteins from other species.

[0051] "Nucleotides" in the context of the present invention include DNA and RNA.

[0052] In the term "isolated nucleic acid molecule," "isolated" refers to a nucleic acid molecule that has been manipulated to remove elements other than the desired components and is not present in the chromosomal genome in which it occurs in nature.

[0053] "Expression vector" or "expression cassette" refers to a nucleotide sequence that contains a gene of interest and gene expression control sequences, including but not limited to promoters, enhancers, terminators, polyadenylation sequences, etc.

[0054] "Vector" refers to a carrier DNA molecule that can carry an exogenous gene into a cell. Examples of vectors include, but are not limited to, plasmids, viral vectors, cosmids, and artificial chromosomes. A vector for introducing an exogenous transformant into a cell and promoting the expression of the transformant is called an "expression vector."

[0055] The term "host cell," as used herein, refers to a cell containing exogenous recombinant DNA, such as an expression vector, and also refers to a cell containing a polypeptide or protein obtained by recombinant DNA expression. Such host cells can be used to produce the trispecific antibodies of the invention. In one embodiment, the host cell is not particularly limited as long as it can be used to express a polypeptide sequence, such as a trispecific antibody of the invention.

[0056] "Tumor" is understood in the broadest sense to refer to abnormal, excessive growth of tissue and is synonymous with "abnormal neoplasm." "Cancer" or "carcinoma" refers to malignant tumors, including sarcomas, carcinomas, lymphomas, leukemias, and gliomas.

[0057] "B-cell-related tumors" refer to tumors characterized by cancerous B cells and can also be called "B-cell leukemias" or "B-cell lymphomas."

[0058] In the present invention, the term "subject" refers to an animal receiving a drug, preferably a vertebrate, more preferably a mammal, for example, a rodent such as a mouse or a rat, or a primate such as a monkey, and most preferably a human.

[0059] "Treatment" refers to the improvement, amelioration, or elimination (i.e., cure) of a disease or its symptoms. In some circumstances, treatment includes prophylactic treatment.

[0060] [Antigen-binding domain] The present invention provides T cell engager antibodies that can simultaneously bind to CD19, CD3, and CD28, also referred to herein as trispecific antibodies or CD19xCD3xCD28 antibodies.

[0061] Specifically, the trispecific antibody of the present invention comprises three binding domains: a first binding domain capable of specifically binding to CD19, a second binding domain capable of specifically binding to and activating CD3 molecules on the surface of T cells, and a third binding domain capable of specifically binding to and activating CD28 molecules on the surface of T cells. The trispecific antibody further comprises a first linker located between the first and second binding domains and a second linker located between the second and third binding domains.

[0062] The human CD19 antigen is a 95 kDa transmembrane glycoprotein and a member of the immunoglobulin superfamily. In addition to being expressed on the surface of normal B lymphocytes, CD19 is also highly expressed in B cell malignancies, and full-length anti-CD19 monoclonal antibodies have already been approved for the treatment of B cell lymphoma. Unless otherwise specified, the CD19 specifically binding to the first binding domain in the context of the present invention is human CD19.

[0063] CD3 is a transmembrane protein, and its transmembrane region is connected to the transmembrane regions of the two peptide chains of TCR via salt bridges to form a TCR-CD3 complex, which jointly participates in T cell antigen recognition and transmits the generated activation signal into the T cell. Unless otherwise specified, the CD3 that specifically binds to the first binding domain in the context of the present invention is human CD3.

[0064] CD28 is a dimeric transmembrane glycoprotein that is expressed in approximately 80% of human CD4 + T cells and 50% CD8 + CD28 is expressed on the surface of T cells. CD28 is a T cell costimulatory molecule that can bind to its ligands CD80 (B7-1) and CD86 (B7-2). It amplifies the first signal transduced by TCR / CD3, maintains T cell survival, and promotes cytokine-induced T cell proliferation and differentiation. Unless otherwise specified, the CD28 that specifically binds to the third binding domain in the context of the present invention is human CD28.

[0065] That is, for B cell-associated tumors, the first binding domain in the trispecific antibody of the present invention can specifically bind to the tumor cell surface-associated antigen CD19, and the second and third binding domains can bind to the T cell surface receptors CD3 and CD28, respectively, thereby "connecting" tumor B cells and immune T cells and simultaneously activating the CD3 signaling pathway and CD28 costimulatory signaling pathway in T cells, thereby enabling the activated immune cells to exert their killing effect on target cells.

[0066] In a preferred embodiment, the trispecific antibody of the present invention is a single-chain fusion protein comprising an scFv that specifically binds to CD19 as a first binding domain, an scFv that specifically binds to CD3 as a second binding domain, and an scFv that specifically binds to CD28 as a third binding domain.

[0067] The scFv sequences for the first, second, and third binding domains can be derived from anti-CD19, anti-CD3, and anti-CD28 antibodies, or antibody mimetics or derivatives known in the art. For example, each binding domain can be an scFv comprising a VH and a VL derived from a known corresponding antibody. The antibody can be, for example, an animal-derived antibody such as a mouse-derived antibody, a chimeric antibody such as a mouse-human chimeric antibody, or a human antibody.

[0068] In a preferred embodiment, the scFv that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises a heavy chain CDR1 represented by SEQ ID NO: 11, a heavy chain CDR2 represented by SEQ ID NO: 12, and a heavy chain CDR3 represented by SEQ ID NO: 13, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 14, a light chain CDR2 represented by SEQ ID NO: 15, and a light chain CDR3 represented by SEQ ID NO: 16, or (b) The VH comprises a heavy chain CDR1 represented by SEQ ID NO: 46, a heavy chain CDR2 represented by SEQ ID NO: 47, and a heavy chain CDR3 represented by SEQ ID NO: 48, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 49, a light chain CDR2 represented by SEQ ID NO: 50, and a light chain CDR3 represented by SEQ ID NO: 51.

[0069] In a further preferred embodiment of the above embodiment, the scFv that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises or consists of an amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 17, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of an amino acid sequence set forth in SEQ ID NO: 19, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 19, wherein all amino acid differences are in non-CDR regions; or (b) the VH comprises or consists of an amino acid sequence set forth in SEQ ID NO: 52, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 52, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 54, wherein all amino acid differences are in non-CDR regions.

[0070] In a further preferred embodiment, the scFv that specifically binds to CD19 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 56, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 56, wherein all amino acid differences are in non-CDR regions.

[0071] Preferably, the amino acid differences are conservative amino acid substitutions.

[0072] In one embodiment, in the scFv that specifically binds to CD19, VH is located at the C-terminus or N-terminus of VL. In a preferred embodiment, in the scFv that specifically binds to CD19, VH is located at the C-terminus of VL, i.e., they are connected in the order of VL-linker sequence-VH.

[0073] In some embodiments, in the scFv that specifically binds to CD19, the VH and VL are connected via an intradomain linker or directly. The intradomain linker is a linker comprising (GGGGS)n or (GGS)n, or (GGGGS)n or (GGS)n, where n is an integer of 1 to 10, preferably an integer of 1 to 5, for example, 1, 2, 3, 4, or 5. In a specific embodiment, in the scFv that specifically binds to CD19, the intradomain linker between the VH and VL is selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and preferably SEQ ID NO: 1.

[0074] In a preferred embodiment, the scFv that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises a heavy chain CDR1 represented by SEQ ID NO: 22, a heavy chain CDR2 represented by SEQ ID NO: 23, and a heavy chain CDR3 represented by SEQ ID NO: 24, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 25, a light chain CDR2 represented by SEQ ID NO: 26, and a light chain CDR3 represented by SEQ ID NO: 27, or (b) The VH comprises a heavy chain CDR1 represented by SEQ ID NO: 57, a heavy chain CDR2 represented by SEQ ID NO: 58, and a heavy chain CDR3 represented by SEQ ID NO: 59, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 60, a light chain CDR2 represented by SEQ ID NO: 61, and a light chain CDR3 represented by SEQ ID NO: 62.

[0075] In a further preferred embodiment of the above embodiment, the scFv that specifically binds to CD3 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises or consists of an amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 28, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of an amino acid sequence represented by SEQ ID NO: 30, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence represented by SEQ ID NO: 30, wherein all amino acid differences are in non-CDR regions; or (b) the VH comprises or consists of an amino acid sequence set forth in SEQ ID NO: 63, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 63, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of an amino acid sequence represented by SEQ ID NO: 65, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence represented by SEQ ID NO: 65, wherein all amino acid differences are in non-CDR regions.

[0076] In a further preferred embodiment, the scFv that specifically binds to CD3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 67, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 67, wherein all amino acid differences are in non-CDR regions.

[0077] Preferably, the amino acid differences are conservative amino acid substitutions.

[0078] In one embodiment, in the scFv that specifically binds to CD3, VH is located at the C-terminus or N-terminus of VL. In a preferred embodiment, in the scFv that specifically binds to CD3, VL is located at the C-terminus of VH, i.e., connected in the order of VH-linker sequence-VL.

[0079] In some embodiments, in the scFv that specifically binds to CD3, the VH and VL are connected via an intradomain linker or directly. The intradomain linker is a linker comprising (GGGGS)n or (GGS)n, or (GGGGS)n or (GGS)n, where n is an integer of 1 to 10, preferably an integer of 1 to 5, for example, 1, 2, 3, 4, or 5. In a specific embodiment, in the scFv that specifically binds to CD3, the intradomain linker between the VH and VL is selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and is preferably SEQ ID NO: 1 or SEQ ID NO: 3.

[0080] In a preferred embodiment, the scFv that specifically binds to CD28 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain CDR1 represented by SEQ ID NO: 33, a heavy chain CDR2 represented by SEQ ID NO: 34, and a heavy chain CDR3 represented by SEQ ID NO: 35, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 36, a light chain CDR2 represented by SEQ ID NO: 37, and a light chain CDR3 represented by SEQ ID NO: 38.

[0081] In a further preferred embodiment of the above embodiment, the scFv that specifically binds to CD28 comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH is an amino acid sequence set forth in SEQ ID NO: 39 or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 39, and wherein all amino acids The VL comprises or consists of an amino acid sequence represented by SEQ ID NO: 41 or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence represented by SEQ ID NO: 41, wherein all amino acid differences are in non-CDR regions.

[0082] In a further preferred embodiment, the scFv that specifically binds to CD28 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 43, wherein all amino acid differences are in non-CDR regions.

[0083] Preferably, the amino acid differences are conservative amino acid substitutions.

[0084] In one embodiment, in the scFv that specifically binds to CD28, VH is located at the C-terminus or N-terminus of VL. In a preferred embodiment, in the scFv that specifically binds to CD28, VL is located at the C-terminus of VH, i.e., connected in the order of VH-linker sequence-VL.

[0085] In some embodiments, in the scFv that specifically binds to CD28, the VH and VL are connected via an intradomain linker or directly. The intradomain linker is a linker comprising (GGGGS)n or (GGS)n, or (GGGGS)n or (GGS)n, where n is an integer of 1 to 10, preferably an integer of 1 to 5, for example, 1, 2, 3, 4, or 5. In a specific embodiment, in the scFv that specifically binds to CD28, the intradomain linker between the VH and VL is selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and preferably SEQ ID NO: 1.

[0086] Those skilled in the art will understand that CDR regions can be defined using any numbering system among the Kabat, IMGT, and Chothia antibody numbering systems. When different numbering systems are used, different CDR regions may be defined for the same variable region amino acid sequence. CDR regions defined using any numbering system are within the scope of the present invention. Unless otherwise stated, the amino acid sequences of specific CDR regions described in this application are defined based on the Kabat antibody numbering system.

[0087] In some embodiments, one or more of the first binding domain, the second binding domain, and the third binding domain of the present application are cross-reactive with corresponding antigens in other species. The other species may be a laboratory animal such as a monkey, e.g., a cynomolgus monkey (Macaca fascicularis). For example, the first binding domain of the present application specifically binds to human CD19 and is also cross-reactive with monkey CD19, i.e., can specifically bind to monkey CD19. For example, the second binding domain of the present application specifically binds to human CD3 and is also cross-reactive with monkey CD3, i.e., can specifically bind to monkey CD3. For example, the third binding domain of the present application specifically binds to human CD28 and is also cross-reactive with monkey CD28, i.e., can specifically bind to monkey CD28. Cross-reactivity with corresponding monkey antigens is useful for antibody research and development.

[0088] [Linker sequence] After determining the binding domains, the design of linkers connecting each binding domain and linkers connecting VH and VL within each binding domain is crucial for the structure and function of triabodies, particularly the former. The linker sequence, together with the functional domains, constitutes the entire trispecific antibody molecule, and affects the structural stability of the entire trispecific antibody molecule. Meanwhile, the length and flexibility of the linker sequence affect the spatial relationship between each binding domain and further affect the binding of each domain to its target. In addition, the overall properties of the trispecific antibody molecule are also affected by the susceptibility of any amino acids in the linker sequence to further modifications, such as dimerization or glycosylation.

[0089] The linker sequence of the present invention may be derived from a naturally occurring sequence or a modified variant thereof, or may be a newly designed artificial sequence. The linker sequence may be derived from a naturally occurring multidomain protein, for example, a linker sequence contained in a natural multidomain protein or a variant thereof. An example of a linker sequence derived from a naturally occurring sequence is a linker sequence derived from the IgD hinge region, which can be used as the second linker sequence in this application. An example of an artificial linker sequence is, for example, (GGGGS) n , (GGGGS) n GGGG, (SGGGG) n ,GGGG(SGGGG) n , (GGS) n , (GGS) n GG, (SGG) n or GG (SGG) n In the present invention, the first inter-domain linker or the intra-domain linker may be a linker sequence based on the following: Another example of an artificial linker sequence is A(EAAAK) n A, A(EAAAK) n ALEA(EAAAK) n Like A (EAAAK) n and can be used as the second linker sequence in the present invention.

[0090] The rigidity / flexibility of the linker is one factor to consider. Flexible linkers allow a certain degree of mobility for the polypeptide sequences attached to both ends. Flexible linkers are typically composed of relatively small amino acids such as glycine (G), serine (S), and threonine (T), and it is these small amino acids that provide the linker with flexibility. Flexible linkers are desirable when it is necessary to allow for relative mobility between the domains at both ends of the linker. However, in some situations, flexible linkers cannot fix the domains attached to both ends, preventing the accurate expression and function of multidomain molecules. From this perspective, rigid linkers may provide superior structural and functional stability to flexible linkers for multifunctional molecules, such as trispecific antibodies that must bind to three targets.

[0091] Specifically, in the trispecific antibody of the present invention, the first domain targets tumor cells, and the second and third domains target immune cells; that is, the adjacent first and second domains necessarily bind to different cells. In this case, it is desirable that there is a certain relative mobility between the first and second domains, so that when binding to different cells, the binding can be made easier by adjusting the position. Therefore, in a preferred embodiment, the first linker sequence of the present invention is a flexible linker, for example, a linker consisting of one or more of glycine (G), serine (S), threonine (T), and lysine (K), such as (GGGGS). n , (SGGGG) n , (GGS) n , (SGG) n GS-rich linkers such as (GGGGS) n , (GGGGS) n GGGG, (SGGGG) n ,GGGG(SGGGG) n , (GGS) n , (GGS) n GG, (SGG) n or GG (SGG) nIn these, n is a positive integer, preferably 1 to 10. The linker connecting VH and VL in each binding domain is also preferably such a flexible linker.

[0092] On the other hand, it is desirable for the engager antibody to bring the two types of cells to be joined as close as possible, which makes the distance of the immune synapse formed between the cells closer to the distance of the immune synapse formed naturally under physiological conditions, thereby further helping the immune cells to exert their killing effect. The distance at which the two types of cells are attracted is affected by the length of the first linker sequence. For example, the length of the first linker sequence does not exceed 30 amino acids, preferably does not exceed 25 amino acids, more preferably does not exceed 20 amino acids, and even more preferably does not exceed 15 amino acids. For example, when the first linker sequence is (GGGGS) n , (SGGGG) n ,GGGG(SGGGG) n In the case where n is any one of the following, preferably n is a positive integer not exceeding 6, preferably a positive integer not exceeding 5, for example, 1, 2, 3, 4 or 5, and the first linker sequence is (GGS) n , (SGG) n or (GGS) n When n is either GG, n is preferably a positive integer not exceeding 10, and preferably a positive integer not exceeding 7, for example, 1, 2, 3, 4, 5, 6, or 7. In a specific embodiment, the first linker sequence is (GGGGS)3 (SEQ ID NO: 1), GGGGS (SEQ ID NO: 2), or (GGS)4GG (SEQ ID NO: 3), and is preferably the amino acid sequence set forth in SEQ ID NO: 1.

[0093] The second linker sequence connects the second and third domains, both of which bind to a receptor on the surface of T cells. A slightly longer second linker sequence is ideal to prevent steric hindrance from affecting the binding of one domain to the same T cell after the other domain binds. Therefore, in a preferred embodiment, the second linker sequence is longer than the first linker sequence. For example, the second linker sequence is at least 1.25 times, at least 1.5 times, at least 2 times, at least 2.25 times, at least 2.5 times, or more than the length of the first linker sequence. For example, the second linker sequence is at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, or more.

[0094] Linkers that bias toward rigidity can also be a preferred choice for the second linker sequence. A more rigid linker also helps to avoid steric hindrance effects. For example, one example of a second linker is (EAAAK) n The linker sequence contains the Glu-Ala-Ala-Ala-Lys structure, which allows the formation of a rigid α-helical structure. n The term "linker sequence comprising" refers to a specific example of a linker sequence comprising A(EAAAK) because such a linker typically further comprises other amino acid residues, such as one or more of alanine (A), leucine (L), and glutamic acid (E) on both sides of the EAAAK overlap. n A, LEA (EAAAK) n ALE, LEA (EAAAK) n ALEA(EAAAK) n ALE, etc., in which n may be any integer from 1 to 10, preferably from 2 to 6. Depending on the required length of the linker sequence, the type of linker can be selected and / or the value of n can be adjusted.

[0095] In a specific embodiment, one of the linker sequences of the invention, eg, the second linker sequence, is a fragment derived from the hinge region of immunoglobulin IgD or a variant thereof.

[0096] Potential glycosylation sites present in the linker sequence may also affect the properties and performance of the trispecific antibody molecule. For example, if the linker sequence contains glycosylation sites, sugar chains formed by glycosylation may unexpectedly mask the active site and reduce the activity of the antibody. During antibody production in cells, sugar chains of various compositions and lengths may be formed, potentially reducing the homogeneity of the antibody product. Common types of glycosylation include N-glycosylation and O-glycosylation. N-glycosylation is a type of glycosylation in which sugar chains are attached to the nitrogen atom of the side chain of amino acids such as asparagine and arginine, while O-glycosylation is a type of glycosylation in which sugar chains are attached to the hydroxyl radical of the side chain of amino acids such as serine, threonine, and tyrosine. When the linker sequence is derived from a native sequence, such as an immunoglobulin hinge region, potential glycosylation sites, such as threonine, can be removed by mutating the native sequence, including by deletion, substitution, or truncation.

[0097] In a specific embodiment, a fragment of an IgD hinge region is used as a linker sequence, e.g., as a second linker sequence. The IgD hinge region is known to contain multiple O-glycosylation sites, such as serine at position 109 (S109) and four threonines (T126, T127, T131, and T132). The 81-amino acid long linker provided herein contains these sites, and the amino acid sequence of the linker sequence is represented by SEQ ID NO:4 or SEQ ID NO:8, in which the one serine and four threonines in the above example are located at positions 20, 37, 38, 42, and 43, respectively. In a preferred embodiment, the IgD hinge region fragment of the present invention does not contain a glycosylation site, e.g., the five glycosylation sites listed above. In one embodiment, a cleavage scheme can be used to remove an IgD hinge region fragment that does not contain a glycosylation site, e.g., at least the five glycosylation sites listed above. For example, all fragments before (and including) the final threonine can be removed, and only all or part of the C-terminal sequence can be retained. Alternatively, the amino acids at the five glycosylation sites can be modified to replace other amino acids.

[0098] Single-chain antibodies form dimers via cysteines in the linker region. A triabody dimer contains twice as many antigen-binding domains. Research has shown that because CD28 naturally exists as a dimer, bivalent antibodies (e.g., IgG antibodies) containing two domains specifically binding to CD28 are more potent than monovalent antibodies (e.g., single-chain antibodies). Similarly, CD28 antibodies exhibit higher potency when cross-linking or aggregation is more pronounced. However, triabodies must simultaneously and precisely bind to three targets to achieve their intended effect. If a triabody only binds to one or two targets on T cells (CD3 and / or CD28) and does not bind to CD19 on B-cell tumors, T cell activation may occur away from the tumor target cells, resulting in "non-target T cell activation." In this situation, a trispecific antibody in a dimeric form is more dangerous because it has a stronger ability to bind to the T cell receptor CD28, is more potent, is more susceptible to cross-linking reactions, generates less responsive off-target activation effects, and increases the risk of cytokine storms. Therefore, in the present invention, the formation of dimers during preparation and / or use of the trispecific antibody is not desired, and for this reason, the linker sequence, particularly the second linker sequence based on the IgD hinge region, has been modified to remove amino acid residues that cause dimer formation, such as cysteine (Cys; C) residues.

[0099] In a preferred embodiment, the linker sequence of the present invention does not contain an amino acid site capable of forming a dimer, e.g., does not contain a cysteine (C). In a specific embodiment, the second linker sequence is derived from the IgD hinge region, and the cysteine (C161) therein has been deleted. For example, the cysteine in the linker sequence is deleted by substituting the cysteine at position 161 with another amino acid. For example, the cysteine in the linker sequence is deleted by substituting the cysteine at position 161 with serine or glycine (substitution of C161S or C161G).

[0100] In a preferred embodiment, the first linker sequence of the trispecific antibody of the invention is a linker comprising (GGGGS)n or (GGS)n, or is (GGGGS)n or (GGS)n, where n is an integer not greater than 6, preferably not greater than 5, for example 1, 2, 3, 4 or 5. In a specific embodiment, the first linker sequence is the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In a more specific embodiment, the first linker sequence is the amino acid sequence set forth in SEQ ID NO: 1.

[0101] In a preferred embodiment, the second linker sequence of the trispecific antibody of the invention is derived from an IgD hinge region and has one or more of the following characteristics:

[0102] (1) Compared to the amino acid sequence represented by SEQ ID NO: 10, it lacks cysteines. (2) Compared with the amino acid sequence represented by SEQ ID NO: 10, it lacks glycosylation sites. (3) It is shorter in length than the amino acid sequence represented by SEQ ID NO: 10.

[0103] In a preferred embodiment, the second linker sequence has the cysteine corresponding to position 72 mutated to another amino acid, preferably serine or glycine, compared to SEQ ID NO: 10. In a specific embodiment, the second linker sequence is the amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9.

[0104] In a preferred embodiment, the second linker sequence is a partial fragment that does not contain a glycosylation site in SEQ ID NO: 4, for example, an N-terminal partial fragment or a C-terminal partial fragment that does not contain a glycosylation site in SEQ ID NO: 4, and is preferably a partial fragment consisting of 38 amino acids located at the C-terminus of the amino acid sequence of SEQ ID NO: 4. The glycosylation sites include at least a serine corresponding to position 20 and threonines corresponding to positions 37, 38, 42, and 43. In a specific embodiment, the second linker sequence is the amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 9.

[0105] In a most preferred embodiment, the second linker sequence of the trispecific antibody of the invention is the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:9.

[0106] In another preferred embodiment, the second linker sequence of the trispecific antibody of the invention is (EAAAK) n A linker sequence comprising, for example, A(EAAAK) n A, LEA (EAAAK) n ALE, LEA (EAAAK) n ALEA(EAAAK) n ALE, wherein n is any integer from 1 to 10, preferably 2 to 6, and more preferably 2 to 4. Specifically, the second linker sequence is a linker sequence containing the amino acid sequence represented by SEQ ID NO: 7, for example, a linker sequence containing SEQ ID NO: 7 as a repeat unit, wherein the repeat unit is repeated 1 to 10 times.

[0107] In a preferred embodiment, the linker sequence connecting VH and VL in each binding domain in the trispecific antibody of the present invention, i.e., the intradomain linker, is a linker comprising (GGGGS)n or (GGS)n, or is (GGGGS)n or (GGS)n, where n is a positive integer not exceeding 10, preferably a positive integer not exceeding 5, for example, 1, 2, 3, 4, or 5. In some embodiments, the linker sequence connecting VH and VL in each binding domain is the same. In some embodiments, the linker sequence connecting VH and VL in each binding domain is different. For example, the intradomain linker sequence is (GGGGS)n n , (SGGGG) n ,GGGG(SGGGG) n wherein n is a positive integer preferably not greater than 6, preferably not greater than 5, for example, 1, 2, 3, 4, or 5. For example, the intradomain linker is (GGS) n , (SGG) n or (GGS) nGG, wherein n is a positive integer preferably not exceeding 10, preferably not exceeding 7, for example 1, 2, 3, 4, 5, 6 or 7.

[0108] In specific embodiments, the intradomain linker is selected from the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In a more specific embodiment, the linkers connecting the VH and VL of each binding domain are all (GGGGS)3 represented by SEQ ID NO: 1. In a more specific embodiment, the three intradomain linkers are (GGGGS)3 represented by SEQ ID NO: 1, (GGS)4GG represented by SEQ ID NO: 3, and (GGGGS)3 represented by SEQ ID NO: 1, respectively.

[0109] [Trispecific antibody] The specific sequence of each antigen-binding domain is not particularly limited, provided that the antigen specificity and sequence order of each antigen-binding domain are determined. The three binding domains can be derived from corresponding antibodies or antibody mimetics known in the art.

[0110] In a preferred embodiment, the trispecific antibody of the present invention can specifically bind to human CD19, human CD3, and human CD28, and comprises a linker sequence such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9. Preferably, the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9 is the linker sequence connecting the binding domain that specifically binds to CD3 and the binding domain that specifically binds to CD28.

[0111] In a specific embodiment, the trispecific antibody of the present invention is a trispecific antibody whose amino acid sequence is set forth in SEQ ID NO: 44 or SEQ ID NO: 68, or a variant thereof. The variant has at least 80% homology to the amino acid sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 68, is capable of specifically binding to CD19, CD3, and CD28, and comprises a sequence such as SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9 as a linker sequence. Preferably, the amino acid sequence of the variant has at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 68.

[0112] [Preparation method] The present invention further relates to a method for preparing said trispecific antibody, which may be a molecular biology method, for example, by preparing one or more nucleotide sequences encoding a single-chain fusion protein such as said trispecific antibody, assembling said one or more encoded nucleotide sequences into one or more expression vectors, and expressing said expression vectors in suitable cells.

[0113] Those skilled in the art will understand that, once the amino acid sequence of a trispecific antibody is determined, different coding nucleotide sequences may be used or optimized due to the existence of codon degeneracy. Methods for optimizing codons in coding sequences are well known to those skilled in the art and involve adjusting codons to those preferred by the host, reducing GC content and / or GC-rich regions, improving mRNA stability, and thereby increasing the expression efficiency of target nucleotides in a particular host.

[0114] In a specific embodiment, said coding nucleotide sequence comprises a nucleotide sequence encoding a first binding domain that specifically binds to CD19 as described in the present invention.

[0115] In a specific embodiment, the coding nucleotide sequence of the first binding domain can comprise: (1) a nucleotide sequence encoding a VH set forth in SEQ ID NO: 18, or a nucleotide sequence which has at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and which encodes the amino acid sequence of a VH set forth in SEQ ID NO: 17, and / or (2) a nucleotide sequence encoding a VL set forth in SEQ ID NO: 20, or a nucleotide sequence which has at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and which encodes the amino acid sequence of a VL set forth in SEQ ID NO: 19. In a specific embodiment, the coding nucleotide sequence of the first binding domain can comprise: (1) a nucleotide sequence encoding a VH represented by SEQ ID NO: 53, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example, at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding the amino acid sequence of a VH represented by SEQ ID NO: 52, and / or (2) a nucleotide sequence encoding a VL represented by SEQ ID NO: 55, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example, at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding the amino acid sequence of a VL represented by SEQ ID NO: 54. In a specific embodiment, the nucleotide sequence encoding the first binding domain further comprises a nucleotide sequence encoding a linker sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0116] In a specific embodiment, the encoding nucleotide sequence comprises a nucleotide sequence encoding a second binding domain that specifically binds to CD3 as described in the present invention. The encoding nucleotide sequence of the second binding domain may comprise: (1) a nucleotide sequence encoding a VH set forth in SEQ ID NO: 29, or a nucleotide sequence that is at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99%, homologous thereto and that encodes the amino acid sequence of a VH set forth in SEQ ID NO: 28, and / or (2) a nucleotide sequence encoding a VL set forth in SEQ ID NO: 31, or a nucleotide sequence that is at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99%, homologous thereto and that encodes the amino acid sequence of a VL set forth in SEQ ID NO: 30. In a specific embodiment, the encoding nucleotide sequence of the first binding domain may comprise: (1) a nucleotide sequence encoding a VH represented by SEQ ID NO: 64, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example, at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding the amino acid sequence of a VH represented by SEQ ID NO: 63, and / or (2) a nucleotide sequence encoding a VL represented by SEQ ID NO: 66, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example, at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding the amino acid sequence of a VL represented by SEQ ID NO: 65. In a specific embodiment, the nucleotide sequence encoding the second binding domain further comprises a nucleotide sequence encoding a linker sequence represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0117] In a specific embodiment, the encoding nucleotide sequence comprises a nucleotide sequence encoding a third binding domain that specifically binds to CD28 as described in the present invention. The encoding nucleotide sequence for the third binding domain may comprise: (1) a nucleotide sequence encoding a VH set forth in SEQ ID NO:40, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and encoding the amino acid sequence of a VH set forth in SEQ ID NO:39, and / or (2) a nucleotide sequence encoding a VL set forth in SEQ ID NO:42, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% identity thereto, and encoding the amino acid sequence of a VL set forth in SEQ ID NO:41. In a specific embodiment, the encoding nucleotide sequence for the third binding domain further comprises a nucleotide sequence encoding a linker sequence set forth in SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0118] In a specific embodiment, a nucleotide sequence encoding a trispecific antibody of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 45, or a nucleotide sequence which is at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, homologous thereto, and which encodes the amino acid sequence set forth in SEQ ID NO: 44. In a specific embodiment, a nucleotide sequence encoding a trispecific antibody of the invention comprises the nucleotide sequence set forth in SEQ ID NO: 69, or a nucleotide sequence which is at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, at least 99%, homologous thereto, and which encodes the amino acid sequence set forth in SEQ ID NO: 68.

[0119] Suitable production cells are known to those skilled in the art. In some embodiments, mammalian cells, such as 293T, CHO, or cell lines derived therefrom, are used as producer cells. In some embodiments, microbial cells, such as bacterial cells, such as E. coli or yeast, or fungal cells, are used as producer cells. In some embodiments, insect cells, such as Sf9, are used as producer cells. For particular production cell lines, the nucleotide sequence encoding the trispecific antibody of the invention can be codon-optimized.

[0120] Preferably, the produced trispecific antibody is purified. The purification of the trispecific antibody can be carried out by a method commonly used in the antibody production field, and the method can include steps such as filtration and chromatography. The filtration step can be one or more of depth filtration, ultrafiltration, permeation filtration, and nanofiltration. The chromatography step can be one or more of affinity chromatography, cation chromatography, anion chromatography, size exclusion chromatography, hydrophobic chromatography, and hydroxyapatite chromatography.

[0121] The antibodies of the present invention can be prepared as liquid or solid formulations such as injections, suspensions, powders, granules, etc. For example, a lyophilized powder injection can be prepared by a common method such as lyophilization, and then reconstituted immediately before administration to prepare an injection.

[0122] [Tumor treatment] The trispecific antibodies of the present invention can be used in therapeutic or diagnostic applications, for example, to treat cancer.

[0123] As an engager antibody, the first binding domain of the trispecific antibody of the invention is responsible for targeting and binding to tumor-associated antigens. If the first binding domain specifically binds to CD19, the trispecific antibody of the invention will target and bind to disease-associated CD19. +It is particularly suitable for targeting target cells that express CD19, such as B cells.

[0124] Thus, the trispecific antibodies of the present invention are particularly suitable for the treatment of diseases associated with B cells, such as B cell-related tumors, including B cell lymphomas and B cell leukemias, particularly relapsed or refractory B-cell malignancies, i.e., the subject has undergone a prior related treatment and has relapsed after successful treatment, or has not responded satisfactorily to the prior related treatment.

[0125] The B-cell lymphoma may be a non-Hodgkin's lymphoma (NHL), such as an infiltrative or indolent non-Hodgkin's lymphoma, or a Hodgkin's lymphoma. Examples of B-cell related diseases include, but are not limited to, classical Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small lymphocytic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt's lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, B-precursor lymphocytic leukemia, hairy cell leukemia, splenic B-cell lymphoma / leukemia, and splenic marginal zone lymphoma.

[0126] Non-Hodgkin's lymphoma (NHL) is one of the most common cancers worldwide and represents a heterogeneous group of malignant lymphomas. The main subtypes are B-cell derived, including diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Currently, treatment options for B-cell derived NHL include anti-CD20 rituximab and the rituximab-combined chemotherapy R-CHOP regimen (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone). While the therapeutic efficacy of rituximab alone or in combination with chemotherapy has improved somewhat in some NHL subtypes, some patients still fail R-CHOP treatment and progress to the relapsed / refractory stage. Rechallenging patients with anti-CD20 monoclonal antibodies has proven challenging, with satisfactory efficacy still not achieved. The therapeutic efficacy of rituximab for B-cell lymphoma depends on the CD20 expression level on tumor cells. However, tumor cell CD20 expression is heterogeneous, and patients with low CD20 expression are insensitive to rituximab and have innate drug resistance. Furthermore, patients with high CD20 expression who are sensitive to rituximab may experience a decrease in CD20 expression to the point of negative expression, leading to acquired drug resistance. Blinatumomab is a bispecific T-cell activating (BiTE) antibody that selectively binds to the B-cell surface antigen CD19 and the invariant CD3 portion of the T-cell receptor (TCR). It has been reported to have anti-lymphoma activity in early proof-of-concept clinical trials for relapsed or refractory B-NHL. Blinatumomab requires continuous intravenous infusion for 28 days, and this difficult dosing regimen is a significant obstacle to its use in treating NHL. Unlike blinatumomab, the trispecific antibody of the present invention can be administered less frequently while providing comparable or better tumor suppression effects. In a specific embodiment, the trispecific antibodies of the invention are used to treat non-Hodgkin's lymphoma.

[0127] Precursor B-cell acute lymphoblastic leukemia (B-ALL) is a tumor of immature B-cell precursors. It typically affects children under the age of six, but also occurs in adolescents and adults. The current primary treatments for B-ALL are chemotherapy and hematopoietic stem cell transplantation (HSCT). While the overall survival (OS) rate for pediatric B-ALL is 80-90%, the OS rate for adult B-ALL is only 30-50%. However, the prognosis for relapsed or refractory ALL is very poor in both age groups. The trispecific antibodies of the present invention offer a novel treatment option for relapsed or refractory ALL. In a specific embodiment, the trispecific antibodies of the present invention are used to treat precursor B-cell acute lymphoblastic leukemia, particularly relapsed or refractory precursor B-cell acute lymphoblastic leukemia.

[0128] [Administration method] The trispecific antibodies of the present invention can be delivered by conventional methods, preferably by systemic administration, for example, intravenous infusion.

[0129] The antibodies of the invention can be used in therapeutically effective amounts. A "therapeutically effective amount" refers to the amount of antibody that, when administered to a subject for treating a disease or at least one clinical symptom of a disease or condition, is sufficient to effect such treatment for the disease, condition, or condition. A "therapeutically effective amount" can vary depending on the antibody, the disease, condition, and / or symptoms of the disease or condition, the severity of the disease, condition, and / or symptoms of the disease or condition, the age of the subject being treated, and / or the weight of the subject being treated.

[0130] In a preferred embodiment of the present application, the antibody dosage (mg / kg) is based on the subject's body weight. For example, a single dose of a trispecific antibody of the present invention may range from about 1 ng / kg body weight to about 5 mcg / kg body weight, preferably from 5 ng / kg body weight to about 2.5 mcg / kg body weight, and more preferably from 0.1 mcg / kg body weight to about 1 mcg / kg body weight. For example, the antibody may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mcg / kg body weight.

[0131] One advantage of the trispecific antibodies of the present invention is that they can activate T cells completely and efficiently, are less likely to induce apoptosis after activation, and induce memory T cells to provide a sustained immune response, allowing for administration at relatively short intervals. In a preferred embodiment, when administered intravenously, the interval between two doses of the trispecific antibodies of the present invention may be one day or more, three days or more, five days or more, or even one week. For example, the trispecific antibodies of the present invention can be administered 7 times a week, 5 times a week, 3 times a week, twice a week, or once a week. The trispecific antibodies of the present invention can be administered in a cycle of 2 to 4 weeks, e.g., 3 weeks, and can be administered in one or several cycles. When administered weekly, the trispecific antibodies of the present invention can exert a higher tumor-suppressing effect than bispecific antibodies such as CD19×CD3, e.g., blinatumomab, at an equivalent dose (e.g., the same molar dose).

[0132] [Drug combinations] The trispecific antibodies of the present invention can be used in combination with other anti-tumor treatments or drugs.

[0133] In some embodiments, the trispecific antibody of the present invention can be used in combination with other antibody-like drugs. The other antibody may be an antibody-like drug that targets a different antigen on target cells, such as B cells. The other antibody-like drug may be an antibody-like drug that specifically binds to CD20, CD22, CD79a, CD38, etc. Specific examples include, but are not limited to, MabThera and Besponsa.

[0134] In some embodiments, the trispecific antibodies of the invention can be used in combination with targeted therapies, such as tyrosine kinase inhibitors (TKIs), including, but not limited to, imatinib, gefitinib, erlotinib, sunitinib, lapatinib, lenatinib, and pyrotinib.

[0135] In some embodiments, the trispecific antibodies of the invention can be used in combination with small molecule chemotherapeutic agents, such as cyclophosphamide, vincristine sulfate, doxorubicin hydrochloride, dexamethasone, etc.

[0136] The present invention further relates to the following items.

[0137] 1. A trispecific antibody that specifically binds to CD19, CD3, and CD28, comprising: (1) a first binding domain that specifically binds to CD19; (2) a first linker sequence; (3) a second binding domain that specifically binds to CD3; (4) a second linker sequence, which is an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9; and (5) A third binding domain that specifically binds to CD28.

[0138] 2. The trispecific antibody of item 1, which is a single-chain fusion protein.

[0139] 3. The trispecific antibody according to item 1 or 2, wherein the first linker sequence comprises (G4S)n or (G2S)n, or is (G4S)n or (G2S)n, wherein n is an integer of 1 to 6, preferably 1 to 3.

[0140] 4. The trispecific antibody of item 3, wherein the first linker sequence is an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0141] 5. The trispecific antibody according to any one of Items 1 to 4, wherein the first binding domain is a single-chain antibody that specifically binds to CD19 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises a heavy chain CDR1 represented by SEQ ID NO: 11, a heavy chain CDR2 represented by SEQ ID NO: 12, and a heavy chain CDR3 represented by SEQ ID NO: 13, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 14, a light chain CDR2 represented by SEQ ID NO: 15, and a light chain CDR3 represented by SEQ ID NO: 16, or (b) The VH comprises a heavy chain CDR1 represented by SEQ ID NO: 46, a heavy chain CDR2 represented by SEQ ID NO: 47, and a heavy chain CDR3 represented by SEQ ID NO: 48, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 49, a light chain CDR2 represented by SEQ ID NO: 50, and a light chain CDR3 represented by SEQ ID NO: 51.

[0142] 6. The trispecific antibody according to item 5, wherein: (a) the VH comprises or consists of an amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 17, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of an amino acid sequence set forth in SEQ ID NO: 19, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 19, wherein all amino acid differences are in non-CDR regions; or (b) the VH comprises or consists of an amino acid sequence set forth in SEQ ID NO: 52, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 52, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 54, wherein all amino acid differences are in non-CDR regions.

[0143] 7. The trispecific antibody according to item 6, wherein the first binding domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 56, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 56, wherein all amino acid differences are located in non-CDR regions.

[0144] 8. The trispecific antibody according to any one of Items 1 to 7, wherein the second binding domain is a single-chain antibody that specifically binds to CD3 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises a heavy chain CDR1 represented by SEQ ID NO: 22, a heavy chain CDR2 represented by SEQ ID NO: 23, and a heavy chain CDR3 represented by SEQ ID NO: 24, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 25, a light chain CDR2 represented by SEQ ID NO: 26, and a light chain CDR3 represented by SEQ ID NO: 27, or (b) The VH comprises a heavy chain CDR1 represented by SEQ ID NO: 57, a heavy chain CDR2 represented by SEQ ID NO: 58, and a heavy chain CDR3 represented by SEQ ID NO: 59, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 60, a light chain CDR2 represented by SEQ ID NO: 61, and a light chain CDR3 represented by SEQ ID NO: 62.

[0145] 9. The trispecific antibody according to item 8, wherein: (a) the VH comprises or consists of an amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 28, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of an amino acid sequence represented by SEQ ID NO: 30, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence represented by SEQ ID NO: 30, wherein all amino acid differences are in non-CDR regions; or (b) the VH comprises or consists of an amino acid sequence set forth in SEQ ID NO: 63, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 63, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of an amino acid sequence represented by SEQ ID NO: 65, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence represented by SEQ ID NO: 65, wherein all amino acid differences are in non-CDR regions.

[0146] 10. The trispecific antibody according to item 9, wherein the scFv that specifically binds to CD3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 67, or an amino acid sequence having at least 80% homology (e.g. at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 67, wherein all amino acid differences are located in non-CDR regions.

[0147] 11. The trispecific antibody according to any one of Aspects 1 to 10, which is a single-chain antibody that specifically binds to CD28, wherein the third binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain CDR1 represented by SEQ ID NO: 33, a heavy chain CDR2 represented by SEQ ID NO: 34, and a heavy chain CDR3 represented by SEQ ID NO: 35, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 36, a light chain CDR2 represented by SEQ ID NO: 37, and a light chain CDR3 represented by SEQ ID NO: 38.

[0148] 12. The trispecific antibody according to item 11, wherein: The VH comprises or consists of an amino acid sequence set forth in SEQ ID NO: 39, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 39, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of an amino acid sequence represented by SEQ ID NO: 41, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence represented by SEQ ID NO: 41, wherein all amino acid differences are in non-CDR regions.

[0149] 13. The trispecific antibody of item 12, wherein the three binding domains comprise or consist of the amino acid sequence set forth in SEQ ID NO: 43 or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 43, wherein all amino acid differences are located in non-CDR regions.

[0150] 14. The trispecific antibody according to any of Items 1 to 13, wherein each binding domain comprises a linker sequence connecting the heavy chain variable region and the light chain variable region, and the linker sequence comprises (G4S)n or (G2S)n, where n is an integer of 1 to 10, preferably 1 to 5, and more preferably 1 to 3.

[0151] 15. The trispecific antibody according to item 14, wherein the amino acid sequence of the linker sequence connecting the heavy chain variable region and the light chain variable region is selected from the amino acid sequences represented by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0152] 16. The trispecific antibody according to any of Items 1 to 15, comprising or consisting of the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 68, or comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 68, or consisting of an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence represented by SEQ ID NO: 44 or SEQ ID NO: 68.

[0153] 17. An isolated nucleic acid molecule encoding the trispecific antibody according to any one of items 1 to 16.

[0154] 18. The isolated nucleic acid molecule according to item 17, wherein the coding nucleotide sequence of the first binding domain comprises the following nucleotide sequence: (1) a nucleotide sequence encoding a VH represented by SEQ ID NO: 18, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, e.g., at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VH represented by SEQ ID NO: 17; and / or (2) A nucleotide sequence encoding a VL represented by SEQ ID NO: 20, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VL represented by SEQ ID NO: 19.

[0155] 19. The isolated nucleic acid molecule according to item 17, wherein the coding nucleotide sequence of the first binding domain comprises the following nucleotide sequence: (1) a nucleotide sequence encoding a VH represented by SEQ ID NO: 53, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, e.g., at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VH represented by SEQ ID NO: 52; and / or (2) A nucleotide sequence encoding a VL represented by SEQ ID NO: 55, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VL represented by SEQ ID NO: 54.

[0156] 20. The isolated nucleic acid molecule according to items 17 to 19, wherein the coding nucleotide sequence of the second binding domain comprises the following nucleotide sequence: (1) a nucleotide sequence encoding a VH represented by SEQ ID NO: 29, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, e.g., at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VH represented by SEQ ID NO: 28; and / or (2) A nucleotide sequence encoding a VL represented by SEQ ID NO: 31, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VL represented by SEQ ID NO: 30.

[0157] 21. The isolated nucleic acid molecule according to items 17 to 19, wherein the coding nucleotide sequence of the second binding domain comprises the following nucleotide sequence: (1) a nucleotide sequence encoding a VH represented by SEQ ID NO: 64, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, e.g., at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VH represented by SEQ ID NO: 63; and / or (2) A nucleotide sequence encoding a VL represented by SEQ ID NO: 66, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VL represented by SEQ ID NO: 65.

[0158] 22. The isolated nucleic acid molecule according to any one of items 17 to 21, wherein the coding nucleotide sequence of the third binding domain comprises the following nucleotide sequence: (1) a nucleotide sequence encoding a VH represented by SEQ ID NO: 40, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, e.g., at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VH represented by SEQ ID NO: 39; and / or (2) A nucleotide sequence encoding a VL represented by SEQ ID NO: 42, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VL represented by SEQ ID NO: 41.

[0159] 23. An isolated nucleic acid molecule according to any one of items 17 to 22, comprising: (1) A nucleotide sequence encoding a first binding domain, comprising a nucleotide sequence encoding a VH represented by SEQ ID NO: 18, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example, at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VH represented by SEQ ID NO: 17, and a nucleotide sequence encoding a VL represented by SEQ ID NO: 20, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example, at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VL represented by SEQ ID NO: 19; (2) A nucleotide sequence encoding a second binding domain, comprising a nucleotide sequence encoding a VH represented by SEQ ID NO: 29, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VH represented by SEQ ID NO: 28, and a nucleotide sequence encoding a VL represented by SEQ ID NO: 31, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VL represented by SEQ ID NO: 30; and (3) A nucleotide sequence encoding a VH represented by SEQ ID NO: 40, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VH represented by SEQ ID NO: 39, and a nucleotide sequence encoding a VL represented by SEQ ID NO: 42, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VL represented by SEQ ID NO: 41. A nucleotide sequence encoding a third binding domain.

[0160] 24. An isolated nucleic acid molecule according to any one of items 17 to 22, comprising: (1) A nucleotide sequence encoding a first binding domain, comprising a nucleotide sequence encoding a VH represented by SEQ ID NO: 53, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example, at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VH represented by SEQ ID NO: 52, and a nucleotide sequence encoding a VL represented by SEQ ID NO: 55, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example, at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VL represented by SEQ ID NO: 54; (2) A nucleotide sequence encoding a second binding domain, comprising a nucleotide sequence encoding a VH represented by SEQ ID NO: 64, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VH represented by SEQ ID NO: 63, and a nucleotide sequence encoding a VL represented by SEQ ID NO: 66, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VL represented by SEQ ID NO: 65; and (3) A nucleotide sequence encoding a VH represented by SEQ ID NO: 40, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VH represented by SEQ ID NO: 39, and a nucleotide sequence encoding a VL represented by SEQ ID NO: 42, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, for example at least 96%, at least 97%, at least 98%, or at least 99% homology thereto, and encoding a VL represented by SEQ ID NO: 41. A nucleotide sequence encoding a third binding domain.

[0161] 25. The isolated nucleic acid molecule according to any one of items 17 to 24, comprising a nucleotide sequence encoding a linker sequence.

[0162] 26. The isolated nucleic acid molecule according to any one of items 17 to 25, comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 45 or SEQ ID NO: 69.

[0163] 27. An expression vector comprising the isolated nucleic acid molecule according to any one of items 17 to 26.

[0164] 28. A host cell comprising the isolated nucleic acid molecule according to any one of items 17 to 26 or the expression vector according to item 27.

[0165] 29. The host cell according to item 28, which is selected from a bacterial cell, a fungal cell, an insect cell or an animal cell.

[0166] 30. The host cell according to item 29, wherein the animal cell is a mammalian cell and is selected from 293T cells, CHO cells or their derived cell lines.

[0167] 31. A method for preparing a trispecific antibody, comprising: (a) providing an isolated nucleic acid molecule according to any one of Items 17 to 26 or an expression vector according to Item 27; (b) introducing the isolated nucleic acid molecule or the expression vector in (a) into a host cell; (c) culturing the host cells obtained in (b) to express the trispecific antibody; and (d) recovering the trispecific antibody from the cell culture.

[0168] 32. A method for preparing a trispecific antibody, comprising: (a) providing a host cell according to item 28 or 29, (b) culturing the host cells to express the trispecific antibody; and (c) recovering the trispecific antibody from the cell culture.

[0169] 33. The method of item 31 or 32, further comprising purifying the recovered trispecific antibody.

[0170] 34. A trispecific antibody obtained by the method according to any one of items 31 to 33.

[0171] 35. Use of a trispecific antibody according to any one of items 1 to 16 or 34 in the preparation of a drug.

[0172] 36. The use according to item 35, wherein the drug is used for the treatment of B-cell-related tumors.

[0173] 37. The use according to item 36, wherein the B-cell-related tumor is a B-cell lymphoma.

[0174] 38. The use according to item 36, wherein the B-cell-related tumor is B-cell leukemia.

[0175] 39. The use according to item 37, wherein the B-cell-related tumor is a refractory or relapsed B-cell tumor.

[0176] 40. The use according to item 37 or 39, wherein the B-cell lymphoma is an aggressive or indolent non-Hodgkin's lymphoma.

[0177] 41. The use according to item 38 or 39, wherein the B-cell leukemia is B-cell acute lymphoblastic leukemia.

[0178] 42. A method for treating a B-cell-related tumor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a trispecific antibody according to any of items 1 to 16 or 34.

[0179] 43. The method of item 42, wherein the B cell-related tumor is a refractory or recurrent B cell tumor.

[0180] 44. The method according to item 42 or 43, wherein the B-cell-associated tumor is a B-cell lymphoma or B-cell leukemia.

[0181] 45. The method according to any of items 42 to 44, wherein the B-cell-associated tumor is an invasive or indolent non-Hodgkin's lymphoma.

[0182] 46. The method according to any one of items 42 to 44, wherein the B-cell-associated tumor is B-cell acute lymphoblastic leukemia.

[0183] 47. The method according to any one of items 42 to 46, wherein the trispecific antibody is administered three times a week, twice a week, or once a week.

[0184] 48. The method of any of items 42 to 47, wherein the trispecific antibody is administered at a dose of from about 1 ng / kg body weight to about 5 mcg / kg body weight, preferably from 10 ng / kg body weight to about 2.5 mcg / kg body weight, more preferably from 0.1 mcg / kg body weight to about 1 mcg / kg body weight.

[0185] 49. A pharmaceutical composition comprising the trispecific antibody according to any one of items 1 to 16 or 34, and a pharmaceutically acceptable vector.

[0186] 50. A pharmaceutical combination comprising a trispecific antibody according to any of items 1 to 16 or 34 and another therapeutic agent.

[0187] 51. The pharmaceutical combination according to item 50, wherein the other therapeutic agent is a therapeutic agent used in the treatment of B-cell-related tumors.

[0188] 52. A linker sequence that is an amino acid sequence derived from the hinge region of IgD, that is 20 to 85 amino acids in length, and that has at least one or two of the following characteristics: (1) Contains no cysteine. (2) does not contain glycosylation sites;

[0189] 53. A linker sequence according to item 52, which is an amino acid sequence opposite the hinge region of IgD, in which cysteines are replaced by other amino acid sequences.

[0190] 54. The linker sequence according to item 53, wherein cysteine is substituted with serine or glycine.

[0191] 55. The linker sequence according to item 53 or 54, wherein the substituted cysteine is a cysteine at position 161.

[0192] 56. The linker sequence according to any of items 52 to 55, wherein the glycosylation sites are serine at position 109 and threonines corresponding to positions 126, 127, 131, and 132.

[0193] 57. A linker sequence according to any of items 52 to 56, in which glycosylation sites have been removed by amino acid substitution or truncation relative to the amino acid sequence of the hinge region of IgD.

[0194] 58. A linker sequence according to any of items 52 to 57, having one or more of the following characteristics: (1) Compared to the amino acid sequence represented by SEQ ID NO: 10, it lacks cysteines. (2) Compared with the amino acid sequence represented by SEQ ID NO: 10, it lacks glycosylation sites. (3) It is shorter in length than the amino acid sequence represented by SEQ ID NO: 10.

[0195] 59. The linker sequence according to any of items 52 to 58, having an amino acid sequence represented by SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9.

[0196] 60. A fusion protein comprising a linker sequence according to any one of items 52 to 59.

[0197] 61. The fusion protein according to item 60, which is a multispecific antibody.

[0198] 62. The fusion protein according to item 61, wherein the linker sequence is used to connect domains with different antigen specificities.

[0199] 64. A linker sequence according to any of items 52 to 59, for use in connecting different domains in a fusion protein. [Example]

[0200] For a more complete understanding and application of the present invention, the present invention will now be described in detail with reference to examples and drawings, which are intended to illustrate the present invention only and are not intended to limit the scope of the present invention, which is specifically limited by the claims appended hereto.

[0201] Example 1. Construction of eukaryotic expression vectors expressing trispecific antibodies LMOAME and MLMOF In this example, a method for constructing expression vectors that express trispecific antibodies and preparing two types of trispecific antibodies, LMOAME and MLMOF, is illustrated.

[0202] The amino acid sequence of LMOAME or MLMOF is represented by SEQ ID NO: 44 or SEQ ID NO: 68 below, respectively, in which each VH and VL sequence is represented in normal font, the three linker sequences connecting each VH and VL within the three binding domains are represented by underlines, and the two linker sequences connecting different binding domains between each binding domain are represented by square frames.

[0203] Complete amino acid sequence of LMOAME (SEQ ID NO: 44) JPEG2025525535000002.jpg85152

[0204] Complete amino acid sequence of MLMOF (SEQ ID NO: 68) JPEG2025525535000003.jpg91153

[0205] As described above, different single-chain antibody sequences are used in the CD19-binding domain and CD3-binding domain in LMOAME and MLMOF, and the CD19-binding domain and CD3-binding domain in MLMOF are cross-reactive with the corresponding monkey antigens. The CD28-binding domain in LMOAME and MLMOF is the same, and the CD28-binding domain is cross-reactive with the corresponding monkey antigen. In LMOAME and MLMOF, five of the six intradomain linkers connecting VH and VL are (G4S)3 (SEQ ID NO: 1), and only the second binding domain (CD3-binding domain) of LMOAME has a linker connecting VH and VL that is (G2S)3G2 (SEQ ID NO: 3). LMOAME and MLMOF use exactly the same interdomain linker, including a first linker sequence G4S (SEQ ID NO: 2) to connect the CD19-binding domain and the CD3-binding domain, and a second linker sequence RNTGRGGE EKKKEKEKEEQEERETKTPE SPSHTQPLGV (SEQ ID NO: 6) to connect the CD3-binding domain and the CD28-binding domain.

[0206] To express the two trispecific antibodies in mammalian cells, the nucleotide sequences encoding the trispecific molecules containing the CD19-, CD3-, and CD28-binding domains were first codon-optimized. The nucleotide sequences encoding the complete trispecific antibodies after optimization are represented by SEQ ID NO: 45 (LMOAME) and SEQ ID NO: 69 (MLMOF), respectively. These two nucleotide sequences encoding the full-length antibody amino acid sequences were then synthesized, and the synthesized sequences were cloned as inserts into the pWNP15 (Novoprotein) expression vector to form antibody expression constructs pWNP15-LMOAME and pWNP15-MLMOF. The inserts were chemically synthesized to contain EcoRI and Sali enzyme cleavage sites at both ends, and the resulting inserts were then cloned into the expression vector pWNP15, which had been linearized with EcoRI and Sali. The resulting recombinant plasmids were then sequenced. The correctly sequenced recombinant plasmids were used to construct stable expression strains of CHO-DG44 (ThermoFisher / Life Technologies, Lot No. 55463WCB2). The constructed recombinant plasmids were linearized with PyuI and transfected into CHO-DG44 using PEI-mediated transfection. Two monoclonal stable cell lines were screened using cell growth, methotrexate (MTX) pressure screening, and cell limiting dilution assays. These were named CHO-DG44-LMOAME and CHO-DG44-MLMOF, respectively.

[0207] Example 2. Expression and purification of trispecific antibodies LMOAME and MLMOF This example continues by describing how to prepare two exemplary trispecific antibodies.

[0208] 2.1 Expression and purification of LMOAME The CHO-DG44 stable expression cell line containing the LMOAME recombinant plasmid obtained in Example 1 was taken, and after a series of expansion in shake flask culture, reactor cell culture, and protein expression and secretion into the culture supernatant, the supernatant was obtained and subjected to clarification filtration. The antibody product obtained in the culture supernatant was purified by the following method.

[0209] The target protein was captured using Capto L filler (purchased from Cytiva), and 3-4 column volumes of the chromatography column were first treated with affinity wash solution. At least 4 column volumes were then balanced with affinity equilibration solution. The clarified cell harvest solution was then sampled in bulk. After sample preparation, 3-4 column volumes were then balanced with affinity equilibration solution. At least 4 column volumes were then washed with affinity wash solution. After washing, the protein was eluted using affinity elution solution. The protein was collected when the UV absorbance value rose to 10-50 mAU / 2 mm and collection was stopped when the UV absorbance value fell to 10-50 mAU / 2 mm. The eluted protein was mixed uniformly by stirring, weighed, and the protein concentration was measured.

[0210] The target protein captured by affinity chromatography is then subjected to a series of purification steps, including low-pH viral inactivation, hydrophobic chromatography, anion chromatography, UF / DF, and nanofiltration, to obtain the purified protein.

[0211] The LMOAME protein purified through multiple steps was analyzed by SDS-PAGE and SEC-HPLC. The results of SDS-PAGE and SEC-HPLC are shown in Figure 2A-B. Five different rod samples yielded bands with the correct molecular weight.

[0212] As shown in Figure 2A, under non-reducing SDS-PAGE conditions, the LMOAME protein prepared in this example shows a single electrophoretic band. The theoretical molecular weight of the LMOAME recombinant protein is 83.8 kDa, and the results indicate that the molecular weight of the purified protein is consistent with the theoretical molecular weight of the monomer. Furthermore, SEC-HPLC analysis (Figure 2B) indicated that the purity of LMOAME after purification using the method and steps of this example was >95%, and no significant misfolding or polymerization of the protein was observed in the cell culture medium.

[0213] Mass spectrometry of the purified LMOAME was performed using a reversed-phase ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometer (XEVO G2-XS-QToF, Waters). The results are shown in Figure 3. As shown in Figure 3, the purified LMOAME contained only one major component, and its molecular weight was consistent with the theoretical molecular weight of the monomer, with no glycosylation modification.

[0214] Furthermore, N-terminal sequence analysis was performed on the purified LMOAME sample, and the results showed that the expressed recombinant protein had no errors in the reading frame.

[0215] To facilitate a comparison of the effects of different linker designs on the physicochemical properties of trispecific antibodies, we performed further affinity chromatography capture and purification using another trispecific antibody previously developed by the inventors, BLMOAM (see Patent Document 1), together with LMOAME as a control. The purification results are shown in Figures 4A-B and 5. BLMOAM is also a trispecific antibody that specifically binds to CD19, CD3, and CD28, but it uses a G4S linker between the first and second binding domains (connecting fragment 1) and a linker spanning 81 amino acids and containing a cysteine between the second and third binding domains (connecting fragment 2, SEQ ID NO: 29 in the patent application). Compared to BLMOAM, the components of LMOAME were homogeneous and contained relatively low amounts of dimers or multimers, indicating that LMOAME is less likely to form dimers or multimers.

[0216] 2.2 Purification of MLMOF Using the same methods and steps as in Experiment 2.1, the antibody product in the cell culture supernatant after transfection with a recombinant plasmid containing the coding MLMOF was purified. The results are shown in Figure 6. The purity was similar to that of LMOAME, and N-terminal sequence analysis showed that the expressed recombinant protein had no errors in the reading frame.

[0217] Example 3. Measurement of antigen binding activity of trispecific antibody LMOAME by ELISA In this example, ELISA was used to evaluate the binding activity of LMOAME to recombinant human CD19 antigen (rhCD19) and recombinant human CD28 antigen (rhCD28).

[0218] Recombinant human CD19 antigen (CD19(C-Fc), Novoprotein, catalog number: C572) and CD28 antigen (CD28(C-Fc), Novoprotein, catalog number: C81) were diluted to 2 μg / mL in protein coating solution and plated at 100 μL per well overnight at 2-8°C. The next day, the plates were washed and sealed with a blocking solution of 1% casein + 3% BSA (PBS + 2% BSA (v / w)).

[0219] LMOAME purified as described in Example 2 was serially diluted with PBS to the indicated concentration range (rhCD19 binding assay: 0.0016-30 μg / mL; rhCD28 binding assay: 0.0006-10 μg / mL). The diluted antibodies were added to antigen-coated 96-well plates and incubated at 37°C for 1 hour. After washing the plates with PBST (PBS + 0.05% Tween-20 (V / V)), measurements were performed using HRP-protein L (GenScript, catalog number: M00098) and TMB (KPL SureBlue®, catalog number: 5120-0075).

[0220] Data were analyzed with GraphPad Prism software and the half maximal effective concentration (EC 50 The values were used to express the binding ability of LMOAME to rhCD19 and rhCD28 antigens, and the results are shown in Figure 7.

[0221] The results showed that the binding ability of LMOAME to rhCD19 and rhCD28 antigens was quite strong, and EC 50 The ranges of values were found to be 0.3607-0.6853 and 0.0571-0.0758 μg / mL, respectively.

[0222] Example 4. Evaluation of the binding activity of trispecific antibody LMOAME and membrane-bound antibodies by in vitro cell binding assay In this example, the binding activity of LMOAME to membrane-bound antigens hCD19, hCD3ε, and hCD28 was evaluated using an in vitro cell binding method.

[0223] 1 x 10 per well 5 Measurement cells (A549-CD19, CHO-CD28, or H9) expressing the target antigen were added to a 96-well plate at a cell density of 1000 μg / well, and negative cells not expressing the target protein were used as controls. Specifically, the cells and negative controls used for measurement of each antigen were as follows:

[0224] To measure the binding activity to hCD19, + CD3 - CD28 - The A549-CD19 cell line, which is a stable A549 cell line expressing CD19, was used, and the A549 cell line that does not express CD19 (purchased from CCTCC, GDC0063) was used as a negative control.

[0225] To measure the binding activity to hCD3, the phenotype was CD19 - CD3 + CD28 - The H9 cell line (purchased from CCTCC, GDC0031), a human T lymphoma cell line expressing CD3, was used, and a CHO cell line that does not express CD19, CD3, or CD28 was used as a negative control.

[0226] To measure the binding activity to hCD28, the phenotype was CD19 - CD3 - CD28 + The CHO-CD28 cell line, a stable CHO cell line expressing CD28, was used, and a CHO cell line that does not express CD19, CD3, or CD28 was used as a negative control.

[0227] LMOAME purified as described in Example 2 was diluted with staining buffer STB (PBS containing 0.5% vol FBS) and added to the 96-well plate at final concentrations of 30, 10, 3, 1, 0.3, 0.1, 0.03, and 0.01 μg / mL. The diluted LMOAME was then co-cultured with the assay cells at 4°C for 1 hour. Unbound antibody was removed by centrifugation. Cells bound to the LMOAME antibody were counted using Biotin Protein L (GenScript, catalog number M00097) and APC streptavidin (Biolegend, catalog number 405207), and mean fluorescence intensity (MFI) was recorded using a Thermo Fisher Attune NxT flow cytometer.

[0228] Data were analyzed with GraphPad Prism software and the half maximal effective concentration (EC 50 The values were used to represent the binding ability of LMOAME to the membrane-bound antigens hCD19, hCD3ε, and hCD28.

[0229] The results showed that the EC2 binding of LMOAME to membrane-bound antigens hCD19, hCD28, and hCD3ε 50 The values were found to be approximately 0.15-0.17 μg / mL, approximately 0.27-0.32 μg / mL, and approximately 5.1-7.5 μg / mL, respectively. The results also showed that there was no detectable binding activity between LMOAME and negative cell lines that do not express the target protein.

[0230] Example 5. In vitro evaluation of trispecific antibody-mediated effector cell activation 5.1 Evaluation of the effect of LMOAME on T cell activation In this example, the ability of LMOAME antibodies to mediate effector cell activation was tested in vitro using human PBMC as an assay system.

[0231] After co-culturing PBMCs with different concentrations of LMOAME, the expression of T cell activation markers CD25, CD69, and GrB in PBMCs was measured, and the activating effect of LMOAME on T cells was observed based on this.

[0232] Specifically, PBMCs from three healthy volunteers were extracted using Ficoll-Paque Plus (GE, Catalog No. 17-1440-02), and 2 × 10 5 The cells were added to a 96-well U-bottom cell culture plate at a density of 1000 cells / well. The purified LMOAME from Example 2 and the commercially available CD19 × CD3 bispecific antibody (BiTE) blinatumomab (Taizhou Baiying Biotechnology Co., Ltd., #20210330T001) were serially diluted in RPMI 1640 complete medium (Gibco, catalog number: C11875500CP) to achieve final LMOAME concentrations of 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 100 ng / mL, and final blinatumomab concentrations of 0.00065, 0.0065, 0.0195, 0.065, 0.195, 0.65, 1.95, 6.5, and 65 ng / mL. A solvent control group containing RPMI 1640 complete medium served as a negative control. LMOAME and blinatumomab were added to 96-well U-bottom cell culture plates and cultured with PBMCs at 37°C for 48 or 120 hours. After washing the plates with STB, the cells were stained with the following antibodies: PerCP / Cyanine5.5 anti-human CD4 antibody: BioLegend, Catalog No.: 317428 FITC anti-human CD8 antibody: BioLegend, Catalog No.: 301006 PE anti-human CD25 antibody: BioLegend, Catalog No.: 356104 BV605 anti-human CD69 antibody: BioLegend, Catalog No.: 310938 APC anti-human / mouse granzyme B antibody: BioLegend, Catalog No.: 372204

[0233] The percentage and number of the following target cell subpopulations in each well of the 96-well plate were then measured using a Thermo Fisher Attune NxT flow cytometer: CD4 + , CD4 + CD25 + , CD4 +CD69 + , CD4 + Granzyme B + , CD8 + , CD8 + CD25 + , CD8 + CD69 + and CD8 + Granzyme B + cell.

[0234] Data statistics were performed using GraphPad Prism software. If one-way ANOVA was statistically significant (P<0.05), further comparative analysis was performed. Dunnett's multiple comparison test was used to analyze differences between LMOAME or blinatumomab and the vehicle control, and Tukey's multiple comparison test was used to analyze differences between LMOAME and blinatumomab at the same molar concentration.

[0235] The results are shown in Figures 8A and 8B. Compared to the vehicle control, after 48 or 120 hours of co-culture of LMOAME with PBMCs, CD25 + , CD69 + and GrB + expressing CD4 + and CD8 + The percentage of cells with LMOAME significantly increased in both groups, and the concentration of LMOAME showed a correlation with the dose.

[0236] The results also showed that LMOAME significantly increased CD25 expression compared to blinatumomab when co-cultured with PBMCs for 48 or 120 hours. + , CD69 + and GrB + expressing CD4 + and CD8 + It was found that the proportion of cell groups in the sham group was higher.

[0237] From the above, it can be seen that the trispecific antibody LMOAME provided by the present invention inhibits CD4 + or CD8 +It can be seen that the expression of CD25 and CD69 on the surface of effector cells can be significantly increased, and the expression of GrB in effector cells can be significantly increased, thereby sufficiently activating effector cells in PBMCs. At the same time, the results also show that the activation effect of the trispecific antibody LMOAME provided by the present invention on effector cells in PBMCs is significantly higher than that of blinatumomab.

[0238] 5.2 Evaluation of the effects of LMOAME, MLMOF, and BLMOAM on inducing T cell activation (Jurkat cell line) In vitro cell biology experiments were performed to measure effector cell activation mediated by different trispecific antibodies. The trispecific antibodies measured included LMOAME and MLMOF prepared in Examples 1 and 2 of the present invention, and BLMOAM, a previously developed antibody of the applicant. A solvent control group containing RPMI1640 complete medium served as a negative control.

[0239] The specific process includes the following: (1) Jurkat cells (ATCC, TIB-152) were taken and cultured in experimental medium at 2.5 × 10 6 After preparing a cell suspension of 1000 cells / mL, inoculate 40 μL of the suspension into each well of a 96-well cell culture plate. (2) Again, take a GFP-stable Nalm-6 cell line (Novoprotein) and inoculate 2.5 × 10 cells / mL in experimental medium. 5After preparing a cell suspension at 1000 cells / mL, 40 μL was inoculated into each well of a 96-well cell culture plate. (3) 20 μL of the gradient diluted sample solution was added per well. (4) After uniform mixing, the plate was incubated at 37°C and 5% CO2 for 24 hours. (5) After washing the plate with 1% FBS-PBS, fluorescent dye 7AAD (Biolegend, catalog number 420404) and Anti-CD69-APC (Biolegend, catalog number 310910) were diluted and added to the 96-well plate. Mix uniformly, and the plate was incubated at 2°C–8°C in the dark for 30 minutes. (6) The plate was washed with 1% FBS-PBS, and 200 μL of 1% FBS-PBS was added per well. After uniform mixing, the percentage of APC-positive cells was measured using a flow cytometer. Data analysis was performed using GraphPad Prism software to determine the 50% maximum effective concentration (EC). 50 The values were used to represent the T cell activation activity of different trispecific antibodies. The results showed that LMOAME, MLMOF, and BLMOAM induced T cell activation. 50 The values were 0.624, 0.871, and 1.505 ng / mL, respectively. These results indicate that the two trispecific antibodies of the present invention can induce T cell activation at lower doses than the conventional trispecific antibody BLMOAM, which uses a different linker sequence, indicating that they have stronger T cell activation ability. Furthermore, the T cell activation ability of LMOAME was slightly higher than that of MLMOF.

[0240] Example 6. In vitro evaluation of trispecific antibody LMOAME-mediated effector cell proliferation In this example, the effect of LMOAME-mediated effector cell proliferation was evaluated. Human PBMC was used as the evaluation system, and after co-culture with PBMC with different concentrations of LMOAME, the CD4 + , CD4 + CFSE dim , CD8 + and CD8 + CFSE dim The percentage and number of cells were measured, and the proliferative effect of LMOAME-induced T cells was examined.

[0241] Specifically, PBMCs from three healthy volunteers were extracted with Ficoll-Paque Plus and incubated with 2 μM carboxyfluorescein diacetate succinimidyl ester (CFSE) at 37°C for 30 minutes. 5 The cells were added to a 96-well U-bottom cell culture plate at a density of 1000 cells / well. The purified LMOAME obtained in Example 2 and commercially available blinatumomab were serially diluted with RPMI1640 complete medium to achieve final LMOAME concentrations of 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 100 ng / mL, and final blinatumomab concentrations of 0.00065, 0.0065, 0.0195, 0.065, 0.195, 0.65, 1.95, 6.5, and 65 ng / mL. A solvent control group containing RPMI1640 complete medium served as a negative control. LMOAME and blinatumomab were added to a 96-well U-bottom cell culture plate, respectively, and cultured with PBMCs at 37°C for 120 hours. Afterwards, they were stained with Pacific Blue anti-human CD4 antibody (Biolegend, Catalog No. 317429) and APC anti-human CD8 antibody (Biolegend, Catalog No. 344722). The CD4 counts in the PBMCs in each well were analyzed using a Thermo Fisher Attune NxT flow cytometer. + , CD4 + CFSE dim , CD8 + and CD8 + CFSE dim The percentage and number of cells were determined.

[0242] The formula for calculating the fold expansion of each cell subpopulation is as follows: CD4 + Cell proliferation fold = CD4 in sample well + Cell count / solvent control well CD4 + cell number; CD4 + CFSE dim Cell proliferation fold = CD4 in sample well + CFSE dim Cell count / solvent control well CD4 + CFSE dim cell number; CD8 + Cell proliferation fold = CD8 in sample well + Cell count / solvent control well CD8 + cell number; CD8 + CFSE dim Cell proliferation fold = CD8 in sample well + CFSE dim Cell count / solvent control well CD8 + CFSE dim number of cells

[0243] Among them, the sample wells refer to wells in which LMOAME or blinatumomab is co-cultured with PBMCs, and the solvent control wells refer to wells in which the solvent control is co-cultured with PBMCs.

[0244] The measured data were expressed as mean ± standard error, and data statistics were performed using GraphPad Prism software. If one-way ANOVA was statistically significant (P < 0.05), further comparative analysis was performed. Dunnett's multiple comparison test was used to analyze the differences between LMOAME or blinatumomab and the vehicle control. Tukey's multiple comparison test was used to analyze the differences between LMOAME and blinatumomab. The results are shown in Figure 9.

[0245] As shown in the figure, LMOAME can induce significant T cell proliferation, and the fold increase in T cell proliferation is dose-dependent on the concentration of LMOAME added.

[0246] The results showed that under the same test conditions, CD4 + and CD8 + It can also be seen that the number of T cells increased was higher in both groups than in the equimolar concentration blinatumomab group. + , CD4 + CFSE dim , CD8 + and CD8 + CFSE dimThe maximum proliferation folds for the cells were 3.58, 798.13, 5.37, and 8639.48, respectively, and the maximum proliferation folds for the above cells at an equimolar concentration of blinatumomab were 1.35, 104.00, 1.84, and 1975.62, respectively. These results demonstrate that the trispecific antibody LMOAME of the present invention has a stronger ability to promote effector cell proliferation than the bispecific antibody blinatumomab.

[0247] Example 7. In vitro evaluation of trispecific antibody LMOAME-mediated effector cell differentiation In this example, we evaluated the ability of LMOAME to induce the differentiation of effector cells. PBMCs from three healthy volunteers were used as the evaluation system. After co-culture of PBMCs with different concentrations of LMOAME, the CD4 + and CD8 + T in T cells CM , T EM , T Effector and T Naive By measuring the proportion and number of cell populations, we are examining the function of LMOAME in inducing T cell differentiation.

[0248] Specifically, PBMCs from three healthy volunteers were extracted using Ficoll-Paque Plus, and 2 × 10 5 LMOAME or blinatumomab was serially diluted in RPIM1640 complete medium to final concentrations of 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 100 ng / mL for LMOAME and 0.00065, 0.0065, 0.0195, 0.065, 0.195, 0.65, 1.95, 6.5, and 65 ng / mL for blinatumomab. The dilutions were then added to the 96-well U-bottom cell culture plates and incubated with PBMCs at 37°C for 48 or 120 hours. Cells were stained with the appropriate antibodies.

[0249] The antibodies used to measure cell phenotypes are specifically as follows: Pacific Blue anti-human CD4 antibody: BioLegend, Catalog No.: 317429 FITC anti-human CD8a antibody: BioLegend, Catalog No.: 301006 PE anti-human CD45RO antibody: BioLegend, Catalog No.: 304206 APC anti-human CCR7 antibody: BioLegend, Catalog No.: 353214

[0250] The percentage and number of target cell subpopulations were measured using a Thermo Fisher Attune NxT flow cytometer, and the results are shown in Figure 10.

[0251] The results showed that co-culture of LMOAME with hPBMC for 48 hours resulted in CD4 + T CM The number of CD4 + T CM After 120 hours of co-culture, the number of LMOAME-induced CD4 + and CD8 + Memory T cells CM and T EM The number of cell populations increased significantly and showed a "bell curve" relationship with the added LMOAME. Compared to the blinatumomab group, LMOAME increased the CD4 + and CD8 + Its ability to induce differentiation of memory T cell populations is much greater than that of blinatumomab.

[0252] Example 8. In vitro evaluation of trispecific antibody LMOAME-mediated killing of target cells In this example, we evaluated the killing activity of LMOAME-mediated target cells. Human PBMCs were used as effector cells, and B-cell lymphoma cell lines Raji-GFP, Ramos, and Daudi, and precursor B-cell leukemia cell line Nalm-6 were used as target cells. After co-culture of PBMCs and target cells with different concentrations of LMOAME, we evaluated the GFP activity in the system. + or CD20 +The percentage and number of cells were measured, thereby evaluating the LMOAME-mediated killing activity of effector cells in PBMCs against different target cells.

[0253] Specifically, in this example, PBMCs were extracted from three healthy volunteers using Ficoll-Paque Plus, and effector and target cells were plated in a 96-well U-bottom cell culture plate at a ratio of 10:1 (PBMCs: 2 × 10 5 Cells / well, target cells: 2 x 10 4 LMOAME was serially diluted in RPIM1640 complete medium to final concentrations of 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 100 ng / mL, and then added to the 96-well U-bottom cell culture plate. After 72 hours of incubation at 37°C, the plate was washed and stained with 7-AAD or FITC anti-human CD20 antibody (BD Pharmingen, Cat. No. 555622). GFP in each well was detected using a Thermo Fisher Attune NxT flow cytometer. + or CD20 + The proportion and number of cells were measured, and the killing efficiency was calculated using the following formula: TIFF2025525535000004.tif17163

[0254] Data were analyzed using GraphPad Prism software, and LMOAME-mediated target cell killing activity in PBMCs was calculated as the 50% maximal effective concentration (EC 50 The results are shown in Figure 11.

[0255] The results showed that LMOAME could mediate PBMC killing of different B-cell lymphoma or B-cell leukemia cell lines in a dose-dependent manner (effect-target ratio 10:1). Among these, the EC 50The values were 1.738–2.071 pM (Raji-GFP), approximately 0.265–0.286 pM (Nalm-6), 0.690–0.864 pM (Ramos), and 0.172–0.173 pM (Daudi), respectively.

[0256] Example 9. In vitro measurement of trispecific antibody LMOAME-mediated hemolysis or agglutination In this example, different concentrations of LMOAME were incubated with a 2% New Zealand rabbit red blood cell suspension using an in vitro test tube method, and the presence of hemolysis or agglutination during this process was evaluated by macroscopic observation. In the test, the 0.9% sodium chloride injection group was used as the negative control, and the deionized water group was used as the positive control, and the observation results of LMOAME were compared with those of the control group.

[0257] Specifically, approximately 5 mL of blood was collected from the jugular vein or central ear artery of male New Zealand rabbits. Red blood cells were extracted and a 2% (v / v) suspension was prepared in 0.9% sodium chloride injection. 2.5 mL of the red blood cell suspension and 0.1-0.5 mL of LMOAME (0.14 mg / mL) purified in Example 2 were added to each test group, and the reaction mixture was adjusted to 5 mL with 0.9% sodium chloride injection. The test and control groups were cultured at 37°C ± 1°C for 3 hours. Observations were made every 15 minutes during the first hour and every hour thereafter, and photographs were taken. Changes in the color of the upper layer solution in the test tube, changes in erythrocyte sedimentation, and the presence or absence of flocculent precipitate were recorded.

[0258] The results showed that LMOAME at final concentrations of 0.0028-0.014 mg / mL did not induce hemolysis of rabbit erythrocytes, and as the incubation time was extended, various degrees of erythrocyte sedimentation were observed in the test tubes, and the upper layer was always colorless and transparent.In addition, the results showed that no reddish-brown or brown flocculent precipitates were observed in the test tubes, indicating that the drug did not induce agglutination.

[0259] Example 10. Tumor suppression effect of LMOAME in an NPG mouse Raji-luc / human PBMC co-implanted subcutaneous tumor model In this example, the tumor suppressive activity of the trispecific antibody LMOAME was measured in an NPG mouse Raji-luc / human PBMC co-implanted subcutaneous tumor model.

[0260] 5 × 10 female immunodeficient NPG mice (Beijing Weitongda Animal Husbandry Co., Ltd.) weighing approximately 21 g were injected subcutaneously into the right skin. 6 Raji-luc cells and 2.5 × 10 6 The Raji-luc cell line used in this study was added to RPMI-1640 medium containing 10% FBS and cultured in an incubator at 37°C with 5% CO2. 5 × 10 6 Suspend 2.5 x 10 cells in 50 µL of PBS and an equal volume of 2.5 x 10 cells. 6 After homogenous mixing with PBMC cells, the mixture was further homogenized with an equal volume of Matrigel and inoculated subcutaneously into the right dorsal region of each mouse in a volume of 200 μL. The animal handling procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Pengli Biosciences.

[0261] Six days after inoculation, 32 mice were selected and randomly divided into four groups (8 mice per group) based on body weight and tumor volume, including a solvent control group (G1) and groups (G2 to G4) receiving 20, 80, and 150 μg / kg of LMOAME purified in Example 2.

[0262] Mice in each group were intravenously administered twice a week (days 1 and 4) for three consecutive weeks (defined as the administration period D1 to D21), for a total of six doses. The body weight and tumor volume of the mice were measured and recorded twice a week, and tumor volume (V), tumor growth inhibition rate (TGI), relative tumor volume (RTV), and tumor weight inhibition rate (IR) were calculated. TW The calculation method for each indicator is as follows: V = (height x width 2 ) / 2 RTV=V t / V0, V t is the tumor volume measured at time point t, and V0 is the tumor volume at the start of treatment. TGI=(1-T / C)×100%, where T and C are the RTVs at a specific time point for the test group and the vehicle control group, respectively. IR TW % = (mean tumor weight of vehicle control group - mean tumor weight of test group) / mean tumor weight of vehicle control group × 100%

[0263] The results are shown in Figures 12A, 12B, and 13.

[0264] [Mouse weight] Compared with the vehicle control (G1), there was no significant difference in the body weight of mice in the 20, 80, and 150 μg / kg LMOAME groups (G2-G4) from D1 to D21 (P>0.05).

[0265] [Tumor volume (V), relative tumor volume (RTV) and tumor growth inhibition rate (TGI)] From D8 to D21, the 20, 80, and 150 μg / kg LMOAME groups (G2–G4) all showed significant tumor-inhibitory effects (P<0.001) compared with the vehicle control (G1), and the reduction in tumor volume was dose-dependent with increasing LMOAME concentration. At the end of treatment, complete disappearance of tumors was detected in all eight mice in the 150 μg / kg LMOAME group (G4).

[0266] At the end of the study, the tumors were excised and weighed, and the tumor weight inhibition rate (IRTW%) was calculated. The IRTW% values at the end of administration for groups G2 to G4 were 87.21%, 95.35%, and 100%, respectively.

[0267] Example 11. Evaluation of the tumor suppressive activity and toxicity of the trispecific antibody LMOAME in a huHSC-NCG mouse Raji-luc subcutaneous tumor model In this example, the tumor suppressive activity and toxicity of the trispecific antibody LMOAME were measured in a Raji-luc subcutaneous tumor model in huHSC-NCG mice.

[0268] The huHSC-NCG mouse is a severely immunodeficient NCG mouse in which a human immune system was reconstituted by transplanting human hematopoietic stem cells (HSCs). 5 × 10 6 Raji-luc cells were inoculated. The average tumor volume was 59.99 mm 3 At the end of the study, 54 mice were selected and randomly divided into nine groups (6 mice per group) based on the peripheral blood hCD45% × hCD3% flow data, mouse body weight, and tumor volume: vehicle control group G1, 100 μg / kg blinatumomab control groups G2–G3, and LMOAME treatment groups G4–G9. Groups G4–G5 received 50 μg / kg, G6–G7 received 150 μg / kg, and G8–G9 received 500 μg / kg. The 100 μg / kg blinatumomab group and the 150 μg / kg LMOAME group had the same molar concentration.

[0269] Mice in groups G1, G2, G4, G6, and G8 were given intravenous injections twice a week (days 1 and 4) for a total of eight injections over four consecutive weeks. Mice in groups G3, G5, G7, and G9 were given intravenous injections once a week for a total of four injections over four consecutive weeks.

[0270] The day of grouping is D1, and the administration period is D1-D28. The body weight and tumor volume of the mice were measured and recorded twice a week, and the tumor volume inhibition rate (TGI) was calculated based on the measured tumor volume. TV At the end of the study, the tumors were excised and weighed, and the tumor weight inhibition rate (TGI TW The drug effect was evaluated by calculating the tumor volume inhibition rate (TGI TV ) and tumor weight inhibition rate (TGI TW The results are expressed as tumor growth inhibition (TGI), which includes the 100% CI, 0.15-0.25 and 0.25-0.45.

[0271] T.G.I. TV The calculation formula is as follows: JPEG2025525535000005.jpg2970V nt : tumor volume on day T of mouse number n; Vn0 : tumor volume on day 0 of mouse number n; RTV n : relative tumor volume on day T of mouse number n; meanRTV treat : Mean RTV of medication group; meanRTV vehicle : Mean RTV of the solvent control group

[0272] T.G.I. TW The calculation formula is as follows: JPEG2025525535000006.jpg1567meanTW treat : Mean tumor weight of the treatment group at the study endpoint; meanTW vehicle : Mean tumor weight in the vehicle control group at the end of the study.

[0273] The pharmacological and toxicological evaluation of LMOAME was conducted based on clinical observation indices such as tumor growth inhibition rate, mouse body weight, 24-hour food and water intake, HE staining of major tissues and organs, human CD3 antigen IHC staining (tumor tissue, spleen, and brain tissue), and mouse peripheral blood flow data analysis. Animal handling procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Jixi Pharmaceutical Biotechnology Co., Ltd.

[0274] Tumor volume tumor inhibition rate (TGI) for each group during the administration period TV The changes in the tumor growth rate are shown in Table 1. The data in Table 1 show that LMOAME inhibited tumor growth in a dose-dependent manner, and that mice administered twice a week showed a higher tumor suppression rate than mice administered once a week. + and hCD20 + By measuring the number of cells in the mouse tumor model hCD19, different doses of LMOAME (50 μg / kg, 150 μg / kg, and 500 μg / kg) and 100 μg / kg of blinatumomab were able to effectively inhibit the proliferation of the model hCD19 in this mouse tumor model. + and hCD20 + It was found that it can exert tumor-suppressing effects by inhibiting cell proliferation.

[0275] [Table 1]

[0276] At the end of the study, the tumors were excised and weighed, and the tumor weight inhibition rate (TGI TW ) was calculated. The results are shown in Table 2 and Figure 14. When the dosing frequency was changed from twice weekly (BIW) to once weekly (QW), the tumor inhibition rate of LMOAME in the G7 group was higher than that of the blinatumomab (G3) group at the same molar concentration and dosing frequency.

[0277] [Table 2]

[0278] During the treatment period, no abnormalities were observed in ear temperature or food and water intake in mice in each treatment group. There was no significant difference in body weight between the treatment groups and the vehicle control group. Spleen weights were significantly reduced in mice in groups G2, G4, and G8. Because the spleen contains a large number of lymphocytes, and blinatumomab and LMOAME have a significant B lymphocyte-depleting effect, the reduction in spleen weight in the treatment groups is thought to be related to the pharmacological effects of the test products. Histopathological studies revealed no drug-related pathological reactions. IHC staining for hCD3 revealed that hCD3 positivity was primarily concentrated in the spleen and tumor tissue, with no hCD3-positive staining in brain tissue.

[0279] Example 12. Tumor-suppressing effect of trispecific antibody LMOAME in a Nalm6-luc tail vein tumor-bearing huHSC-NCG mouse model 1 x 10 cells into a female huHSC-NCG mouse 6 Nalm6-luc cells were inoculated via the tail vein. The average photon count was 5.25 × 10 4 p / sec / cm 2At the time of reaching / sr, based on the flow data of hCD45% × hCD3% cells in the peripheral blood, mouse body weight, and tumor photon count, 32 mice were randomly divided into four groups (8 mice / group), including a solvent control group (G1) and 50, 120, and 300 μg / kg dose groups (G2 to G4) of LMOAME purified in Example 2.

[0280] Mice in each group received intravenous dosing twice a week (days 1 and 4) for a total of eight doses over four consecutive weeks (the dosing period was defined as days 1 to 28). Mouse weights and tumor photon counts were measured and recorded twice a week, and tumor growth inhibition (TGI) was calculated based on the measured photon counts. The TGI calculation formula is as follows: JPEG2025525535000009.jpg2475R nt : tumor photon count on day t of mouse number n; R n0 : tumor photon count on day 0 of mouse number n; RR n : tumor relative photon count on day t of mouse number n; meanRR treat : Mean value of medication group RR; meanRTV vehicle : Mean value of RR in the vehicle control group.

[0281] Animal handling procedures were reviewed and approved by the Laboratory Animal Care and Use Committee (IACUC) of Jiaozhou Pharmaceutical Biotechnology Co., Ltd.

[0282] [12.1 Mouse weight] As shown in FIG. 15A, the body weight of all mice in each group decreased, and tumor growth is thought to be the main cause of the weight loss.

[0283] [12.2 Survival curves and mortality statistics] No mice were euthanized during the study period. The survival status of mice by group is shown in Table 3 and Figure 16. In group G1, mouse deaths began on day 15, with only one mouse remaining by day 20. By day 21, all mice in group G1 had died. In groups G2 to G4, mouse deaths began on day 20, with four, three, and seven surviving mice remaining at the end of the study, respectively. Combined with tumor fluorescence imaging data, mouse deaths were likely due to tumor burden. Compared with the vehicle control, LMOAME at different doses significantly extended the mean survival time of mice (P<0.001***).

[0284] [Table 3]

[0285] [12.3 Tumor Photon Count and Tumor Growth Inhibition Rate (TGI)] The mean tumor photon counts for mice in each group are shown in Table 4 and FIG. 15B, and the tumor growth inhibition rate (TGI) values are shown in Table 5.

[0286] [Table 4]

[0287] [Table 5]

[0288] Tables 4 and 5 show that tumor-inhibitory effects were observed in the 50, 120, and 300 μg / kg LMOAME groups (G2 to G4) on D5, D10, and D15, and the survival time of mice was significantly extended. The TGI values for the G2 to G4 groups on D5 were 67.12%, 76.18%, and 86.08%, respectively; the TGI values on D10 were 83.86%, 86.11%, and 94.38%, respectively; and the TGI values on D15 were 66.77%, 79.12%, and 92.86%, respectively.

[0289] 12.4 Flow data analysis of human immune cells in mouse peripheral blood On days 3 and 28, peripheral blood samples were collected from the mice and analyzed by flow cytometry to measure the percentage and number of mCD45, hCD45, hCD19, hCD20, hCD3, hCD4, and hCD8 cells. The data showed that the percentage and number of hCD19 cells in the peripheral blood of mice in the 50, 120, and 300 μg / kg LMOAME groups (G2 to G4) were significantly higher than those in the control group. + and hCD20 + The number of cells was found to be significantly reduced from the levels at D3, demonstrating that LMOAME can target and eliminate human B cells.

[0290] Example 13. Phase I Clinical Study of LMOAME This example describes a Phase I clinical study of the trispecific antibody of the present invention, in which the LMOAME trispecific antibody was formulated as a lyophilized powder injection and its safety, pharmacokinetics (PK), and immunogenicity were measured in subjects with relapsed and refractory (r / r) B-cell-related tumors, specifically B-cell non-Hodgkin's lymphoma (NHL) and B-cell acute lymphoblastic leukemia (B-ALL) that express CD19. Patients must have progressed after receiving currently available, approved, or established therapies with known clinical benefit.

[0291] [13.1 Participant group] Relapsed / refractory B-ALL and B-cell lymphoma.

[0292] [13.2 Research purpose] Main research objectives: The safety of LMOAME will be evaluated and, based on dose-limiting toxicity (DLT) evaluation, a recommended Phase II dose (RP2D) of LMOAME in adult patients will be established.

[0293] Secondary research objectives: -To evaluate the drug-related toxicity of LMOAME and its correlation with dose and PK of LMOAME. -To evaluate the pharmacokinetic (PK) parameters and immunogenicity of LMOAME in patients with CD19-expressing B-cell malignancies. - Identify safe and biologically active dosages and treatment plans for use in Phase II clinical trials. -To evaluate any objective tumor response rate of LMOAME in B-cell related tumors.

[0294] Exploratory research objectives: - To explore the correlation between PK parameters and B cell depletion, serum cytokine levels, CRS, and clinical treatment effects. -Perform immune classification on peripheral blood, including CD4+ and CD8+ T cell activation, CD19, and CD20 percentages. -The MRD status of ALL patients who have achieved CR / CRi will be assessed using a multiparameter flow cytometer.

[0295] [13.3 Planning Design] This study is a Phase I, multicenter, open-label, dose-escalation design. The study is divided into two phases. Patient recruitment and sample size in Phase I followed the method of Simon R et al., while Phase II followed a conventional 3+3 dose-escalation design.

[0296] Phase I: Rapid Titration Design The primary objective of Phase I is to establish the priming dose level. Other objectives include safety, PK, and immunogenicity.

[0297] There are five dose groups in total: 0.3mcg, 0.6mcg, 1.2mcg, 2.4mcg, and 4.8mcg, with one patient per dose group.

[0298] The evaluation period for dose-limiting toxicity (DLT) is 21 days.

[0299] In the first dosing cycle, the patient will be given the drug once a week in the first week, and twice a week in the second and third weeks, and the DLT observation period will be 21 days.

[0300] From the second dosing cycle onwards, the drug is administered twice weekly, with the cycle length being 3 weeks, and continues until disease progression (progressive disease; PD) or intolerance.

[0301] All five dose groups will undergo accelerated dose escalation, and if one DLT or grade 2 or higher adverse event (AE) occurs within 21 days, the dose group will be expanded to three patients and the dose escalation will be changed to a 3+3 design.

[0302] If one case of Grade 3 or higher cytokine release syndrome (CRS) or two cases of Grade 2 CRS occur at a given dose level, a second priming dose will be added to the first cycle of each dose level to designate the "intermediate" priming dose. The intermediate priming dose will be one level higher than the confirmed initial priming dose level. The DLT period will be extended to 28 days, and will include a priming dose plan (one cycle of the initial priming dose and one cycle of the intermediate priming dose) and two-week escalation dose levels. The confirmation and optimization of the priming dose schedule (initial and intermediate doses) will be based on all available data, including safety, efficacy, PK, PD (including cytokine data), and other data reviewed by the SMC. Pharmacokinetic (PD) analyses to confirm the active biological dose will include B-cell depletion, initial Grade 2 CRS, and clinical response.

[0303] If the first patient treated with 0.3 mcg develops Grade 3 or higher cytokine release syndrome (CRS) or neurotoxicity, one patient will be treated with 0.15 mcg using a 3 + 3 method. If no Grade 3 or higher CRS occurs at 0.15 mcg or the maximum tolerated dose (MTD) is not reached, the dose will be increased to 0.3 mcg.

[0304] If no instances of DLT or grade 2 or higher AEs are reported (excluding exogenous causes or disease progression), patients at the next dose level will be enrolled 21 days after the previous dose group. If a DLT or AE occurs at any time during the accelerated portion of the study, the study will revert to a 3+3 design.

[0305] Phase II: Traditional 3+3 dose-escalation design The main objective of Phase II is to study safety, PK, immunogenicity, and first-stage therapeutic efficacy, and to determine the recommended Phase II dose (RP2D).

[0306] There were five dose groups, namely 9.6mcg, 16mcg, 24mcg, 34mcg, and 45mcg, with three patients in each dose group.

[0307] In the first dosing cycle, the patient will be given the drug once a week in the first week, and twice a week in the second and third weeks, and the DLT observation period will be 21 days.

[0308] From the second dosing cycle onwards, the dose will be administered twice weekly for a 3-week cycle, continuing until PD or intolerance occurs.

[0309] Patients will be admitted to the hospital for the first 72 hours after recruitment to receive the initial dose, intermediate doses (if applicable), and titration. The first dose (Day 1 of Cycle 1, C1D1) will be administered by slow infusion within 3 hours. If there is no Grade 2 or higher infusion reaction (IRR), the next dose (Day 8 of Cycle 1, C1D8) can be administered within 2 hours, and if there is no IRR, the third and subsequent doses can be administered within 1 hour.

[0310] Premedication with an antihistamine and acetaminophen must be administered 1 hour before the three previous LMOAME doses (e.g., before the first dose of C1D1 and before the two previous BIW doses of C1D8 and C1D11). Dexamethasone may be added to subsequent infusions based on clinical indications. Investigators may reduce or cancel subsequent preoperative doses of dexamethasone as appropriate. After the infusion, patients will be monitored for twice the duration of the infusion. If CRS occurs during the three previous doses, premedication with dexamethasone 20 mg, an antihistamine, and acetaminophen must be administered at least 1 hour before the subsequent dose. If two CRS events of grade 2 or higher occur during the study period, or one CRS event of grade 3 or higher occurs, all patients will receive prophylactic treatment with acetaminophen 650 mg (oral), diphenhydramine 50 mg (oral or intravenous), and tocilizumab 8 mg / kg (intravenous over 1 hour), followed by an initial escalating dose.

[0311] On day 8 of cycle 1, patients receive their first dose at the escalating dose level on a BIW schedule over a 2-week period. The DLT period is 3 weeks (1 week of warm-up dose, 2 weeks of escalation dose). If one grade 3 or higher CRS event or two grade 2 CRS events occur at a given dose level, a second interim dose will be added to cycle 1 of each dose level, as in Phase I (see above). If all patients at the previous dose level have passed the DLT evaluation period and the MTD has not been exceeded, the escalation dose will be increased. From cycle 2 onwards, patients will begin dosing BIW on day 1. These cycles are 3 weeks long.

[0312] Patients will continue treatment until disease progression, DLT, or intolerable toxicity occurs, at which point the investigator will make a decision to discontinue treatment and withdraw informed consent, consistent with the patient's best interests.

[0313] After reviewing all safety, PK, PD, and preliminary efficacy data, the RP2D will be determined, and the dose level will then be expanded to 6-10 patients with NHL and 6-10 patients with B-ALL at the estimated RP2D.

[0314] [13.4 Number of employees to be hired] TIFF2025525535000013.tif61138 TIFF2025525535000014.tif61138

[0315] After the maximum tolerated dose (MTD) is determined, an additional 6 to 10 patients from the NHL and B-ALL groups will be recruited for an extension study to further determine the safety and efficacy at the MTD dose.

[0316] 13.5 Medication Methods The lyophilized powder of LMOAME is reconstituted and administered intravenously at the dose and frequency described in 13.3 above.

[0317] [13.6 Recruitment criteria] Male or female patients aged 18 years or older who met the following criteria were selected for inclusion in this study:

[0318] -Histologically confirmed one of several B-cell malignancies, including lymphocytes or lymphoma cells with positive CD19 antigen expression confirmed by immunohistochemistry and / or flow cytometry. If the patient has received prior CD19-directed therapy, post-treatment CD19 expression must be confirmed.

[0319] Non-Hodgkin's Lymphoma (NHL): Aggressive B-cell NHL includes the following: Diffuse large B-cell lymphoma (DLBCL), NOS Primary mediastinal large B-cell lymphoma High-grade B-cell lymphoma with MYC and BCL2 and / or BCL6 rearrangements High-grade B-cell lymphoma, NOS High-grade B-cell lymphoma caused by inactive NHL Mantle cell lymphoma (which can present as indolent or aggressive disease) Follicular lymphoma, class 3B

[0320] Inactive B-cell NHL includes: Follicular lymphoma (class 1-3A) Splenic marginal zone lymphoma Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma, excluding gastric MALT lymphoma)

[0321] Other requirements for recruitment of NHL patients include: NHL patients entering the Phase II study must have at least one measurable target lesion as defined by the Lugano criteria (nodal SAD must be ≥ 15 mm, and extranodal LD must be ≥ 10 mm). Patients with indolent lymphoma are included in treatment only if they meet the systematic treatment criteria. Patients with rapidly progressing, aggressive NHL will be included in the study only if bioactive dose levels are reached.

[0322] Aggressive NHL: Patients must have received prior treatment with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP, R-EPOCH, or equivalent anti-CD20-containing therapy). Patients must have received at least two lines of therapy and have received or are intolerant to all other standard therapies that are recognized to provide clinical benefit.

[0323] Indolent NHL: Refractory or relapsed after two or more prior lines of systemic therapy, including rituximab or other anti-CD20 monoclonal antibodies alone or in combination with chemotherapy (e.g., bendamustine, CHOP, CVP), and has received or is intolerant to all other clinically effective standard therapies.

[0324] B-cell acute lymphoblastic leukemia (B-ALL): - Philadelphia chromosome-positive or -negative B-cell leukemia that is refractory or has relapsed following treatment with first-line therapy (induction / consolidation / maintenance), salvage therapy (e.g., blinatumomab, inotuzumab ozogamicin, chemotherapy), and all available tyrosine kinase inhibitors directed against Bcr-Ab1. - Receiving or intolerant to all other standard therapies known to provide clinical benefit. -Relapsed or refractory CD19+ B-cell ALL that has failed all available therapies known to have clinical efficacy. - No active acute or chronic graft-versus-host disease in the 2 months prior to recruitment and no immunosuppressive therapy at the time of recruitment. Peripheral blood counts must be <10,000 / mm3 at the time of first dose. This count can be maintained with prednisone and / or hydroxycarbamide during the screening period. Patients with B-ALL may be recruited into the dose-upgrade portion of the study only if their dose level reaches the bioactive dose level. The bioactive dose level will be determined based on the initial CRS of grade 2 or greater and the dose level of peripheral B-cell depletion.

[0325] Both NHL and ALL patients must meet the following criteria: -ECOG status 0-2 and life expectancy greater than 3 months. - Clinical biomarkers meet the following criteria during the screening period: Total bilirubin should be less than 1.5 times the upper limit of normal (ULN). If the elevation is reasonably attributable to the presence of metastatic disease in the liver or in patients with a history of Gilbert syndrome (unconjugated hyperbilirubinemia), total bilirubin may be elevated up to 3x ULN. Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) must be less than 3 x ULN. AST and ALT may be elevated up to 5 x ULN if the elevation is reasonably attributable to the presence of metastatic liver disease. Calculated creatinine clearance >50 mL / min (Cockcroft-Gault equation) Hemoglobin must be ≥ 8g / dL. Neutrophil count >1500 / mm 3 For ALL, where neutrophil depletion is thought to be due to leukemic cell involvement in the BM, there is no standard for neutrophil counts. Platelet count: 75,000 / mm 3 For patients with ALL, if leukemia cells are thought to be engulfing the BM and causing thrombocytopenia, there is no standard platelet count. At the investigator's discretion, platelets can be transfused as needed. All patients had a peripheral blood count of ≤10,000 / mm before first use of LMOAME. 3 This can be achieved by taking dexamethasone at 20-40 mg / m² for four consecutive days. A dose of hydroxycarbamide can also be used to control the white blood cell count. These measures should result in a white blood cell count of 10,000 / mm² or less. 3 If the level does not decrease below this, the patient will not be recruited. The prothrombin time international normalized ratio (PT-INR) must be ≦1.5. - If female, have a negative urine pregnancy test or have been menopausal for at least 1 year prior to screening. Fertile female patients or male patients with fertile partners must begin using one or more methods of contraception at screening and continue for the duration of study medication until 3 months after the last dose. Methods of contraception include: a. Total abstinence (if consistent with the patient's preferred usual lifestyle). Cyclic abstinence (calendar, ovulatory, sympathetic, postovulatory, etc.) and withdrawal are not acceptable methods of contraception. b. Female sterilization (bilateral salpingectomy with or without hysterectomy) or tubal ligation performed at least 12 weeks prior to study treatment. c. Male infertility (at least 6 months prior to screening). For female patients participating in this study, the only male partner must have had a vasectomy. d. Use of oral, injectable, or implantable hormonal contraceptive methods, or other hormonal contraceptive methods with equivalent therapeutic efficacy (failure rate <1%), such as an implantable intrauterine device (IUD) or intrauterine system (IUS), hormonal contraceptive ring, or transdermal hormonal contraceptive methods. e. If female patients are taking oral contraceptives, they must be on the same medication stably for at least 3 months prior to study treatment. -Able to understand and provide written informed consent and comply with prescribed visit and study procedures.

[0326] [13.7 Exclusion criteria] -Having received systematic anti-cancer treatment (including I / O therapy) within 4 weeks prior to starting LMOAME treatment. - Having received radiation therapy within 2 weeks prior to study entry. - Receiving CAR-T or transplant therapy within 3 months prior to study entry. - Had a serious infection within 14 days prior to study entry that required antibiotic treatment. -Patients who have previously received anti-CD19 targeted therapy may participate in this study only if their tumor cells are shown to still express CD19 after completing the CD19 targeted therapy. -Patients with brain metastases or other significant neurological disease, except for patients with brain metastases who were asymptomatic and radiologically stable within 2 weeks prior to first use of LMOAME and not requiring steroids. ALL patients must not have overt CNS disease. - Have received corticosteroids (>10 mg prednisone per day or equivalent) or immunosuppressant therapy within 7 days prior to first use of LMOAME, except in the following circumstances: Topical, ophthalmic, intra-articular, intranasal or inhaled corticosteroids Dexamethasone is used to reduce peripheral blood cell counts in ALL - Have received any investigational product (including cell or gene therapy) within 5 half-lives of the drug or 4 weeks prior to starting LMOAME (whichever is shorter). -Currently have an autoimmune disease or have a history of an underlying autoimmune disease of the central nervous system. -You are allergic to any ingredient in the drug formulation (including polysorbate 80). - Admits to or has evidence of illegal drug use, drug abuse, or alcohol abuse. - Any cerebrovascular accident (CVA), transient ischemic attack (TIA), myocardial infarction (MI), unstable angina, or New York Heart Association (NYHA) Class I or IV heart failure occurring within 6 months prior to employment, or uncontrolled arrhythmia occurring within 3 months after employment. - Major surgery within 4 weeks or minor surgery within 2 weeks prior to screening. The wound must be fully healed (minor surgery such as catheter placement is not an exclusion criterion). - History of level 3-4 anaphylaxis following other monoclonal antibody treatments or known allergies to protein drugs or recombinant proteins or known cofactors contained in LMOAME formulations. - Congenital long QT syndrome, QTcF > 480 milliseconds, use of a pacemaker, left ventricular ejection fraction (LVEF) < 50%, clinically significant arrhythmia requiring intervention, cardiac troponin I or T > 2.0 ULN, poorly controlled diabetes (HbA1c > 9%), or hypertension (systolic blood pressure > 160 mmHg or diastolic blood pressure > 100 mmHg) is present. - Any other significant underlying disease (e.g., active gastric ulcer, uncontrolled seizure, cerebrovascular event, gastrointestinal bleeding, severe signs and symptoms of blood coagulation and blood coagulation disorders, cardiac conditions), mental, psychological, family or geographical situations, in some cases by the judgment of the researcher, may interfere with the planned staging, treatment and follow-up, affect patient compliance, or pose a high risk of causing treatment-related complications to the patient. - Patients who have been infected with COVID-19 and have tested negative by virus test after isolation can participate. - Patients who had other malignancies within 5 years before enrollment. However, it excludes patients with well-treated cervical intraepithelial neoplasia, local squamous cell skin cancer, basal cell carcinoma, local prostate cancer, non-invasive ductal breast cancer or urothelial cancer < T1. - Have received an organ or allogeneic stem cell / bone marrow transplant before. Patients who have received autologous stem cell transplant up to 6 months before the study screening are eligible if their hematological parameters meet the inclusion criteria. - Have a history of grade 3 - 4 immune-related adverse events (irAEs) or irAEs that required discontinuation of previous treatment. However, it excludes endocrine diseases within grade 3 managed by hormone replacement therapy. - Require regular drainage or medical intervention for pleural effusion, pericardial effusion, or ascites. - Have received live vaccines within 4 weeks before study participation. - Active hepatitis B or C. HBV carriers without active disease (HBV DNA titer < 1000 cps / mL or 200 IU / mL), or patients with cured hepatitis C (negative HCV RNA test) can participate. - Known HIV infection.

[0327] 13.8 Study Endpoints Safety Study Endpoints -Proportion of patients with serious adverse events (SAEs) and adverse events (AEs) at each dose level; -Type, frequency and dose-relatedness of DLTs; - Incidental clinical or laboratory findings.

[0328] PK Study Endpoints: AUC, C max , C min PK parameters including initial elimination rate, volume of distribution and elimination half-life

[0329] PD Study Endpoints: Depletion of B cells, release of cytokines (including IL-2, IL-6, IFNg, and TNFa)

[0330] Immunogenicity study endpoints: Percentage of patients with anti-drug antibodies (ADA), neutralizing antibodies.

[0331] Treatment endpoints: Objective remission rate (ORR) Sequence information

[0332] [Table 6]

[0333] [Table 7] TIFF2025525535000017.tif245157 TIFF2025525535000018.tif242157 TIFF2025525535000019.tif245105

[0334] [Table 8] TIFF2025525535000021.tif244157 TIFF2025525535000022.tif243157 TIFF2025525535000023.tif245108

Claims

1. A trispecific antibody that specifically binds to CD19, CD3, and CD28, wherein the trispecific antibody is a single-chain fusion protein; and (1) a first binding domain that specifically binds to CD19; (2) a first linker sequence; (3) a second binding domain that specifically binds to CD3; (4) a linker sequence II, which is an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:9; (5) a third binding domain that specifically binds to CD28; A trispecific antibody comprising:

2. The first linker sequence is (G 4 S)n or (G 2 S)n, or the first linker sequence comprises (G 4 S)n or (G 2 S) n, where n is an integer from 1 to 6, preferably from 1 to 3.

3. The trispecific antibody of claim 2, wherein the first linker sequence is selected from the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3.

4. The first binding domain is a single chain antibody that specifically binds to CD19, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises a heavy chain CDR1 represented by SEQ ID NO: 11, a heavy chain CDR2 represented by SEQ ID NO: 12, and a heavy chain CDR3 represented by SEQ ID NO: 13, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 14, a light chain CDR2 represented by SEQ ID NO: 15, and a light chain CDR3 represented by SEQ ID NO: 16; or (b) the VH comprises a heavy chain CDR1 represented by SEQ ID NO: 46, a heavy chain CDR2 represented by SEQ ID NO: 47, and a heavy chain CDR3 represented by SEQ ID NO: 48, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 49, a light chain CDR2 represented by SEQ ID NO: 50, and a light chain CDR3 represented by SEQ ID NO: 51; The trispecific antibody according to any one of claims 1 to 3.

5. (a) the VH comprises or consists of an amino acid sequence represented by SEQ ID NO: 17, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence represented by SEQ ID NO: 17, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of an amino acid sequence represented by SEQ ID NO: 19, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) with the amino acid sequence represented by SEQ ID NO: 19, wherein all amino acid differences are in non-CDR regions; or (b) the VH comprises or consists of an amino acid sequence represented by SEQ ID NO: 52, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence represented by SEQ ID NO: 52, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of an amino acid sequence represented by SEQ ID NO: 54, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) with the amino acid sequence represented by SEQ ID NO: 54, wherein all amino acid differences are in non-CDR regions. The trispecific antibody of claim 4.

6. 6. The trispecific antibody of claim 5, wherein the first binding domain comprises or consists of an amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:56, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:56, wherein all amino acid differences are located in non-CDR regions.

7. The second binding domain is a single-chain antibody that specifically binds to CD3, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the VH comprises a heavy chain CDR1 represented by SEQ ID NO: 22, a heavy chain CDR2 represented by SEQ ID NO: 23, and a heavy chain CDR3 represented by SEQ ID NO: 24, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 25, a light chain CDR2 represented by SEQ ID NO: 26, and a light chain CDR3 represented by SEQ ID NO: 27; or (b) the VH comprises a heavy chain CDR1 represented by SEQ ID NO: 57, a heavy chain CDR2 represented by SEQ ID NO: 58, and a heavy chain CDR3 represented by SEQ ID NO: 59, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 60, a light chain CDR2 represented by SEQ ID NO: 61, and a light chain CDR3 represented by SEQ ID NO: 62; The trispecific antibody according to any one of claims 1 to 6.

8. (a) the VH comprises or consists of an amino acid sequence represented by SEQ ID NO:28, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence represented by SEQ ID NO:28, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of an amino acid sequence represented by SEQ ID NO: 30, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) with the amino acid sequence represented by SEQ ID NO: 30, wherein all amino acid differences are in non-CDR regions; or (b) the VH comprises or consists of an amino acid sequence represented by SEQ ID NO: 63, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence represented by SEQ ID NO: 63, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of an amino acid sequence represented by SEQ ID NO: 65 or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence represented by SEQ ID NO: 65, wherein all amino acid differences are in non-CDR regions. The trispecific antibody of claim 7.

9. 9. The trispecific antibody of claim 8, wherein the scFv that specifically binds to CD3 comprises, or consists of, the amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 67, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 67, wherein all amino acid differences are located in non-CDR regions.

10. The trispecific antibody of any one of claims 1 to 9, wherein the third binding domain is a single-chain antibody that specifically binds to CD28 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain CDR1 represented by SEQ ID NO: 33, a heavy chain CDR2 represented by SEQ ID NO: 34, and a heavy chain CDR3 represented by SEQ ID NO: 35, and the VL comprises a light chain CDR1 represented by SEQ ID NO: 36, a light chain CDR2 represented by SEQ ID NO: 37, and a light chain CDR3 represented by SEQ ID NO:

38.

11. The VH comprises or consists of an amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence represented by SEQ ID NO: 39, wherein all amino acid differences are in non-CDR regions; and The VL comprises or consists of an amino acid sequence represented by SEQ ID NO: 41, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, and even more preferably at least 96%, at least 97%, at least 98%, or at least 99% homology) to the amino acid sequence represented by SEQ ID NO: 41, wherein all amino acid differences are in non-CDR regions. The trispecific antibody of claim 10.

12. 12. The trispecific antibody of claim 11 , wherein the three binding domains comprise or consist of the amino acid sequence set forth in SEQ ID NO: 43, or an amino acid sequence having at least 80% homology (e.g., at least 85% homology, preferably at least 90% homology, more preferably at least 95% homology, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homology) to the amino acid sequence set forth in SEQ ID NO: 43, wherein all amino acid differences are in non-CDR regions.

13. The trispecific antibody of any of claims 1 to 12, comprising an amino acid sequence represented by SEQ ID NO:44 or SEQ ID NO:68, or consisting of an amino acid sequence represented by SEQ ID NO:44 or SEQ ID NO:68, or comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence represented by SEQ ID NO:44 or SEQ ID NO:68, or consisting of an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence represented by SEQ ID NO:44 or SEQ ID NO:

68.

14. An isolated nucleic acid molecule encoding the trispecific antibody of any one of claims 1 to 13.

15. 15. The isolated nucleic acid molecule of claim 14, comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 45 or SEQ ID NO:

69.

16. 16. An expression vector comprising the isolated nucleic acid molecule of claim 14 or 15.

17. A host cell comprising the isolated nucleic acid molecule of claim 14 or 15 or the expression vector of claim 16.

18. (a) providing an isolated nucleic acid molecule according to claim 14 or 15 or an expression vector according to claim 16; (b) introducing the isolated nucleic acid molecule or the expression vector in (a) into a host cell; (c) culturing the host cells obtained in (b) to express the trispecific antibodies; and (d) recovering the trispecific antibodies from the cell culture. A method for preparing a trispecific antibody, comprising:

19. (a) providing a host cell according to claim 17; (b) culturing the host cells to express the trispecific antibody; and (c) recovering the trispecific antibody from the cell culture. A method for preparing a trispecific antibody, comprising:

20. Use of the trispecific antibody of any one of claims 1 to 13 in the preparation of a drug.

21. 21. The use according to claim 20, wherein the drug is used for the treatment of B-cell related tumors such as B-cell lymphoma, B-cell leukemia, refractory or relapsed B-cell neoplasms, aggressive or inactive non-Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia, etc.

22. A pharmaceutical composition comprising the trispecific antibody of any one of claims 1 to 13 and a pharmaceutically acceptable vector.

23. A pharmaceutical combination comprising the trispecific antibody of any of claims 1 to 13 and another therapeutic agent, such as a therapeutic agent for the treatment of B-cell-related tumors.

24. A linker sequence having an amino acid sequence represented by SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, or SEQ ID NO:9.

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