Tri-specific antibodies and uses thereof

Abstract

The present invention relates to a trispecific antibody that specifically binds to CD19, CD3 and CD28, said trispecific antibody comprising a fragment derived from the IgD hinge region as a linker. The invention also relates to the use of said trispecific antibodies for the treatment of tumors, such as malignant hematological tumors.

Description

Trispecific antibodies and their uses

[0001] Related applications

[0002] This application claims priority to the Chinese invention patent application with application number 202210822959.8, filed on July 11, 2022, and entitled “Trispecific antibodies and their uses”, and the contents of that application are incorporated by reference in their entirety. Technical Field

[0003] The present invention belongs to the field of antibody pharmaceuticals. It provides a trispecific antibody that specifically binds to CD19, CD3, and CD28, comprising a linker sequence derived from the hinge region of IgD. The antibody is particularly suitable for use as a tumor therapeutic, particularly for treating relapsed or refractory B-cell malignancies. Background Art

[0004] Antibody immunotherapy drugs are popular drugs in the field of tumor treatment. Tumor immunotherapy specifically eliminates tiny residual tumor lesions and inhibits tumor growth by activating immune cells in the body and enhancing the body's anti-tumor immune response. Due to its small side effects and significant therapeutic effects, it is gradually becoming the development direction of future tumor treatment. Immune Checkpoint Blockage (ICB) can induce lasting responses in various types of solid tumors and is considered to be the most successful immunotherapy at present, but most cancer patients still do not respond to treatment. In these patients, tumor immunogenicity and inherent MHC antigen presentation function are defective. Therefore, it is necessary to develop other methods that can promote immune cell-mediated tumor cell killing.

[0005] Bispecific T-cell engagers (BiTEs) are composed of two single-chain variable domain fragments (scFvs), one of which targets the CD3ε chain of the T-cell receptor (TCR) on T cells and is linked to a second scFv targeting a tumor-associated antigen (TAA). BiTEs can induce polyclonal T cells to lyse cancer cells in an MHC-independent manner. Bispecific T-cell engager drugs have an effective dose that is over 10,000-fold lower than that of typical antibodies, but they also have some limitations, such as dose-limiting toxicity, poor pharmacokinetic properties, and issues with the durability of therapeutic efficacy.

[0006] The transition of naive T lymphocytes from quiescence to full activation requires the synergistic action of two activation signals. After the antigen / MHC complex is recognized by the TCR, the first signal, transmitted via CD3, is insufficient to fully activate the T cell and requires signals from costimulatory molecules as a supplement. The lack of costimulatory signals can easily lead to T cell dysfunction or activation-induced T cell apoptosis (Schietinger A and Greenberg PD. Tolerance and exhaustion: defining mechanisms of T cell dysfunction. Trends in Immunology. 2014; 35: 51-60).

[0007] Among the T cell co-activating molecules discovered so far, CD28 and B7 family are the most important co-stimulatory molecules, which can bind to ligands CD80 (B7-1) and CD86 (B7-2) and amplify the first signal transmitted by TCR / CD3, maintain T cell survival, and promote cytokine-induced T cell proliferation and differentiation. CD28 is a dimeric transmembrane glycoprotein that is present in about 80% of human CD4 + T cells and 50% of CD8 + It is expressed on the surface of T cells and is therefore considered a good target for T cell activation (Jeong S and Park SH. Costimulatory receptors in cancers and their implications for cancer immunotherapy. Immune Netw. 2020; 20(1): e3; Esensten JH, Helou YA, Chopra G, et al. CD28 costimulation: from mechanism to therapy. Immunity. 2017; 44: 973-988).

[0008] CD28 costimulation can prolong T cell survival through both exogenous and endogenous mechanisms. The exogenous mechanism primarily promotes cytokine release, particularly IL-2, a key cytokine in T cell growth. The endogenous mechanism is manifested by enhancing expression of the anti-apoptotic protein Bcl-XL (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). Bcl-XL expression is weak in the presence of CD3 or CD28 alone, but is significantly enhanced in the presence of both signals (Watts TH. Staying alive: T cell costimulation, CD28, and Bcl-XL. J Immunol. 2010;185:3785–3787). In addition, CD28 co-stimulatory signals can promote the proliferation of effector T cells and memory T cells. Memory T cells can be quickly activated when the body encounters the same tumor cells again, thereby effectively eliminating tumor cells.

[0009] The importance of co-activation signals for T cell activation is also clearly reflected in the development of chimeric antigen receptor T cell (CAR-T) drugs (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.; 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.). The structure of the first-generation CAR molecule consists only of an extracellular scFv targeting tumor-associated antigens and an intracellular CD3ζ chain for T cell activation. Due to the limited expansion capacity of T cells loaded with this CAR molecule and the poor persistence of pharmacological effects, it has not been successfully verified in clinical practice. The second and third generation CAR molecules further introduced co-stimulatory signal domains (such as CD28, 4-1BB, OX40, etc.), which significantly enhanced the activation effect and persistence of pharmacological effects of CAR-T cells loaded with such CAR molecules. At present, cell therapies designed based on the second generation CAR have been successfully used in the treatment of certain hematological tumors (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.; 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.).

[0010] Based on BiTE, the present invention applies the principle of dual-signal activation of T cells to the design of multifunctional antibodies. It develops scFv domains that dual-target CD3 and CD28 of T cells, respectively, and adds scFv domains targeting tumor cell-associated antigens (TAA) to form trispecific antibodies, so as to simultaneously achieve tumor cell targeting and dual-signal activation of T cells.

[0011] The inventor discloses a trispecific antibody that combines CD19, CD3 and CD28 simultaneously in the prior application CN106589129A. In the trispecific antibody of the application, an IgD hinge region sequence of up to 81 amino acids is used as a linker between the second and third domains, and the cysteine ​​contained in the linker sequence is used to enable the trispecific antibody to form a dimer. The application verifies the expression of monomers and dimers of trispecific antibodies, antigen binding activity, cell engagement experiments (the ability of antibodies to connect CD19-positive target cells and CD3 / CD28-positive effector cells together into agglomerates) and cell killing ability (by co-culturing antibodies with lymphoma cells and CIK cells to observe the death of target cells) in vitro experiments. However, the application does not verify the efficacy of this antibody as a tumor therapeutic drug by any in vivo experiments.

[0012] In fact, although such trispecific antibody molecules can achieve better effects in principle, due to the complexity of the molecular structure, how to ensure that each binding domain can exert the expected effect is still a matter of considerable creative work in this field to improve the structure and function of trispecific antibody molecules, thereby providing a safe and effective new anti-tumor drug.

[0013] Summary of the Invention

[0014] Based on previous work, the inventors further developed a new T-cell engager antibody that can simultaneously bind to CD19, CD3, and CD28, and designed a new linker sequence for connecting the second and third domains, thereby completing the present invention.

[0015] Therefore, in a first aspect, the present invention relates to a trispecific antibody comprising:

[0016] (1) The first binding domain that specifically binds to CD19;

[0017] (2) a first linker sequence;

[0018] (3) a second binding domain that specifically binds to CD3;

[0019] (4) a second linker sequence; and

[0020] (5) The third binding domain specifically binds to CD28.

[0021] 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 Linker sequence.

[0022] In a preferred embodiment, the amino acid sequence of the second linker sequence does not contain cysteine, so that the trispecific antibody does not 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 derived from the amino acid sequence of the IgD hinge region or is an amino acid sequence comprising (EAAAK) n amino acid sequence.

[0023] In a second aspect, the present invention provides an isolated nucleic acid molecule encoding the nucleotide sequence of the trispecific antibody of the first aspect.

[0024] In a third aspect, the present invention provides an expression vector comprising the isolated nucleic acid molecule of the second aspect.

[0025] In a fourth aspect, the present invention provides a host cell comprising the expression vector of the third aspect.

[0026] In a fifth aspect, the present invention provides a method for preparing the multispecific antibody of the first aspect.

[0027] In a sixth aspect, the present invention provides use of the trispecific antibody of the first aspect in the preparation of a medicament for treating B cell-related diseases, particularly B cell-related tumors, 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 can be non-Hodgkin's lymphoma (NHL) or Hodgkin's lymphoma (HL). Examples of B cell-related diseases include, but are not limited to, classical Hodgkin lymphoma, nodular 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-PLL), precursor B cell lymphoblastic leukemia (PLL), and leukemia. B-cell lymphoblastic leukemia), hairy cell leukemia (HCL), splenic B-cell lymphoma / leukemia, splenic marginal zone lymphoma (SMZL).

[0028] In a seventh aspect, the present invention relates to a method for treating tumors, comprising using the trispecific antibody of the first aspect.

[0029] In an eighth aspect, the present invention relates to a pharmaceutical composition comprising the trispecific antibody of the present invention and a pharmaceutically acceptable carrier.

[0030] In a ninth aspect, the present invention relates to the use of the trispecific antibody of the present invention in combination with another treatment or drug.

[0031] In a tenth aspect, the present invention relates to use of an amino acid fragment derived from the hinge region of IgD as a linker sequence in a fusion protein.

[0032] The trispecific antibodies of the present invention have at least the following advantages.

[0033] (1) Long-lasting T cell activation provided by dual-signal mechanism

[0034] The trispecific antibodies of the present invention can simultaneously activate two T cell stimulation signals, CD3 and CD28. This "dual signal" activation mechanism can better regulate TCR / CD3 signals, enabling the body to produce a long-term and effective immune response. That is, it exerts its effects in the body by inducing activated T cells rather than the drug itself. Therefore, even when the drug half-life is short (relative to antibodies containing an Fc region), the frequency of administration can be reduced, and there is no need for frequent continuous infusions to maintain long-term drug activation of T cells.

[0035] CD19 / CD3 bispecific antibodies, which are also T cell engager-type Compared with blinatumomab, the trispecific antibody of the present invention is more convenient in terms of administration. Blinatumomab, due to the lack of co-stimulatory signals, can easily lead to T cell dysfunction or activation-induced T cell apoptosis, thus requiring frequent continuous infusion to maintain the drug's persistent activation of T cells.

[0036] In addition, compared with CAR-T cell therapy (such as CD19CAR-T), the trispecific antibodies of the present invention can be industrially produced through production processes and approaches similar to monoclonal antibodies and can be widely used in different individuals. On the contrary, approved CAR-T products and the vast majority of CAR-T clinical trials use autologous CAR-T, that is, the patient's own T cells are used as the starting material. The production cost of this "privately customized" therapy is high, and the high price limits its market potential. In addition, the quality stability of CAR-T therapy is relatively poor. The patient's own T cells usually have quality and quantity defects. The quality differences between different batches of products are large, making it difficult to achieve large-scale industrial production and standardized quality control. Individualized preparation and administration are required for different patients.

[0037] (2) Formation of immune synapses closer to the natural state

[0038] Under physiological conditions, the TCR on the T cell recognizes the antigenic peptide presented by the MHC of the tumor cell, thereby forming an immune synapse (IS), which is a key step in the T cell activation process. After the formation of the immune synapse under physiological conditions, the distance between the T cell and the tumor cell is usually about 15 nm. Although some bispecific antibodies can bind to T cells and tumor cells at the same time, they are limited by their own structure and the antigen binding epitopes they recognize, and cannot bring the tumor cell and the T cell closer to a distance of about 15 nm. The trispecific antibodies of the present application are structurally optimized and designed to make the immune synapse formed between the T cell and the tumor cell closer to the distance in the natural state, so that it has extremely high efficacy in mediating the process of polyclonal T cell killing of tumors (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.).

[0039] (3) Convenience based on scFv structure

[0040] Trispecific antibodies formed by scFv also have advantages such as small molecular weight and strong tumor penetration ability. Although antibodies with scFv structures have a shorter half-life compared to antibodies with Fc structures, the "dual signal" activation mechanism makes the T cells activated by trispecific antibodies persistent and less likely to undergo apoptosis after activation. In addition, trispecific antibodies using the "dual signal" activation mechanism can fully activate T cells at extremely low drug concentrations, enhance T cell survival, and achieve long-term killing of target cells by inducing the differentiation of memory T cell populations.

[0041] (4) Inducing memory T cells to achieve long-term immune response

[0042] Existing preclinical research data show that the trispecific antibody of the present invention can significantly enhance immune memory and induce the differentiation of memory T cells with self-renewal and replication capabilities, long survival time in the body, and the ability to kill tumor cells in a long-term manner, mainly manifested as effector memory T cells (T EM ) and central memory T cells (T CM ) increases in number and proportion, thereby maintaining a long-term immune response in the body and preventing tumor recurrence.

[0043] (5) Advantages of novel linker sequences

[0044] The present invention uses a novel linker sequence to connect the second and third binding domains. This linker sequence is derived from a fragment of the hinge region of IgD, obtained by modifying the hinge region to remove glycosylation sites, remove cysteines that may lead to dimer formation, and / or shorten the overall length of the hinge region. These modifications enable the resulting linker sequence, when used in trispecific antibodies, to produce more homogeneous antibody products, with fewer structural isomers or aggregates, and reduce the possibility of off-target activation. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIG1 is a schematic diagram of the structure of the trispecific antibody of the present invention.

[0046] 2A-B are the analysis results of SDS-PAGE (silver staining method) (A) and SEC-HPLC (B) of the purified LMOAME in Example 2.

[0047] FIG3 is a mass spectrum of the purified LMOAME in Example 2.

[0048] 4A-B are the results of SEC-HPLC analysis of LMOAME (A) captured by affinity chromatography and a trispecific antibody (BLMOAM) (B) as a control.

[0049] FIG5 shows the SDS-PAGE analysis results of LMOAME captured by affinity chromatography and a trispecific antibody (BLMOAM) as a control (Coomassie Brilliant Blue staining).

[0050] FIG6 is a SEC-HPLC spectrum of the purified MLMOF in Example 2.

[0051] FIG. 7 is a graph showing the binding curves of LMOAME to hCD19 and hCD28 antigens measured in Example 3.

[0052] Figures 8A-B show the results of the T cell activation activities of LMOAME and Blinatumomab measured in Example 5. (A) CD4 + Cell population; (B) CD8 + Cell clusters.

[0053] FIG. 9 shows the results of the assay in Example 6 showing the effects of LMOAME and Blinatumomab on the proliferation of effector cells.

[0054] FIG10 shows the results of two batches of LMOAME and Blinatumomab tested in Example 7 in mediating effector cell differentiation.

[0055] FIG11 shows the results of the two batches of LMOAME and Blinatumomab tested in Example 8 in terms of their ability to mediate killing of target cells.

[0056] Figure 12A-B is a curve showing the change of mouse body weight over time (mean ± SEM, g) (A) and the change of tumor volume over time (mean ± SEM, mm) measured in the animal experiment in Example 10. 3 ) (B) Statistical analysis of tumor volume and G1 in each group was performed using Dunnett's multi-comparison method. *: P < 0.05, **: P < 0.01 indicate significant differences.

[0057] 13A-B are photos of mice and tumors at the end point of dosing (day 21) determined in the animal experiment in Example 10. (A) Photo of mice at the end point of dosing; (B) Photo of the removed tumor at the end point of dosing.

[0058] Figure 14 shows the average tumor weight of mice in each group at the end of the dosing period (day 28) in the animal experiment in Example 11. Tumor weights are expressed as mean ± SEM. The independent sample T test was used to analyze the tumor weights of different groups. When compared with G1, *: P < 0.05, **: P < 0.01, ***: P < 0.001 indicate significant differences; when G2 is compared with G6, and when G3 is compared with G7, ^ :P<0.05 indicates significant difference.

[0059] FIG15A-B is a curve showing the change of mouse body weight over time after administration (mean ± SEM, g) (A) and the change of tumor photon count over time (mean ± SEM, p / sec / cm 2 The number of tumor photons in each treatment group was statistically analyzed compared with that in the G1 group using Dunnett's multi-comparison test. *: P < 0.05; **: 0.001 ≤ P < 0.01; ***: P < 0.001 indicates significant difference.

[0060] FIG16 is a survival curve of mice in each group after administration determined in the animal experiment in Example 12.

[0061] Detailed Description of the Invention

[0062] definition

[0063] In the context of the present invention, the term "antibody" is interpreted in a broad sense, i.e., it includes not only traditional IgG antibodies, but also antibody analogs and derivatives, such as scFv. For example, IgG antibodies are immunoglobulins with a Y-shaped structure that are produced by the immune system in response to foreign substances such as pathogens. The classical structure of an antibody is a homodimer, with each monomer containing a heavy chain and a light chain connected by disulfide bonds. The light chain consists of a variable region (V L ) and a constant region (C L ) and the heavy chain consists of a variable region (V H ) and three constant regions (C H 1. C H 2 and C H 3) Composition. Each variable region contains three "complementarity determining regions (CDRs)" that form the antigen binding site responsible for complementarity with the antigen. L 、C L 、V H and C H The antigen-binding fragment composed of 1 is called "Fab". The remaining part, the "trunk" part below the Y-shaped structure, is called the "Fc region", which is composed of the constant region C of the antibody heavy chain. H 2 and C H The hinge region is located at the C of IgG, IgA and IgG class immunoglobulin molecules. H 1 and C H 2, which connects the Fab and Fc regions.

[0064] "scFv" refers to a single chain variable region fragment (single chain fragment variable), sometimes also referred to as a single chain antibody, which is a fusion protein composed of the heavy chain variable region (VH) and light chain variable region (VL) of an antibody. In scFv, the connection order of VH and VL can be swapped, and VH can be placed at the N-terminus or VL can be placed at the N-terminus. VH and VL can be connected by a "linker" sequence. Single chain variable region fragments can also include heavy chain variable region fragments (Single variable domain on a heavy chain (VHH)), or nano antibodies for short. Due to the lack of light chains, nano antibodies only have three antigen recognition regions (CDR regions) belonging to the heavy chain. The molecular weight is about one-tenth of that of traditional antibodies and about one-half of that of antigen binding fragments (scFV, VH-VL).

[0065] "IgD" is one of the isotypes of immunoglobulins and has a Y-shaped structure like IgG. In the present invention, a fragment of the hinge region of IgD is used as a linker sequence.

[0066] A "multispecific antibody" is an antibody or antibody analog that is capable of simultaneously and specifically binding to multiple targets.

[0067] "Trispecific antibody" refers to an antibody or antibody analog that can simultaneously and specifically bind to three targets. In the context of the present invention, a trispecific antibody specifically refers to a single-chain fusion protein that is formed by three domains with binding specificity for different antigenic targets in series, such as three different scFvs in series.

[0068] "Specific binding" or "specific binding to..." means that the antibody exhibits preferential binding to a particular target compared to other proteins, but such specificity need not be absolute binding specificity. An antibody is considered "specific" for its intended target if its binding can determine the presence of the target protein in a sample, e.g., without producing undesirable results, such as false positives. The antibodies or antigen-binding fragments thereof of the present invention will bind to the target protein with an affinity that is at least 2 times higher, preferably at least 10 times higher, more preferably at least 20 times higher, and most preferably at least 100 times higher, than to non-target proteins. Alternatively or in addition thereto, the antibodies or antigen-binding fragments thereof of the present invention will have a binding affinity for their target protein, such as a binding affinity of less than 1x10 -7 M, 1x10 -8 M, less than 1x10 -9 M (1nM), less than 1x10 -10 M, less than 1x10 -11 M, or even lower than 1x10 -12M (1pM) K D If the antibody of the present application binds to a polypeptide comprising a given amino acid sequence (e.g., human CD19, human CD3, human CD28) but does not bind to a protein lacking the sequence, it is said to specifically bind to a polypeptide comprising the given amino acid sequence.

[0069] "Binding domain" or "antigen binding domain" in the context of multispecific antibodies herein refers to a polypeptide structure that can specifically recognize and bind to a target, sometimes also referred to as a "domain".

[0070] In the context of the present invention, a "linker sequence" refers to a peptide segment used to connect two binding domains, and also refers to a peptide segment used to connect VH and VL within a single binding domain. A "first linker sequence" specifically refers to a linker used to connect the first binding domain and the second binding domain, and a "second linker sequence" specifically refers to a linker used to connect the second binding domain and the third binding domain. The first linker sequence and the second linker sequence are both linkers used to connect different binding domains, and are therefore collectively referred to as "interdomain linkers." If a linker also exists between the VH and VL contained in a domain, it is referred to as an "intradomain linker."

[0071] "T cell engager antibodies" are antibodies that can specifically bind to two cells and bring them closer together, thereby "engaging" them in a desired manner.

[0072] A "CD19×CD3×CD28 antibody" herein refers to a trispecific antibody comprising a binding domain that specifically binds CD19, a binding domain that specifically binds CD3, and a binding domain that specifically binds CD28.

[0073] "G4S" refers to Gly-Gly-Gly-Gly-Ser, a pentapeptide unit consisting of four glycine residues and one serine residue. One or more of these units are commonly used linker sequences in recombinant fusion proteins. Similarly, "G2S" refers to Gly-Gly-Ser.

[0074] "Rigid" and "flexible" are used herein to describe the rigidity or flexibility of molecular conformations. Specifically, when used to describe a peptide linker sequence, a "flexible linker" refers to a peptide linker that allows the two linked polypeptides or functional domains to change their relative positions or interact to a certain extent, while a "rigid linker" generally does not have a similar effect and provides a more fixed conformation.

[0075] "Percent homology" or "% homology" is used in the context of the present invention 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 can be calculated as follows: after aligning the two sequences, the number of positions where the residues are identical is divided by the total length of the aligned sequences and multiplied by 100%. Methods and tools for aligning two amino acid sequences or nucleotide sequences are well known in the art, for example, the BLAST suite available on the NCBI website (Altschul, SF et al. (1990) J. Mol. Biol. 215: 403-410).

[0076] "Conservative substitutions" of amino acids are well known in the art and generally refer to changing one amino acid residue to another amino acid residue having a structurally or functionally similar side chain. For example, an exemplary conservative substitution list is provided in the table below.

[0077] "Cross-reactivity" refers to the ability of an antibody that specifically binds to one antigen to bind to another antigen. If the two antigens are from the same species, this cross-reactivity may be referred to as "intra-species cross-reactivity" or "inter-target cross-reactivity." If the two antigens are from different species, this 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, that is, the ability to react with homologous or orthologous proteins from other species.

[0078] "Nucleotide" in the context of the present invention includes DNA and RNA.

[0079] In the term "isolated nucleic acid molecule", "isolated" means that a manipulation has been performed to remove factors other than the desired component, and the isolated nucleic acid molecule no longer exists in its naturally occurring state, such as no longer exists in a chromosomal genome.

[0080] "Expression construct" or "expression cassette" refers to a nucleotide sequence comprising a target gene and gene expression regulatory sequences, including but not limited to promoters, enhancers, terminators, polyadenylation sequences, and the like.

[0081] "Vector" refers to a DNA molecule that can carry exogenous genes into cells. Examples of vectors include, but are not limited to, plasmids, viral vectors, cosmids, and artificial chromosomes. A vector used to introduce an exogenous transgene into a cell and facilitate its expression is called an "expression vector."

[0082] "Host cell" 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 expressed from the recombinant DNA. Such host cells can be used to produce the trispecific antibodies of the present invention. In one embodiment, the host cell is not particularly limited as long as it is capable of expressing a polypeptide sequence, such as the trispecific antibodies of the present invention.

[0083] "Tumor" is understood in its broadest sense to mean an abnormal overgrowth of tissue and is synonymous with "abnormal neoplasm." "Cancer" or "cancer" refers to malignant tumors, including sarcomas, carcinomas, lymphomas, leukemias, and gliomas.

[0084] "B-cell-related tumors" refer to tumors characterized by cancerous B cells, and may also be called "B-cell leukemia" or "B-cell lymphoma."

[0085] The term "subject" as used herein refers to an animal to be administered, preferably a vertebrate, more preferably a mammal, such as a rodent, eg, mouse, rat; a primate, eg, monkey; and most preferably, a human.

[0086] "Treatment" refers to the improvement, alleviation, or elimination (ie, cure) of a disease or its symptoms. In some cases, treatment includes prophylactic treatment.

[0087] Antigen binding domain

[0088] The present invention provides a T cell engager antibody that can simultaneously bind to CD19, CD3 and CD28, also referred to herein as a trispecific antibody or a CD19×CD3×CD28 antibody.

[0089] Specifically, the trispecific antibody of the present invention comprises three binding domains, namely a first binding domain that specifically binds to CD19, a second binding domain that specifically binds to and activates CD3 molecules on the surface of T cells, and a third binding domain that specifically binds to and activates CD28 molecules on the surface of T cells. In addition, the trispecific antibody further comprises a first linker positioned between the first and second binding domains, and a second linker positioned between the second and third binding domains.

[0090] 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. Full-length anti-CD19 monoclonal antibodies have been approved for the treatment of B-cell lymphomas. Unless otherwise specified, the CD19 specifically bound by the first binding domain in the context of the present invention is human CD19.

[0091] CD3 is a transmembrane protein whose transmembrane region connects to the transmembrane regions of the two peptide chains of the TCR via a salt bridge, forming a TCR-CD3 complex. Together, they participate in T cell antigen recognition and transduction of the resulting activation signal into the T cell. Unless otherwise specified, the CD3 specifically bound by the first binding domain in the context of this invention is human CD3.

[0092] CD28 is a dimeric transmembrane glycoprotein that is present in approximately 80% of human CD4 + T cells and 50% of CD8 +Expressed on the surface of T cells. CD28 is a T cell co-stimulatory molecule that binds to its ligands CD80 (B7-1) and CD86 (B7-2) and amplifies the primary signal transmitted by the TCR / CD3, maintaining T cell survival and promoting cytokine-induced T cell proliferation and differentiation. Unless otherwise specified, the CD28 specifically bound by the third binding domain in the context of the present invention is human CD28.

[0093] Therefore, for B cell-related tumors, the first binding domain in the trispecific antibody of the present invention can specifically bind to the tumor cell surface-related antigen CD19, and the second and third binding domains can bind to the T cell surface receptors CD3 and CD28, respectively, thereby "connecting" the tumor B cells and immune T cells together, and simultaneously activating the CD3 signaling pathway and CD28 co-stimulatory signaling pathway of the T cells, so that the activated immune cells can exert a killing effect on the target cells.

[0094] In a preferred embodiment, the trispecific antibody of the present invention is a single-chain fusion protein comprising: a scFv that specifically binds to CD19 as a first binding domain; a scFv that specifically binds to CD3 as a second binding domain; and a scFv that specifically binds to CD28 as a third binding domain.

[0095] The scFv sequences serving as the first, second, and third binding domains can be derived from anti-CD19, anti-CD3, and anti-CD28 antibodies, or antibody analogs and derivatives known in the art, respectively. For example, each binding domain can be an scFv comprising VH and VL sequences derived from known corresponding antibodies. The antibodies can be animal-derived antibodies, such as murine antibodies, chimeric antibodies, such as human-mouse chimeric antibodies, or human or humanized antibodies.

[0096] 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

[0097] (a) the VH comprises a heavy chain CDR 1 as set forth in SEQ ID NO: 11, a heavy chain CDR2 as set forth in SEQ ID NO: 12, and a heavy chain CDR3 as set forth in SEQ ID NO: 13, and the VL comprises a light chain CDR 1 as set forth in SEQ ID NO: 14, a light chain CDR2 as set forth in SEQ ID NO: 15, and a light chain CDR3 as set forth in SEQ ID NO: 16; or

[0098] (b) the VH comprises a heavy chain CDR 1 as shown in SEQ ID NO: 46, a heavy chain CDR2 as shown in SEQ ID NO: 47, and a heavy chain CDR3 as shown in SEQ ID NO: 48, and the VL comprises a light chain CDR 1 as shown in SEQ ID NO: 49, a light chain CDR2 as shown in SEQ ID NO: 50, and a light chain CDR3 as shown in SEQ ID NO: 51.

[0099] 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

[0100] (a) the VH comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 80% homologous (e.g., at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homologous) to the amino acid sequence of SEQ ID NO: 17 and in which all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; and

[0101] The VL comprises the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 19 and all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; or

[0102] (b) the VH comprises the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence that is at least 80% homologous (e.g., at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homologous) to the amino acid sequence of SEQ ID NO: 52 and wherein all of the amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; and

[0103] The VL comprises the amino acid sequence shown in SEQ ID NO: 54, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 54 and all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence.

[0104] In a further preferred embodiment, the scFv that specifically binds to CD19 comprises an amino acid sequence as shown in SEQ ID NO: 21 or SEQ ID NO: 56, or an amino acid sequence that has at least 80% homology (such as 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 as shown in SEQ ID NO: 21 or SEQ ID NO: 56 and all the amino acid differences are in non-CDR regions; or consists of such an amino acid sequence.

[0105] Preferably, the above amino acid differences are conservative substitutions of amino acids.

[0106] 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., connected in the order of VL-linker sequence-VH.

[0107] In some embodiments, in the scFv that specifically binds to CD19, VH and VL are connected by an intradomain linker or are directly connected. The intradomain linker is a linker containing (GGGGS)n or (GGS)n, or (GGGGS)n or (GGS)n, and n is an integer of 1-10, preferably an integer of 1-5, for example 1, 2, 3, 4 or 5. In a specific embodiment, in the scFv that specifically binds to CD19, the intradomain linker between VH and VL is selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, preferably SEQ ID NO: 1.

[0108] 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

[0109] (a) the VH comprises a heavy chain CDR1 as set forth in SEQ ID NO: 22, a heavy chain CDR2 as set forth in SEQ ID NO: 23, and a heavy chain CDR3 as set forth in SEQ ID NO: 24, and the VL comprises a light chain CDR1 as set forth in SEQ ID NO: 25, a light chain CDR2 as set forth in SEQ ID NO: 26, and a light chain CDR3 as set forth in SEQ ID NO: 27; or

[0110] (b) the VH comprises a heavy chain CDR 1 as shown in SEQ ID NO: 57, a heavy chain CDR2 as shown in SEQ ID NO: 58, and a heavy chain CDR3 as shown in SEQ ID NO: 59, and the VL comprises a light chain CDR 1 as shown in SEQ ID NO: 60, a light chain CDR2 as shown in SEQ ID NO: 61, and a light chain CDR3 as shown in SEQ ID NO: 62.

[0111] 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

[0112] (a) the VH comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 80% homologous (e.g., at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homologous) to the amino acid sequence of SEQ ID NO: 28, and all of the amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; and

[0113] The VL comprises the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 30 and all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; or

[0114] (b) the VH comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 80% homologous (e.g., at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homologous) to the amino acid sequence of SEQ ID NO: 63 and wherein all of the amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; and

[0115] The VL comprises the amino acid sequence shown in SEQ ID NO: 65, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 65 and all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence.

[0116] In a further preferred embodiment, the scFv that specifically binds to CD3 comprises the amino acid sequence as shown in SEQ ID NO: 32 or SEQ ID NO: 67, or an amino acid sequence that has at least 80% homology (such as 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 as shown in SEQ ID NO: 32 or SEQ ID NO: 67 and all the amino acid differences are in non-CDR regions; or consists of such an amino acid sequence.

[0117] Preferably, the above amino acid differences are conservative substitutions of amino acids.

[0118] 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.

[0119] In some embodiments, in the scFv that specifically binds to CD3, VH and VL are connected by an intradomain linker or directly connected. The intradomain linker is a linker containing (GGGGS)n or (GGS)n, or (GGGGS)n or (GGS)n, and n is an integer of 1-10, preferably an integer of 1-5, for example 1, 2, 3, 4 or 5. In a specific embodiment, in the scFv that specifically binds to CD3, the intradomain linker between VH and VL is selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, preferably SEQ ID NO: 1 or SEQ ID NO: 3.

[0120] 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 CDR 1 as shown in SEQ ID NO: 33, a heavy chain CDR2 as shown in SEQ ID NO: 34, and a heavy chain CDR3 as shown in SEQ ID NO: 35, and the VL comprises a light chain CDR 1 as shown in SEQ ID NO: 36, a light chain CDR2 as shown in SEQ ID NO: 37, and a light chain CDR3 as shown in SEQ ID NO: 38.

[0121] 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 comprises the amino acid sequence as shown in SEQ ID NO: 39, or an amino acid sequence that has at least 80% homology (such as 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 as shown in SEQ ID NO: 39 and all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; and the VL comprises the amino acid sequence as shown in SEQ ID NO: 41, or an amino acid sequence that has at least 80% homology (such as 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 as shown in SEQ ID NO: 39; The amino acid sequence shown in NO:41 has at least 80% homology (such as 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) and all amino acid differences are in the amino acid sequence of non-CDR regions, or consists of such an amino acid sequence.

[0122] In a further preferred embodiment, the scFv that specifically binds to CD28 comprises the amino acid sequence as shown in SEQ ID NO:43, or an amino acid sequence that has at least 80% homology (such as 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 as shown in SEQ ID NO:43 and all the amino acid differences are in non-CDR regions; or consists of such an amino acid sequence.

[0123] Preferably, the above amino acid differences are conservative substitutions of amino acids.

[0124] 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.

[0125] In some embodiments, in the scFv that specifically binds to CD28, VH and VL are connected by an intradomain linker or are directly connected. The intradomain linker is a linker containing (GGGGS)n or (GGS)n, or is (GGGGS)n or (GGS)n, and n is an integer of 1-10, preferably an integer of 1-5, for example 1, 2, 3, 4 or 5. In a specific embodiment, in the scFv that specifically binds to CD28, the intradomain linker between VH and VL is selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, preferably SEQ ID NO: 1.

[0126] Those skilled in the art will appreciate that the determination of the CDR regions can be determined using any of the Kabat, IMGT, and Chothia antibody numbering systems. When using different numbering systems, different CDR regions may be delineated for the amino acid sequence of the same variable region. Regardless of which numbering system is used to determine the CDR regions, they all fall within the scope of protection of the present invention. Unless otherwise stated, the amino acid sequences of the specific CDR regions recorded in this application are determined according to the Kabat antibody numbering system.

[0127] In some embodiments, one or more of the first binding domain, the second binding domain, and the third binding domain of the present application have cross-reactivity with corresponding antigens in other species. The other species can be laboratory animals, such as monkeys, such as crab-eating macaques (Macaca fascicularis). For example, the first binding domain of the present application, on the basis of specific binding to human CD19, also has cross-reactivity with monkey CD19, that is, it can also specifically bind to monkey CD19. For example, the second binding domain of the present application, on the basis of specific binding to human CD3, also has cross-reactivity with monkey CD3, that is, it can also specifically bind to monkey CD3. For example, the third binding domain of the present application, on the basis of specific binding to human CD28, also has cross-reactivity with monkey CD28, that is, it can also specifically bind to monkey CD28. Cross-reactivity with corresponding monkey antigens will facilitate antibody research and development.

[0128] Adapter sequence

[0129] On the basis of determining the binding domains, how to design the linkers used to connect the various binding domains, as well as the linkers used to connect the VH and VL in each binding domain, is also crucial for the structure and function of trispecific antibodies, especially the former. On the one hand, the linker sequence and the functional domain together constitute the entire trispecific antibody molecule, and the linker sequence will affect the structural stability of the entire trispecific antibody molecule. On the other hand, the length and flexibility of the linker sequence will affect the spatial position relationship between the various binding domains, and thus affect the binding of each domain to its target. In addition, whether certain amino acids in the linker sequence are prone to further modification, such as dimerization and glycosylation, will also affect the overall properties of the trispecific antibody molecule.

[0130] The linker sequence of the present invention can be derived from a naturally occurring sequence or a modified variant thereof, or can be an artificial sequence designed from scratch. The linker sequence can be derived from a naturally occurring multi-domain protein, for example, a linker sequence contained in a natural multi-domain protein or a variant thereof. An example of a linker sequence derived from a naturally occurring sequence is a linker sequence from the hinge region of IgD, which can be used as a second linker sequence in the present application. An example of an artificial linker sequence is, for example, based on (GGGGS) n 、(GGGGS) n GGGG, (SGGGG) n 、GGGG(SGGGG) n 、(GGS) n 、(GGS) n GG, (SGG) n or GG(SGG) n A linker sequence, which can be used as a first interdomain linker or as an intradomain linker in the present invention. Another example of an artificial linker sequence is one comprising (EAAAK) n Linker sequence, such as A(EAAAK) n A, A(EAAAK) n ALEA(EAAAK) n A, which can serve as the second linker sequence in the present invention.

[0131] The rigidity / flexibility of the linker is a factor that needs to be considered. A flexible linker allows the polypeptide sequences connected at both ends to move within a certain range. Flexible linkers are usually composed of smaller amino acids such as glycine (G), serine (S), and threonine (T), and it is these small amino acids that give the linker flexibility. In cases where it is desired to retain the possibility of some relative movement for the domains at both ends of the linker, a flexible linker is preferably used. However, in some cases, flexible linkers cannot fix the domains connected at both ends, making it impossible for multi-domain molecules to express or function correctly. From this perspective, for multifunctional molecules, such as trispecific antibodies that need to bind to three targets, rigid linkers may be able to provide better structural and functional stability than flexible linkers.

[0132] Specifically for the trispecific antibodies of the present invention, the first domain targets tumor cells, while the second and third domains target immune cells, that is, the adjacent first and second domains will inevitably bind to different cells. In this case, it is hoped that the first domain and the second domain have a certain ability to move relative to each other, so that the binding can be made easier by adjusting the position when binding to different cells. Therefore, in a preferred embodiment, the first linker sequence of the present invention is a flexible linker, such as a linker composed of one or more of glycine (G), serine (S), threonine (T) and lysine (K), such as a GS-rich linker, such as (GGGGS). n 、(SGGGG) n 、(GGS) n 、(SGG) n Connectors, such as (GGGGS) n 、(GGGGS) n GGGG, (SGGGG) n 、GGGG(SGGGG) n 、(GGS) n 、(GGS) n GG, (SGG) n or GG(SGG) n , wherein n is a positive integer, preferably 1 to 10. The linker used to connect VH and VL in each binding domain is also preferably such a flexible linker.

[0133] On the other hand, as an adapter antibody, it is hoped that the two cells to be connected will be brought as close as possible, so that the distance of the immune synapse formed between the cells will be closer to the distance of the immune synapse formed naturally under physiological conditions, which is more conducive to the immune cells to play a killing role. The distance between the two cells 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, if the first linker sequence is (GGGGS) n 、(SGGGG) n 、GGGG(SGGGG) n If the first linker sequence is (GGS), then preferably n is a positive integer not exceeding 6, preferably a positive integer not exceeding 5, such as 1, 2, 3, 4 or 5; n 、(SGG) n or (GGS) n GG, then preferably n is a positive integer not exceeding 10, 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), preferably the amino acid sequence shown in SEQ ID NO: 1.

[0134] In another embodiment, the second joint sequence is connected to the second and third domains, and these two domains are all combined to the receptor on T cell surface.In order to prevent that after one of them domain combines, the steric hindrance effect of generation affects another domain and is combined on the same T cell, and slightly longer second joint sequence is more ideal.Therefore, in a preferred embodiment, the second joint sequence is longer than the first joint sequence.For example, the length of the second joint 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 longer than the length of the first structural sequence.For example, the length of the second joint sequence is at least 15 amino acid, at least 20 amino acid, at least 25 amino acid, at least 30 amino acid, at least 35 amino acid, at least 40 amino acid or longer.

[0135] In addition, a linker that is more rigid can also be used as a preferred second linker sequence. A more rigid linker is also beneficial to avoid steric hindrance effects. For example, an example of a second linker is (EAAAK) n The Glu-Ala-Ala-Ala-Lys structure in this linker can form a rigid α-helical structure. nThe reason for the "linker sequence" is that such linkers usually contain other amino acid residues, such as one or more of alanine (A), leucine (L), and glutamic acid (E) on both sides of the EAAAK repeat. Specific examples include A(EAAAK) n A.LEA(EAAAK) n ALE, LEA (EAAAK) n ALEA(EAAAK) n ALE, etc., wherein n can be any integer from 1 to 10, preferably from 2 to 6. The type of linker can be selected and / or the value of n can be adjusted according to the required linker sequence length.

[0136] In a specific embodiment, one of the linker sequences of the present invention, such as the second linker sequence, is a fragment from the hinge region of immunoglobulin IgD or a variant thereof.

[0137] 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 a glycosylation site, the sugar chains formed by glycosylation may mask the active site in an unexpected manner, reducing the activity of the antibody. When the antibody is produced in cells, sugar chains of different compositions and lengths may be formed, resulting in a decrease in the homogeneity of the antibody product. Common types of glycosylation include N-linked glycosylation and O-linked glycosylation. N-linked glycosylation is a type of glycosylation in which the sugar chain is attached to the nitrogen atom of the side chain of amino acids such as asparagine and arginine, while O-linked glycosylation is a type of glycosylation in which the sugar chain is attached to the oxygen atom of the side chain hydroxyl of amino acids such as serine, threonine, and tyrosine. When the linker sequence is derived from a natural sequence, such as from the hinge region of an immunoglobulin, potential glycosylation sites in the sequence, such as threonine, can be removed by performing mutations including deletion, substitution, and truncation of the natural sequence.

[0138] In a specific embodiment, a fragment of the IgD hinge region is used as a linker sequence, such as a second linker sequence. It is known that the IgD hinge region includes multiple O-linked glycosylation sites, such as the serine at position 109 (S109) and four threonines (T126, T127, T131, and T132). These sites are included in the 81-amino acid long linker provided herein, and the amino acid sequence of the linker sequence is shown in SEQ ID NO:4 or SEQ ID NO:8, wherein the one serine and four threonines mentioned above 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 include a glycosylation site, such as the five glycosylation sites mentioned above. In one embodiment, the IgD hinge region fragment that does not include a glycosylation site can be truncated, such as by removing at least the five glycosylation sites mentioned above. For example, the entire fragment before (including) the last threonine can be removed, retaining only all or part of the sequence at its C-terminus. As another alternative, the amino acids at the five glycosylation sites may be substituted with other amino acids through modification.

[0139] Single-chain antibodies can form dimers through the presence of cysteines in their linker regions. Trispecific antibody dimers contain twice the antigen-binding domains. Studies have shown that because CD28 naturally exists as a dimer, bivalent antibodies (e.g., IgG antibodies) containing two domains that specifically bind to CD28 are more potent than monovalent antibodies (e.g., single-chain antibodies). Similarly, CD28 antibodies exhibit increased potency when cross-linked or when aggregated. However, trispecific antibodies must simultaneously and correctly bind to all three targets to exert their intended effect. If a trispecific antibody binds only to one or two targets on T cells (CD3 and / or CD28) but not CD19 on B cell tumors, it may lead to T cell activation at sites distant from the tumor target cells, a phenomenon known as "off-target T cell activation." In this scenario, trispecific antibodies in dimer form are more dangerous due to their stronger and more potent binding to the T cell receptor CD28, as well as their susceptibility to cross-linking, which can lead to more difficult-to-manage off-target activation and increase the risk of a cytokine storm. Therefore, the present invention does not want the trispecific antibodies to form dimers during preparation and / or use, and for this purpose, the linker sequence, especially the second linker sequence based on the hinge region of IgD, is modified to remove amino acid residues that may cause dimer formation, such as cysteine ​​(Cys; C) residues.

[0140] In a preferred embodiment, the linker sequence of the present invention does not contain amino acid sites capable of forming dimers, such as cysteine ​​(C). In a specific embodiment, the second linker sequence is derived from the IgD hinge region and cysteine ​​(C161) is removed. For example, the cysteine ​​in the linker sequence is removed by replacing the cysteine ​​at position 161 with another amino acid. For example, the cysteine ​​in the linker sequence is removed by replacing the cysteine ​​at position 161 with serine or glycine (C161S or C161G substitution).

[0141] In a preferred embodiment, the first linker sequence of the trispecific antibody of the present invention is a linker containing (GGGGS)n or (GGS)n, or (GGGGS)n or (GGS)n, and n is an integer not exceeding 6, preferably an integer not exceeding 5, such as 1, 2, 3, 4 or 5. In a specific embodiment, the first linker sequence is the amino acid sequence shown 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 shown in SEQ ID NO: 1.

[0142] In a preferred embodiment, the second linker sequence of the trispecific antibody of the invention is derived from the IgD hinge region and has one or more of the following characteristics:

[0143] (1) Compared with the amino acid sequence shown in SEQ ID NO: 10, it lacks cysteine;

[0144] (2) lacking a glycosylation site compared to the amino acid sequence shown in SEQ ID NO: 10;

[0145] (3) Compared with the amino acid sequence shown in SEQ ID NO: 10, it has a shorter length.

[0146] In a preferred embodiment, the second linker sequence is compared with SEQ ID NO: 10, and the cysteine ​​at position 72 is mutated to another amino acid, preferably serine or glycine. In a specific embodiment, the second linker sequence is the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 9.

[0147] In a preferred embodiment, the second linker sequence is a partial fragment of SEQ ID NO: 4 that does not contain a glycosylation site, for example, an N-terminal partial fragment or a C-terminal partial fragment of SEQ ID NO: 4 that does not contain a glycosylation site, preferably a partial fragment consisting of the C-terminal 38 amino acids of the amino acid sequence of SEQ ID NO: 4; the glycosylation site contains at least a serine corresponding to position 20 and threonine corresponding to positions 37, 38, 42, and 43. In a specific embodiment, the second linker sequence is the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 9.

[0148] In the most preferred embodiment, the second linker sequence of the trispecific antibody of the present invention is the amino acid sequence shown in SEQ ID NO:6 or SEQ ID NO:9.

[0149] In another preferred embodiment, the second linker sequence of the trispecific antibody of the present invention comprises (EAAAK) n Linker sequence, such as 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 from 2 to 6, more preferably from 2 to 4. Specifically, the second linker sequence is a linker sequence comprising the amino acid sequence shown in SEQ ID NO: 7, for example, a linker sequence comprising SEQ ID NO: 7 as a repeating unit, wherein the repeating unit is repeated 1 to 10 times.

[0150] In a preferred embodiment, the linker sequence for connecting VH and VL in each binding domain of the trispecific antibody of the present invention, i.e., the intradomain linker, is a linker containing (GGGGS)n or (GGS)n, or (GGGGS)n or (GGS)n, and n is a positive integer not exceeding 10, preferably a positive integer not exceeding 5, such as 1, 2, 3, 4 or 5. In some embodiments, the linker sequence for connecting VH and VL in each binding domain is the same. In some embodiments, the linker sequence for connecting VH and VL in each binding domain is different. For example, the intradomain linker sequence is (GGGGS) n 、(SGGGG) n 、GGGG(SGGGG) n Any of, wherein preferably n is a positive integer not exceeding 6, preferably a positive integer not exceeding 5, such as 1, 2, 3, 4 or 5. For example, the intradomain linker is (GGS) n、(SGG) n or (GGS) n GG, wherein preferably n is a positive integer not exceeding 10, preferably a positive integer not exceeding 7, such as 1, 2, 3, 4, 5, 6 or 7.

[0151] In a specific embodiment, the intradomain linker is selected from the sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3. In a more specific embodiment, the linker used to connect VH and VL in each binding domain is (GGGGS)3 as shown in SEQ ID NO: 1. In a more specific embodiment, the three intradomain linkers are (GGGGS)3 as shown in SEQ ID NO: 1, (GGS)4GG as shown in SEQ ID NO: 3, and (GGGGS)3 as shown in SEQ ID NO: 1.

[0152] Trispecific antibodies

[0153] Once the antigen specificity and arrangement order of each antigen binding domain are determined, there is no particular limitation on the specific sequence of each binding domain. The three binding domains can be derived from corresponding antibodies or antibody analogs known in the art.

[0154] In a preferred embodiment, the trispecific antibody of the present invention is capable of specifically binding to human CD19, human CD3, and human CD28, and comprises a linker sequence as set forth in 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 a linker sequence connecting a binding domain that specifically binds to CD3 and a binding domain that specifically binds to CD28.

[0155] In a specific embodiment, the trispecific antibody of the present invention is a trispecific antibody or a variant thereof having an amino acid sequence as set forth in SEQ ID NO:44 or SEQ ID NO:68; the variant has at least 80% homology to the amino acid sequence as 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 linker sequence as set forth in 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 the variant has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology to the amino acid sequence as set forth in SEQ ID NO:44 or SEQ ID NO:68.

[0156] Preparation method

[0157] The present invention also relates to a method for preparing the trispecific antibody. The method can be a molecular biology method. For example, the method comprises preparing one or more nucleotide sequences encoding the trispecific antibody single-chain fusion protein, constructing the one or more encoding nucleotide sequences into one or more expression vectors, and expressing the expression vectors in appropriate cells for preparation.

[0158] Those skilled in the art will appreciate that, given the amino acid sequence of the trispecific antibody is determined, different encoding nucleotide sequences can be used due to codon degeneracy, and the encoding nucleotide sequence can also be optimized. Methods for codon optimization of coding sequences are known to those skilled in the art and include adjusting codons to the host's preferred codons, reducing GC content and / or reducing GC-rich regions, and improving mRNA stability, thereby increasing the expression efficiency of the target nucleotide in a specific host.

[0159] In a specific embodiment, the encoding nucleotide sequence comprises a nucleotide sequence encoding a first binding domain that specifically binds to CD19 as described in the present invention.

[0160] In a specific embodiment, the nucleotide sequence encoding the first binding domain may comprise: (1) a nucleotide sequence encoding VH as shown in SEQ ID NO: 18, or a nucleotide sequence having 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% homology thereto and encoding the amino acid sequence of VH as shown in SEQ ID NO: 17; and / or (2) a nucleotide sequence encoding VL as shown in SEQ ID NO: 20, or a nucleotide sequence having 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% homology thereto and encoding the amino acid sequence of VL as shown in SEQ ID NO: 19. In a specific embodiment, the nucleotide sequence encoding the first binding domain may comprise: (1) a nucleotide sequence encoding VH as shown in SEQ ID NO: 53, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto and encoding the amino acid sequence of VH as shown in SEQ ID NO: 52; and / or (2) a nucleotide sequence encoding VL as shown in SEQ ID NO: 55, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto and encoding the amino acid sequence of VL as shown in 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 as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0161] In a specific embodiment, the encoding nucleotide sequence comprises a nucleotide sequence encoding a second binding domain that specifically binds to CD3 as described herein. The encoding nucleotide sequence of the second binding domain may comprise: (1) a nucleotide sequence encoding VH as shown in SEQ ID NO: 29, or a nucleotide sequence having 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% homology thereto and encoding the amino acid sequence of VH as shown in SEQ ID NO: 28; and / or (2) a nucleotide sequence encoding VL as shown in SEQ ID NO: 31, or a nucleotide sequence having 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% homology thereto and encoding the amino acid sequence of VL as shown in SEQ ID NO: 30. In a specific embodiment, the nucleotide sequence encoding the first binding domain may comprise: (1) a nucleotide sequence encoding VH as shown in SEQ ID NO: 64, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto and encoding the amino acid sequence of VH as shown in SEQ ID NO: 63; and / or (2) a nucleotide sequence encoding VL as shown in SEQ ID NO: 66, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto and encoding the amino acid sequence of VL as shown in 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 as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0162] In a specific embodiment, the encoding nucleotide sequence comprises a nucleotide sequence encoding a third binding domain that specifically binds to CD28 as described herein. The encoding nucleotide sequence of the third binding domain may comprise: (1) a nucleotide sequence encoding VH as shown in SEQ ID NO: 40, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto and encoding the amino acid sequence of VH as shown in SEQ ID NO: 39; and / or (2) a nucleotide sequence encoding VL as shown in SEQ ID NO: 42, or a nucleotide sequence having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto and encoding the amino acid sequence of VL as shown in SEQ ID NO: 41. In a specific embodiment, the encoding nucleotide sequence of the third binding domain further comprises a nucleotide sequence encoding a linker sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0163] In a specific embodiment, the nucleotide sequence encoding the trispecific antibody of the present invention comprises the nucleotide sequence as shown in SEQ ID NO: 45, or a nucleotide sequence having 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% homology thereto and encoding the amino acid sequence as shown in SEQ ID NO: 44. In a specific embodiment, the nucleotide sequence encoding the trispecific antibody of the present invention comprises the nucleotide sequence as shown in SEQ ID NO: 69, or a nucleotide sequence having 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% homology thereto and encoding the amino acid sequence as shown in SEQ ID NO: 68.

[0164] 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 production cells. In some embodiments, microbial cells, such as bacterial cells or fungal cells, are used as production cells, such as E. coli or yeast. In some embodiments, insect cells, such as Sf9, are used as production cells. The nucleotide sequence encoding the trispecific antibodies of the present invention can be codon-optimized for a specific production cell line.

[0165] The trispecific antibody produced is preferably purified. Purification of the trispecific antibody can be performed using conventional methods in the field of antibody production, which may include filtration, chromatography, and other steps. The filtration step can be selected from one or more of depth filtration, ultrafiltration, diafiltration, and nanofiltration. The chromatography step can be selected from one or more of affinity chromatography, cationic chromatography, anionic chromatography, size exclusion chromatography, hydrophobic chromatography, and hydroxyapatite chromatography.

[0166] The antibodies of the present invention can be prepared into liquid or solid preparations, such as injections, suspensions, powders, and granules. For example, they can be prepared into lyophilized powder injections by conventional methods such as freeze drying, and then reconstituted into injections for administration on the spot by reconstitution before administration.

[0167] Cancer treatment

[0168] The trispecific antibodies of the invention can be used in therapeutic or diagnostic applications, such as the treatment of cancer.

[0169] As an adapter antibody, the first binding domain of the trispecific antibody of the present invention is responsible for targeting and binding to tumor-associated antigens. When the first binding domain specifically binds to CD19, the trispecific antibody of the present invention is particularly suitable for targeting target cells expressing CD19, such as CD19 associated with diseases. + B cells.

[0170] Therefore, the trispecific antibodies of the present invention are particularly suitable for treating diseases involving B cells, such as B cell-related tumors, including B cell lymphomas and B cell leukemias. In particular, the B cell-related tumors are relapsed or refractory B cell malignancies, that is, the subject has experienced a prior relevant treatment and achieved a therapeutic benefit, or has not produced the expected response to the prior relevant treatment.

[0171] The B cell lymphoma can be non-Hodgkin lymphoma (NHL), such as aggressive or indolent non-Hodgkin lymphoma, or Hodgkin lymphoma. Examples of the B cell-related diseases include, but are not limited to, classical Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, diffuse large B cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue lymphoma, small lymphocytic lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, Burkitt lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-prolymphocytic leukemia, precursor B cell lymphocytic leukemia, hairy cell leukemia, splenic B cell lymphoma / leukemia, and splenic marginal zone lymphoma.

[0172] Non-Hodgkin's lymphoma (NHL) is one of the most common cancers worldwide, representing a heterogeneous group of lymphoid malignancies. The major subtypes are B-cell-derived, including diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Current treatment strategies for B-cell-derived NHL include anti-CD20 rituximab and the R-CHOP regimen (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), a rituximab-chemotherapy combination. Although treatment efficacy has improved with rituximab alone or in combination with chemotherapy in some NHL subtypes, some patients still fail R-CHOP and progress to relapsed / refractory disease. Re-treatment with anti-CD20 monoclonal antibodies is difficult for these patients to achieve satisfactory results. The therapeutic efficacy of rituximab for B-cell lymphoma depends on the level of CD20 expression on tumor cells. However, CD20 expression on tumor cells is heterogeneous. Patients with low CD20 expression are insensitive to rituximab and develop innate resistance. Furthermore, patients with high CD20 expression, who are sensitive to rituximab, may experience decreased or even negative CD20 expression after treatment, leading to acquired resistance. Blinatumomab is a bispecific T-cell activator (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 shown to exhibit anti-lymphoma activity in early proof-of-concept clinical trials for relapsed or refractory B-NHL. Blinatumomab requires 28 days of continuous intravenous infusion, a difficult administration regimen that is a major obstacle to its application in the treatment of NHL. Unlike blinatumomab, the trispecific antibodies of the present invention can be administered less frequently and provide comparable or superior tumor suppression. In a specific embodiment, the trispecific antibodies of the present invention are used to treat non-Hodgkin's lymphoma.

[0173] Precursor B-cell acute lymphoblastic leukemia (B-ALL) is a tumor of an immature B-cell precursor that typically affects children under the age of 6, but also occurs in adolescent and adult populations. Currently, the main treatment strategies for B-ALL are chemotherapy and hematopoietic stem cell transplantation (HSCT), with an overall survival rate (OS) of 80-90% for pediatric B-ALL and an OS rate of 30-50% for adult B-ALL. However, the prognosis for relapsed or refractory ALL is very poor in both age groups. The trispecific antibodies of the present invention provide a new 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.

[0174] Dosage

[0175] The trispecific antibodies of the invention can be delivered by conventional methods, preferably systemically, for example by intravenous infusion.

[0176] The antibodies of the present invention can be administered in a therapeutically effective amount. A "therapeutically effective amount" refers to an amount of an antibody that, when administered to a subject to treat a disease, or at least one clinical symptom of a disease or condition, is sufficient to effect such treatment of the disease, condition, or symptom. 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 to be treated, and / or the weight of the subject to be treated.

[0177] In a preferred embodiment of the present application, the dosage (mg / kg) of the antibody is based on the weight of the subject. For example, a single dose of the trispecific antibody of the present invention can be in the range of about 1 ng / kg body weight to about 5 mcg / kg body weight, preferably in the range of 5 ng / kg body weight to about 2.5 mcg / kg body weight, more preferably in the range of 0.1 mcg / kg body weight to about 1 mcg / kg body weight. For example, the antibody can 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.

[0178] An advantage of the trispecific antibodies of the present invention is that they can efficiently and completely activate T cells, and are not prone to post-activation apoptosis. In addition, memory T cells can be induced to provide a sustained immune response, so that administration can be performed at a relatively low dosing interval. In a preferred embodiment, when administered by intravenous infusion, the interval between two administrations of the trispecific antibodies of the present invention can be no less than 1 day, no less than 3 days, no less than 5 days, or even one week apart. 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 2-4 weeks, for example, 3 weeks, and administered for one cycle or multiple cycles. The trispecific antibodies of the present invention, when administered at a frequency of once a week, can produce better tumor suppression effects than other antibodies such as CD19xCD3 bispecific antibodies such as Blinatumomab at equal doses (such as at the same molar dose).

[0179] Drug combination

[0180] The trispecific antibodies of the present invention can be used in combination with other anti-tumor treatments or drugs.

[0181] In some embodiments, the trispecific antibodies of the present invention can be used in combination with another antibody drug. The other antibody can be an antibody drug that targets a different antigen on a target cell, such as a B cell. The other antibody drug can be an antibody drug that specifically binds to CD20, CD22, CD79a, CD38, or the like. Specific examples include, but are not limited to, rituximab and inotuzumab.

[0182] 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, neratinib, and pyrotinib.

[0183] In some embodiments, the trispecific antibodies of the present invention can be used in combination with small molecule chemotherapeutic agents, such as cyclophosphamide, vincristine sulfate, doxorubicin hydrochloride, dexamethasone, and the like.

[0184] The present invention also relates to the following:

[0185] 1. A trispecific antibody that specifically binds to CD19, CD3, and CD28, comprising:

[0186] (1) The first binding domain that specifically binds to CD19;

[0187] (2) a first linker sequence;

[0188] (3) a second binding domain that specifically binds to CD3;

[0189] (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

[0190] (5) The third binding domain specifically binds to CD28.

[0191] 2. The trispecific antibody according to item 1, which is a single-chain fusion protein.

[0192] 3. The trispecific antibody according to item 1 or 2, wherein the first linker sequence comprises (G4S)n or (G2S)n, or the first linker sequence is (G4S)n or (G2S)n, wherein n is an integer of 1-6, preferably 1-3.

[0193] 4. The trispecific antibody according to claim 3, 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.

[0194] 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),

[0195] (a) the VH comprises a heavy chain CDR 1 as set forth in SEQ ID NO: 11, a heavy chain CDR2 as set forth in SEQ ID NO: 12, and a heavy chain CDR3 as set forth in SEQ ID NO: 13, and the VL comprises a light chain CDR 1 as set forth in SEQ ID NO: 14, a light chain CDR2 as set forth in SEQ ID NO: 15, and a light chain CDR3 as set forth in SEQ ID NO: 16; or

[0196] (b) the VH comprises a heavy chain CDR 1 as shown in SEQ ID NO: 46, a heavy chain CDR2 as shown in SEQ ID NO: 47, and a heavy chain CDR3 as shown in SEQ ID NO: 48, and the VL comprises a light chain CDR 1 as shown in SEQ ID NO: 49, a light chain CDR2 as shown in SEQ ID NO: 50, and a light chain CDR3 as shown in SEQ ID NO: 51.

[0197] 6. The trispecific antibody according to item 5, wherein:

[0198] (a) the VH comprises the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 80% homologous (e.g., at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homologous) to the amino acid sequence of SEQ ID NO: 17 and in which all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; and

[0199] The VL comprises the amino acid sequence shown in SEQ ID NO: 19, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 19 and all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; or

[0200] (b) the VH comprises the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence that is at least 80% homologous (e.g., at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homologous) to the amino acid sequence of SEQ ID NO: 52 and wherein all of the amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; and

[0201] The VL comprises the amino acid sequence shown in SEQ ID NO: 54, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 54 and all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence.

[0202] 7. The trispecific antibody according to claim 6, wherein the first binding domain comprises the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 56, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 21 or SEQ ID NO: 56 and all amino acid differences are in non-CDR regions; or consists of such an amino acid sequence.

[0203] 8. The trispecific antibody according to any one of items 1 to 7, wherein the second binding domain is a single-chain antibody specifically binding to CD3 comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0204] (a) the VH comprises a heavy chain CDR1 as set forth in SEQ ID NO: 22, a heavy chain CDR2 as set forth in SEQ ID NO: 23, and a heavy chain CDR3 as set forth in SEQ ID NO: 24, and the VL comprises a light chain CDR1 as set forth in SEQ ID NO: 25, a light chain CDR2 as set forth in SEQ ID NO: 26, and a light chain CDR3 as set forth in SEQ ID NO: 27; or

[0205] (b) the VH comprises a heavy chain CDR 1 as shown in SEQ ID NO: 57, a heavy chain CDR2 as shown in SEQ ID NO: 58, and a heavy chain CDR3 as shown in SEQ ID NO: 59, and the VL comprises a light chain CDR 1 as shown in SEQ ID NO: 60, a light chain CDR2 as shown in SEQ ID NO: 61, and a light chain CDR3 as shown in SEQ ID NO: 62.

[0206] 9. The trispecific antibody according to item 8, wherein

[0207] (a) the VH comprises the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence that is at least 80% homologous (e.g., at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homologous) to the amino acid sequence of SEQ ID NO: 28, and all of the amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; and

[0208] The VL comprises the amino acid sequence shown in SEQ ID NO: 30, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 30 and all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; or

[0209] (b) the VH comprises the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence that is at least 80% homologous (e.g., at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homologous) to the amino acid sequence of SEQ ID NO: 63 and wherein all of the amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; and

[0210] The VL comprises the amino acid sequence shown in SEQ ID NO: 65, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 65 and all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence.

[0211] 10. The trispecific antibody according to claim 9, wherein the scFv that specifically binds to CD3 comprises the amino acid sequence shown in SEQ ID NO: 32 or SEQ ID NO: 67, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 32 or SEQ ID NO: 67 and all amino acid differences are in non-CDR regions; or consists of such an amino acid sequence.

[0212] 11. The trispecific antibody according to any one of items 1 to 10, 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 CDR 1 as shown in SEQ ID NO: 33, a heavy chain CDR2 as shown in SEQ ID NO: 34, and a heavy chain CDR3 as shown in SEQ ID NO: 35, and the VL comprises a light chain CDR 1 as shown in SEQ ID NO: 36, a light chain CDR2 as shown in SEQ ID NO: 37, and a light chain CDR3 as shown in SEQ ID NO: 38.

[0213] 12. The trispecific antibody according to item 11, wherein

[0214] The VH comprises the amino acid sequence shown in SEQ ID NO: 39, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 39 and all the amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; and

[0215] The VL comprises the amino acid sequence shown in SEQ ID NO: 41, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 41 and all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence.

[0216] 13. The trispecific antibody according to claim 12, wherein the third binding domain comprises the amino acid sequence shown in SEQ ID NO: 43, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 43 and all amino acid differences are in non-CDR regions; or consists of such an amino acid sequence.

[0217] 14. A trispecific antibody according to any one of items 1 to 13, wherein each binding domain comprises a linker sequence for connecting the heavy chain variable region and the light chain variable region, and the linker sequence comprises (G4S)n or (G2S)n, wherein n is an integer of 1-10, preferably 1-5, and more preferably 1-3.

[0218] 15. The trispecific antibody according to item 14, wherein the amino acid sequence of the linker sequence for connecting the heavy chain variable region and the light chain variable region is selected from the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0219] 16. The trispecific antibody according to any one of items 1 to 15, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 44 or SEQ ID NO: 68; or comprising or consisting of an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the amino acid sequence shown in SEQ ID NO: 44 or SEQ ID NO: 68.

[0220] 17. An isolated nucleic acid molecule encoding the trispecific antibody of any one of items 1 to 16.

[0221] 18. The isolated nucleic acid molecule of claim 17, wherein the nucleotide sequence encoding the first binding domain comprises the following nucleotide sequence:

[0222] (1) a nucleotide sequence encoding a VH as shown in SEQ ID NO: 18, or a nucleotide sequence encoding a VH as shown in SEQ ID NO: 17 having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto; and / or

[0223] (2) a nucleotide sequence encoding a VL as shown in SEQ ID NO: 20, or a nucleotide sequence encoding a VL as shown in SEQ ID NO: 19 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto.

[0224] 19. The isolated nucleic acid molecule of claim 17, wherein the nucleotide sequence encoding the first binding domain comprises the following nucleotide sequence:

[0225] (1) a nucleotide sequence encoding a VH as shown in SEQ ID NO: 53, or a nucleotide sequence encoding a VH as shown in SEQ ID NO: 52 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto; and / or

[0226] (2) a nucleotide sequence encoding a VL as shown in SEQ ID NO: 55, or a nucleotide sequence encoding a VL as shown in SEQ ID NO: 54 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto.

[0227] 20. The isolated nucleic acid molecule of items 17-19, wherein the nucleotide sequence encoding the second binding domain comprises the following nucleotide sequence:

[0228] (1) a nucleotide sequence encoding a VH as shown in SEQ ID NO: 29, or a nucleotide sequence encoding a VH as shown in SEQ ID NO: 28 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto; and / or

[0229] (2) a nucleotide sequence encoding a VL as shown in SEQ ID NO: 31, or a nucleotide sequence encoding a VL as shown in SEQ ID NO: 30 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto.

[0230] 21. The isolated nucleic acid molecule of items 17-19, wherein the nucleotide sequence encoding the second binding domain comprises the following nucleotide sequence:

[0231] (1) a nucleotide sequence encoding a VH as shown in SEQ ID NO: 64, or a nucleotide sequence encoding a VH as shown in SEQ ID NO: 63 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto; and / or

[0232] (2) a nucleotide sequence encoding a VL as shown in SEQ ID NO: 66, or a nucleotide sequence encoding a VL as shown in SEQ ID NO: 65 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto.

[0233] 22. The isolated nucleic acid molecule of any one of items 17 to 21, wherein the nucleotide sequence encoding the third binding domain comprises the following nucleotide sequence:

[0234] (1) a nucleotide sequence encoding a VH as shown in SEQ ID NO: 40, or a nucleotide sequence encoding a VH as shown in SEQ ID NO: 39 having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto; and / or

[0235] (2) a nucleotide sequence encoding a VL as shown in SEQ ID NO: 42, or a nucleotide sequence encoding a VL as shown in SEQ ID NO: 41 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto.

[0236] 23. The isolated nucleic acid molecule of any one of items 17 to 22, comprising:

[0237] (1) a nucleotide sequence encoding a first binding domain, comprising a nucleotide sequence encoding a VH as shown in SEQ ID NO: 18, or a nucleotide sequence encoding a VH as shown in SEQ ID NO: 17 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto; and a nucleotide sequence encoding a VL as shown in SEQ ID NO: 20, or a nucleotide sequence encoding a VL as shown in SEQ ID NO: 19 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto;

[0238] (2) a nucleotide sequence encoding a second binding domain, which comprises a nucleotide sequence encoding a VH as shown in SEQ ID NO: 29, or a nucleotide sequence encoding a VH as shown in SEQ ID NO: 28 having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homologous thereto; and a nucleotide sequence encoding a VL as shown in SEQ ID NO: 31, or a nucleotide sequence encoding a VL as shown in SEQ ID NO: 30 having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homologous thereto;

[0239] (3) a nucleotide sequence encoding a third binding domain, which comprises a nucleotide sequence encoding VH as shown in SEQ ID NO:40, or a nucleotide sequence encoding VH as shown in SEQ ID NO:39 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto; and a nucleotide sequence encoding VL as shown in SEQ ID NO:42, or a nucleotide sequence encoding VL as shown in SEQ ID NO:41 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto.

[0240] 24. The isolated nucleic acid molecule of any one of items 17 to 22, comprising:

[0241] (1) a nucleotide sequence encoding a first binding domain, comprising a nucleotide sequence encoding a VH as shown in SEQ ID NO: 53, or a nucleotide sequence encoding a VH as shown in SEQ ID NO: 52 having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homologous thereto; and a nucleotide sequence encoding a VL as shown in SEQ ID NO: 55, or a nucleotide sequence encoding a VL as shown in SEQ ID NO: 54 having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homologous thereto;

[0242] (2) a nucleotide sequence encoding a second binding domain, which comprises a nucleotide sequence encoding a VH as shown in SEQ ID NO: 64, or a nucleotide sequence encoding a VH as shown in SEQ ID NO: 63 having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homologous thereto; and a nucleotide sequence encoding a VL as shown in SEQ ID NO: 66, or a nucleotide sequence encoding a VL as shown in SEQ ID NO: 65 having at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homologous thereto;

[0243] (3) a nucleotide sequence encoding a third binding domain, which comprises a nucleotide sequence encoding VH as shown in SEQ ID NO:40, or a nucleotide sequence encoding VH as shown in SEQ ID NO:39 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto; and a nucleotide sequence encoding VL as shown in SEQ ID NO:42, or a nucleotide sequence encoding VL as shown in SEQ ID NO:41 that has at least 85%, preferably at least 90%, more preferably at least 95%, such as at least 96%, at least 97%, at least 98%, or at least 99% homology thereto.

[0244] 25. The isolated nucleic acid molecule of any one of items 17 to 24, comprising a nucleotide sequence encoding a linker sequence.

[0245] 26. The isolated nucleic acid molecule of any one of items 17 to 25, comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 45 or SEQ ID NO: 69.

[0246] 27. An expression vector comprising the isolated nucleic acid molecule of any one of items 17-26.

[0247] 28. A host cell comprising the isolated nucleic acid molecule of any one of items 17 to 26 or the expression vector of item 27.

[0248] 29. The host cell of item 28, which is selected from bacterial cells, fungal cells, insect cells or animal cells.

[0249] 30. The host cell of item 29, wherein the animal cell is a mammalian cell and is selected from 293T cells, CHO cells or their derivative cell lines.

[0250] 31. A method for preparing a trispecific antibody, comprising:

[0251] (a) providing the isolated nucleic acid molecule of any one of items 17 to 26 or the expression vector of item 27,

[0252] (b) introducing the isolated nucleic acid molecule or the expression vector described in (a) into a host cell,

[0253] (c) culturing the host cell obtained in (b) to express the trispecific antibody, and

[0254] (d) recovering the trispecific antibody from the culture of the cells.

[0255] 32. A method for preparing a trispecific antibody, comprising:

[0256] (a) providing the host cell of item 28 or 29,

[0257] (b) culturing the host cell to express the trispecific antibody, and

[0258] (c) recovering the trispecific antibody from the culture of the cells.

[0259] 33. The method of item 31 or 32 further comprises purifying the recovered trispecific antibody.

[0260] 34. A trispecific antibody obtained by the method of any one of items 31-33.

[0261] 35. Use of the trispecific antibody of any one of items 1 to 16 or 34 in the preparation of a medicament.

[0262] 36. The use described in item 35, wherein the drug is used to treat B cell-related tumors.

[0263] 37. The use according to claim 36, wherein the B cell-related tumor is a B cell lymphoma.

[0264] 38. The use described in item 36, wherein the B cell-related tumor is B cell leukemia.

[0265] 39. The use described in item 37, wherein the B cell-related tumor is a refractory or relapsed B cell tumor.

[0266] 40. The use of item 37 or 39, wherein the B-cell lymphoma is an aggressive or indolent non-Hodgkin's lymphoma.

[0267] 41. The use of item 38 or 39, wherein the B cell leukemia is B cell acute lymphoblastic leukemia.

[0268] 42. A method of treating a B cell-associated tumor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the trispecific antibody of any one of items 1-16 or 34.

[0269] 43. The method of claim 42, wherein the B cell-related tumor is a refractory or relapsed B cell tumor.

[0270] 44. The method of item 42 or 43, wherein the B cell-related tumor is a B cell lymphoma or a B cell leukemia.

[0271] 45. The method of any one of items 42-44, wherein the B cell-related tumor is an aggressive or indolent non-Hodgkin's lymphoma.

[0272] 46. ​​The method of any one of items 42-44, wherein the B cell-related tumor is B cell acute lymphoblastic leukemia.

[0273] 47. The method of any one of items 42 to 46, wherein the trispecific antibody is administered at a frequency of 3 times per week, 2 times per week, or once per week.

[0274] 48. The method of any one of items 42 to 47, wherein the trispecific antibody is administered at a dose of about 1 ng / kg body weight to about 5 mcg / kg body weight, preferably 10 ng / kg body weight to about 2.5 mcg / kg body weight, more preferably 0.1 mcg / kg body weight to about 1 mcg / kg body weight.

[0275] 49. A pharmaceutical composition comprising the trispecific antibody of any one of items 1-16 or 34, and a pharmaceutically acceptable carrier.

[0276] 50. A pharmaceutical combination comprising the trispecific antibody of any one of items 1-16 or 34, and another therapeutic agent.

[0277] 51. The drug combination of claim 50, wherein the other therapeutic agent is a therapeutic agent for treating B cell-related tumors.

[0278] 52. A linker sequence derived from the amino acid sequence of the hinge region of IgD, having a length of 20-85 amino acids and having at least one or two of the following characteristics:

[0279] (1) Does not contain cysteine;

[0280] (2) It does not contain glycosylation sites.

[0281] 53. The linker sequence of item 52, wherein cysteine ​​is replaced by other amino acids relative to the amino acid sequence of the hinge region of IgD.

[0282] 54. The linker sequence of claim 53, wherein cysteine ​​is replaced by serine or glycine.

[0283] 55. The linker sequence of item 53 or 54, wherein the substituted cysteine ​​is the cysteine ​​at position 161.

[0284] 56. The linker sequence of any one of items 52 to 55, wherein the glycosylation site is serine at position 109 and threonine corresponding to positions 126, 127, 131, and 132.

[0285] 57. The linker sequence of any one of items 52 to 56, wherein the glycosylation site is removed by amino acid substitution or truncation relative to the amino acid sequence of the hinge region of IgD.

[0286] 58. The linker sequence of any one of items 52 to 57, which has one or more of the following features:

[0287] (1) Compared with the amino acid sequence shown in SEQ ID NO: 10, it lacks cysteine;

[0288] (2) lacking a glycosylation site compared to the amino acid sequence shown in SEQ ID NO: 10;

[0289] (3) Compared with the amino acid sequence shown in SEQ ID NO: 10, it has a shorter length.

[0290] 59. The linker sequence of any one of items 52 to 58, which has an amino acid sequence as shown in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:9.

[0291] 60. A fusion protein comprising the linker sequence of any one of items 52 to 59.

[0292] 61. The fusion protein of claim 60, which is a multispecific antibody.

[0293] 62. The fusion protein of claim 61 , wherein the linker sequence is used to connect domains with different antigen specificities.

[0294] 64. Use of the linker sequence of any one of items 52 to 59 in a fusion protein for connecting different domains. Example

[0295] In order to more fully understand and apply the present invention, the present invention will be described in detail below with reference to the embodiments and accompanying drawings. The embodiments are intended to illustrate the present invention by way of example only and are not intended to limit the scope of the present invention. The scope of the present invention is specifically defined by the appended claims.

[0296] Example 1. Construction of eukaryotic expression vectors expressing trispecific antibodies LMOAME and MLMOF

[0297] This example exemplifies the preparation of two trispecific antibodies, LMOAME and MLMOF, and particularly describes the construction of expression vectors for expressing the trispecific antibodies.

[0298] The amino acid sequences of LMOAME or MLMOF are shown below as SEQ ID NO:44 or SEQ ID NO:68, respectively, wherein the VH and VL sequences are indicated in plain font; the three linker sequences within the three binding domains used to connect the respective VH and VL are underlined; and the two linker sequences between the binding domains used to connect different binding domains are marked with boxes.

[0299] The complete amino acid sequence of LMOAME (SEQ ID NO: 44)

[0300] The complete amino acid sequence of MLMOF (SEQ ID NO: 68)

[0301] As shown above, different single-chain antibody sequences are used in the CD19- and CD3-binding domains of LMOAME and MLMOF, with the CD19- and CD3-binding domains of MLMOF cross-reactive with their corresponding monkey antigens. The CD28-binding domains of LMOAME and MLMOF are identical and cross-reactive with their corresponding monkey antigens. In LMOAME and MLMOF, five of the six intradomain linkers connecting the VH and VL domains are (G4S)3 (SEQ ID NO: 1). Only the second binding domain (CD3-binding domain) of LMOAME uses the (G2S)3G2 (SEQ ID NO: 3) linker connecting the VH and VL domains. LMOAME and MLMOF used exactly the same inter-domain linkers, including the first linker sequence G4S (SEQ ID NO: 2) for connecting the CD19 binding domain and the CD3 binding domain, and the second linker sequence RNTGRGGE EKKKEKEKEEQEERETKTPE SPSHTQPLGV (SEQ ID NO: 6) for connecting the CD3 binding domain and the CD28 binding domain.

[0302] To express the two trispecific antibodies described above in mammalian cells, the nucleotide sequences encoding the trispecific molecules comprising the CD19-binding domain, the CD3-binding domain, and the CD28-binding domain were first codon-optimized. The optimized nucleotide sequences encoding the complete trispecific antibodies are shown in SEQ ID NO:45 (LMOAME) and SEQ ID NO:69 (MLMOF), respectively. These two nucleotide sequences encoding the full-length amino acid sequences of the antibodies were synthesized and then cloned as inserts into the pWNP15 (Novoprotein) expression vector to form the antibody expression constructs pWNP15-LMOAME and pWNP15-MLMOF. The inserts were chemically synthesized with EcoRI and SalI restriction sites at both ends for cloning into the pWNP15 expression vector, which had been linearized with EcoRI and SalI. The resulting recombinant plasmids were sequenced and verified. The recombinant plasmids that had been sequenced correctly were used to construct stable expression strains in CHO-DG44 (Thermo Fisher / Life Technologies, lot number 55463WCB2). The constructed recombinant plasmids were linearized with PyuI and transfected into CHO-DG44 cells using PEI. Through cell expansion, methotrexate (MTX) pressure selection, and limiting dilution methods, two monoclonal stable strains expressing the constructs were identified, designated CHO-DG44-LMOAME and CHO-DG44-MLMOF, respectively.

[0303] Example 2. Expression and purification of trispecific antibodies LMOAME and MLMOF

[0304] This example goes on to describe methods for making two exemplary trispecific antibodies.

[0305] 2.1 Expression and purification of LMOAME

[0306] The CHO-DG44 stable cell line containing the recombinant plasmid encoding LMOAME obtained in Example 1 was amplified through a series of shake flask culture, reactor cell culture and protein expression and secretion into the culture supernatant. The supernatant was harvested and clarified and filtered, and the antibody product obtained in the culture supernatant was purified according to the following method.

[0307] CaptoL filler (purchased from Cytiva) was used to capture the target protein. First, the chromatography column was treated with affinity cleaning solution for 3-4 column volumes. Equilibrate with affinity equilibration solution for at least 4 column volumes. The clarified cell harvest was loaded at one time. After loading, equilibrate with affinity equilibration solution for 3-4 column volumes. Then, rinse with affinity rinsing solution for at least 4 column volumes. After rinsing, the protein was eluted with affinity elution solution. When the UV absorbance value rose to 10-50mAU / 2mm, the protein was collected. When the UV absorbance value dropped to 10-50mAU / 2mm, the collection was terminated. The eluted protein was stirred and weighed to determine the protein concentration.

[0308] The target protein captured by affinity chromatography is then purified through a series of purification steps including low pH virus inactivation, hydrophobic chromatography, anion chromatography, UF / DF, and nanofiltration to obtain the purified protein.

[0309] 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 Figures 2A-B , showing that bands with the correct molecular weight were obtained for all five samples from different batches.

[0310] As shown in Figure 2A, the LMOAME protein prepared in this example exhibited a single electrophoretic band under non-reducing SDS-PAGE conditions. The theoretical molecular weight of the recombinant LMOAME 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, as shown in Figure 2B, the SEC-HPLC analysis shows that after purification using the methods and steps of this example, the LMOAME purity exceeded 95%, with no significant presence of misfolded or aggregated proteins observed in the cell culture medium.

[0311] The purified LMOAME was analyzed by mass spectrometry using reversed-phase ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (XEVO G2-XS-QToF, Waters), and the results are shown in Figure 3. As shown in Figure 3, the purified LMOAME has only one major component, and its molecular weight is consistent with the theoretical molecular weight of the monomer, without glycosylation modification.

[0312] The N-terminal sequence analysis of the purified LMOAME sample was further performed, and the results showed that the expressed recombinant protein had the correct reading frame.

[0313] To facilitate comparison of the effects of different linker designs on the physicochemical properties of trispecific antibodies, another trispecific antibody previously developed by the inventors, BLMOAM (see Chinese Patent Application Publication No. CN106589129A), was used as a control and subjected to one-step affinity chromatography capture purification along with LMOAME. 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 uses a G4S linker between the first and second binding domains (linker segment 1) and an 81-amino acid linker containing cysteine ​​between the second and third binding domains (linker segment 2, SEQ ID NO: 29 in the same application). The results show that compared to BLMOAM, LMOAME has a more uniform composition and contains fewer dimers or multimers, indicating that LMOAME is less likely to form dimers or multimers.

[0314] 2.2 Purification of MLMOF

[0315] The antibody product from the cell culture supernatant after transfection with the recombinant plasmid encoding MLMOF was purified using methods and steps similar to those in Experiment 2.1. The results are shown in Figure 6 , and a purity similar to that of LMOAME was obtained. N-terminal sequence analysis showed that the expressed recombinant protein was in the correct reading frame.

[0316] Example 3. ELISA detection of antigen binding activity of trispecific antibody LMOAME

[0317] In this example, ELISA analysis was used to evaluate the binding activity of LMOAME to recombinant human CD19 antigen (rhCD19) and recombinant human CD28 antigen (rhCD28).

[0318] Recombinant human CD19 antigen (CD19 (C-Fc), Novoprotein, Cat#: C572) and CD28 antigen (CD28 (C-Fc), Novoprotein, Cat#: C81) were diluted to 2 μg / mL in protein coating buffer, 100 μL per well, and coated overnight at 2-8°C. The next day, the plates were washed and blocked with 1% Casein + 3% BSA blocking buffer (PBS + 2% BSA (v / w)).

[0319] The LMOAME purified in Example 2 was serially diluted with PBS to the specified concentration range (rhCD19 binding assay: 0.0016-30 μg / mL; rhCD28 binding assay: 0.0006-10 μg / mL). The diluted antibodies were added to 96-well flat-bottom plates coated with antigens and incubated at 37°C for 1 hour. After washing with PBST (PBS + 0.05% Tween-20 (V / V)), the plates were stained with HRP-protein L (GenScript, Cat#: M00098) and TMB (KPL SureBlue). TM , Cat#:5120-0075) for detection.

[0320] Data were analyzed using GraphPad Prism software and the half-maximal effect concentration (EC) was used. 50 The values ​​represent the binding ability of LMOAME to rhCD19 and rhCD28 antigens. The results are shown in Figure 7.

[0321] The results showed that LMOAME had a strong binding ability with rhCD19 and rhCD28 antigens. 50 The values ​​ranged from 0.3607-0.6853 and 0.0571-0.0758 μg / mL, respectively.

[0322] Example 4. In vitro cell binding assay to evaluate the binding activity of the trispecific antibody LMOAME to membrane-bound antigens

[0323] In this example, the binding activity of LMOAME to membrane-bound antigens hCD19, hCD3e, and hCD28 was evaluated using an in vitro cell binding assay.

[0324] 1×10 per well 5 Target antigen-expressing cells (A549-CD19, CHO-CD28, or H9) were added to a 96-well plate at a density of 10 cells / well. A negative control containing cells that do not express the target protein was used as a control. Specifically, the cells and negative controls used for each antigen were as follows.

[0325] To detect the binding activity with hCD19, the A549-CD19 cell line was used, which is a CD19-expressing A549 stable cell line with a CD19 phenotype. + CD3 - CD28 - ; and the A549 cell line that does not express CD19 (purchased from CCTCC, GDC0063) was used as a negative control.

[0326] To detect the binding activity to hCD3, the H9 cell line (purchased from CCTCC, GDC0031) was used, which is a human T lymphoma cell line expressing CD3 and has a CD19 phenotype. - CD3 + CD28 - ; and a CHO cell line that does not express CD19, CD3 and CD28 was used as a negative control.

[0327] To detect the binding activity with hCD28, the CHO-CD28 cell line was used, which is a stable CHO cell line expressing CD28 with a CD19 phenotype. - CD3 - CD28 + ; and a CHO cell line that does not express CD19, CD3 and CD28 was used as a negative control.

[0328] LMOAME purified in Example 2 was diluted with STB staining buffer (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 cells were incubated with the assay cells at 4°C for 1 hour. Unbound antibody was removed by centrifugation. Cells bound to the LMOAME antibody were detected using Biotin-Protein L (GenScript, Cat#: M00097) and APC-Streptavidin (Biolegend, Cat#: 405207). Mean fluorescence intensity (MFI) was measured using a Thermo Fisher Attune NxT flow cytometer.

[0329] Data were analyzed using GraphPad Prism software and the half-maximal effect concentration (EC) was used. 50 The values ​​represent the binding capacity of LMOAME to membrane-bound antigens hCD19, hCD3e, and hCD28.

[0330] The results showed that the EC binding of LMOAME to membrane-bound antigens hCD19, hCD28 and hCD3e 50 The values ​​were about 0.15-0.17 μg / mL, about 0.27-0.32 μg / mL, and about 5.1-7.5 μg / mL, respectively. The results also showed that LMOAME had no detectable binding activity to negative cell lines that did not express the target protein.

[0331] Example 5. In vitro evaluation of trispecific antibody-mediated effector cell activation

[0332] 5.1 Evaluation of the effect of LMOAME on T cell activation

[0333] This example uses human PBMC as an evaluation system to test the ability of LMOAME antibodies to mediate effector cell activation in in vitro experiments.

[0334] After incubating PBMCs with LMOAME at different concentrations, the expressions of T cell activation markers CD25, CD69, and GrB in PBMCs were detected to observe the activation effect of LMOAME on T cells.

[0335] Specifically, PBMCs from three healthy volunteers were extracted with Ficoll-Paque Plus (GE, Cat#: 17-1440-02) and the concentration was 2×10 5 Cells were added to a 96-well U-bottom cell culture plate at a density of 10 cells / well. The LMOAME purified in Example 2 and the commercially available CD19xCD3 bispecific antibody (BiTE) Blinatumomab (Taizhou Baiying Biotechnology Co., Ltd., #20210330T001) were serially diluted with RPMI1640 complete medium (Gibco, Cat#: C11875500CP): the final concentrations of LMOAME were set to 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 100 ng / mL; the final concentrations of Blinatumomab were set to 0.00065, 0.0065, 0.0195, 0.065, 0.195, 0.65, 1.95, 6.5, 65 ng / mL. A vehicle control group of RPMI 1640 complete medium was used as a negative control. LMOAME and Blinatumomab were added to 96-well U-bottom cell culture plates and incubated with PBMC at 37°C for 48 hours or 120 hours. After washing with STB, the cells were stained with detection antibodies. The following detection antibodies were used:

[0336] PerCP / Cyanine5.5 anti-human CD4 antibody: BioLegend, Cat#:317428

[0337] FITC anti-human CD8 antibody: BioLegend, Cat#:301006

[0338] PE anti-human CD25 antibody: BioLegend, Cat#: 356104

[0339] BV605 anti-human CD69 antibody: BioLegend, Cat#: 310938

[0340] APC anti-human / mouse Granzyme B antibody: BioLegend, Cat#:372204.

[0341] The ratio and number of the following target cell subsets in each well of the 96-well plate were then detected using a Thermo Fisher Attune NxT flow cytometer: + 、CD4 + CD25 + 、CD4 + CD69 + 、CD4 + Granzyme B + 、CD8 + 、CD8 + CD25 + 、CD8 + CD69 + and CD8 + Granzyme B + cell.

[0342] Data were analyzed using GraphPad Prism software. If one-way ANOVA was statistically significant (P < 0.05), further comparative analysis was performed. Dunnett's Multiple Test was used to analyze differences between LMOAME or blinatumomab and the vehicle control; Tukey's Multiple Test was used to analyze differences between LMOAME and blinatumomab at the same molar concentration.

[0343] The results are shown in Figures 8A and 8B. Compared with the vehicle control, LMOAME and PBMC were co-incubated for 48 h or 120 h and expressed CD25 + 、CD69 + and GrB + CD4 + and CD8 + The cell proportions of the 247 cells were significantly increased and showed a dose-related relationship with the concentration of LMOAME.

[0344] In addition, the results showed that after incubation with PBMC for 48 h or 120 h, LMOAME expressed CD25 compared with Blinatumomab. + 、CD69 + and GrB + CD4 + and CD8 + A higher proportion of cell populations.

[0345] In summary, the trispecific antibody LMOAME provided by the present invention can significantly increase CD4 + or CD8 +The expression of CD25 and CD69 on the effector cell surface and GrB expression in the effector cells were significantly increased, fully activating the effector cells in PBMCs. The results also showed that the trispecific antibody LMOAME provided by the present invention had a significantly higher activation effect on effector cells in PBMCs than blinatumomab.

[0346] 5.2 Evaluation of the Effects of LMOAME, MLMOF, and BLMOAM on T Cell Activation (Jurkat Cell System)

[0347] In vitro cell biology experiments were conducted to examine effector cell activation mediated by different trispecific antibodies. The trispecific antibodies tested included LMOAME and MLMOF prepared in Examples 1 and 2 of the present invention, as well as BLMOAM from the applicant's prior application. A vehicle control group containing complete RPMI 1640 medium served as a negative control.

[0348] The specific process includes: (1) Jurkat cells (ATCC, TIB-152) were taken and prepared into 2.5×10 6 After the cell suspension was prepared into 10 cells / mL, 40 μL was inoculated into each well of a 96-well cell culture plate; (2) the GFP stable transfectant of Nalm-6 cells (Novoprotein) was taken and the experimental culture medium was used to prepare 2.5×10 5 After the cell suspension was prepared at a concentration of 10 cells / mL, 40 μL was inoculated into a 96-well cell culture plate at each well; (3) 20 μL of the test solution was added to each well in a gradient dilution; (4) the plate was mixed and incubated at 37°C and 5% CO2 for 24 h; (5) the plate was washed with 1% FBS-PBS, and the fluorescent dye 7AAD (Biolegend, Cat# 420404) and Anti-CD69-APC (Biolegend, Cat# 310910) were diluted and added to the 96-well plate, mixed and incubated at 2°C to 8°C in the dark for 30 min; (6) the plate was washed with 1% FBS-PBS, 200 μL / well of 1% FBS-PBS was added and mixed, and the percentage of APC-positive cell population was detected by flow cytometry. GraphPad Prism software was used for data analysis, and the 50% maximum effect concentration EC was used. 50 The values ​​represent the T cell activation activity of different trispecific antibodies. The results show that LMOAME, MLMOF and BLMOAM induced EC activation of T cells. 50The values ​​were 0.624, 0.871, and 1.505 ng / mL, respectively. This result demonstrates that the two trispecific antibodies of the present invention can induce T cell activation at lower dosages compared to the previously described trispecific antibody BLMOAM, which uses a different linker sequence, indicating a stronger T cell activation capacity. Furthermore, LMOAME exhibited slightly higher T cell activation capacity than MLMOF.

[0349] Example 6. In vitro evaluation of effector cell proliferation mediated by the trispecific antibody LMOAME

[0350] This example evaluated the effect of LMOAME-mediated effector cell proliferation, wherein human PBMC was used as the evaluation system, and different concentrations of LMOAME were co-incubated with PBMC to detect the CD4 + 、CD4 + CFSE dim 、CD8 + and CD8 + CFSE dim The proportion and number of cells were measured to investigate the proliferation of T cells induced by LMOAME.

[0351] Specifically, PBMCs from three healthy volunteers were extracted with Ficoll-Paque Plus and incubated with 2 μM hydroxyfluorescein diacetate succinimidyl ester (CFSE) at 37°C for 30 min. 5 Cells were plated at a density of 10 cells / well in a 96-well U-bottom cell culture plate. LMOAME purified in Example 2 and commercially available blinatumomab were serially diluted in RPMI 1640 complete medium: final concentrations of LMOAME were set at 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 100 ng / mL; final concentrations of blinatumomab were set at 0.00065, 0.0065, 0.0195, 0.065, 0.195, 0.65, 1.95, 6.5, and 65 ng / mL. A vehicle control group containing RPMI 1640 complete medium was used as a negative control. LMOAME and Blinatumomab were added to 96-well U-bottom cell culture plates, incubated with PBMCs at 37°C for 120 h, and then stained with Pacific Blue anti-human CD4 antibody (BioLegend, Cat#: 317429) and APC anti-human CD8 antibody (BioLegend, Cat#: 344722). The CD4 expression in PBMCs in each well was detected using a Thermo Fisher Attune NxT flow cytometer. + 、CD4 + CFSE dim 、CD8+ and CD8 + CFSE dim The proportion and number of cells.

[0352] The calculation formula for the proliferation fold of each cell subpopulation is as follows:

[0353] CD4 + Cell proliferation fold = sample well CD4 + Cell number / vehicle control well CD4 + Cell number;

[0354] CD4 + CFSE dim Cell proliferation fold = sample well CD4 + CFSE dim Cell number / vehicle control well CD4 + CFSE dim Cell number;

[0355] CD8 + Cell proliferation fold = sample well CD8 + Cell number / vehicle control well CD8 + Cell number;

[0356] CD8 + CFSE dim Cell proliferation fold = sample well CD8 + CFSE dim Cell number / vehicle control well CD8 + CFSE dim Cell number.

[0357] The sample wells refer to the wells where LMOAME or Blinatumomab was co-incubated with PBMC; the vehicle control wells refer to the wells where the vehicle control was co-incubated with PBMC.

[0358] Data are presented as mean ± standard error, and statistics were analyzed using GraphPad Prism software. If statistically significant (P < 0.05) was found in one-way ANOVA, further comparative analysis was performed. Dunnett's Multiple Comparison Test was used to analyze differences between LMOAME or blinatumomab and the vehicle control. Tukey's Multiple Comparison Test was used to analyze differences between LMOAME and blinatumomab. The results are shown in Figure 9.

[0359] As shown in the figure, LMOAME can induce significant T cell proliferation, and the proliferation fold of T cells is dose-dependent with the added LMOAME concentration.

[0360] The results also showed that under the same experimental conditions, the CD4 + and CD8 + The number of T cells increased was higher than that of the equimolar concentration blinatumomab group. + 、CD4 + CFSE dim 、CD8 + and CD8 + CFSE dim The maximum proliferation folds of the cells were 3.58, 798.13, 5.37, and 8639.48, respectively. The maximum proliferation folds of the cells at equimolar concentrations 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.

[0361] Example 7. In vitro evaluation of effector cell differentiation mediated by the trispecific antibody LMOAME

[0362] This example evaluated the ability of LMOAME to induce effector cell differentiation. PBMCs from three healthy volunteers were used as the evaluation system. Different concentrations of LMOAME were co-incubated with PBMCs to detect CD4 + With CD8 + T cells in T CM 、T EM 、T Effector and T Naive The proportion and number of cell populations were measured to investigate the function of LMOAME in inducing T cell differentiation.

[0363] Specifically, PBMCs from three healthy volunteers were extracted with Ficoll-Paque Plus and the concentration was 2 × 10 5 Cells were plated at a density of 10 cells / well in a 96-well U-bottom cell culture plate. LMOAME or blinatumomab was serially diluted in RPIM 1640 complete medium: final concentrations of LMOAME were set to 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 100 ng / mL; final concentrations of blinatumomab were set to 0.00065, 0.0065, 0.0195, 0.065, 0.195, 0.65, 1.95, 6.5, and 65 ng / mL. The cells were then added to the 96-well U-bottom cell culture plate and incubated with PBMCs at 37°C for 48 or 120 hours. The cells were stained with detection antibodies.

[0364] The cell phenotype detection antibodies used are as follows:

[0365] Pacific Blue anti-human CD4 antibody: BioLegend, Cat#: 317429

[0366] FITC anti-human CD8a antibody BioLegend, Cat#:301006

[0367] PE anti-human CD45RO antibody: BioLegend, Cat#: 304206

[0368] APC anti-human CCR7 antibody: BioLegend, Cat#: 353214

[0369] The ratio and number of target cell subpopulations were measured using a Thermo Fisher Attune NxT flow cytometer. The results are shown in Figure 10.

[0370] The results showed that after LMOAME was co-incubated with hPBMC for 48 h, CD4 + T CM The number of CD4 + T CM After 120h of co-incubation, LMOAME induced CD4 + and CD8 + Memory T cells CM and T EM The number of cell populations increased significantly and showed a bell-shaped curve relationship with the addition of LMOAME. Compared with the Blinatumomab group, LMOAME induced differentiation into CD4 + and CD8 + The capacity of memory T cell population is much higher than that of Blinatumomab.

[0371] Example 8. In vitro evaluation of the killing activity of the trispecific antibody LMOAME mediated against target cells

[0372] This example evaluates the LMOAME-mediated killing activity against target cells. Human PBMCs were used as effector cells, and B-cell lymphoma cell lines Raji-GFP, Ramos, and Daudi, as well as a precursor B-cell leukemia cell line Nalm-6, were used as target cells. The GFP expression in the system was detected by incubating different concentrations of LMOAME with PBMCs and target cells. + or CD20 +The proportion and number of cells were measured to evaluate the killing activity of LMOAME-mediated effector cells in PBMCs against different target cells.

[0373] Specifically, in this example, PBMCs from three healthy volunteers were extracted with Ficoll-Paque Plus, and effector cells and target cells were added to a 96-well U-bottom cell culture plate at a ratio of 10:1 (PBMC: 2×10 5 cells / well, target cells: 2×10 4 LMOAME was serially diluted in RPIM 1640 complete medium to a final concentration of 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, and 100 ng / mL, and then added to the above-mentioned 96-well U-bottom cell culture plate. After incubation at 37°C for 72 h, the plate was washed and stained with 7-AAD or FITC anti-human CD20 antibody (BD Pharmingen, Cat#: 555622). GFP expression in each well was detected using a Thermo Fisher Attune NxT flow cytometer. + or CD20 + The ratio and number of cells were used to calculate the killing efficiency. The killing efficiency calculation formula is as follows:

[0374] The data were analyzed using GraphPad Prism software. The activity of LMOAME-mediated PBMC killing of target cells was expressed as 50% maximal effect concentration (EC). 50 The results are shown in Figure 11.

[0375] The results showed that LMOAME could dose-dependently mediate PBMC killing of different B-cell lymphoma or B-cell leukemia cell lines (efficacy-target ratio was 10:1). 50 The 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.

[0376] Example 9. In vitro detection of hemolysis or coagulation mediated by the trispecific antibody LMOAME 聚 反 answer

[0377] 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. The presence of hemolysis or agglutination was assessed visually. A 0.9% sodium chloride injection group served as a negative control, and a deionized water group served as a positive control. The LMOAME observations were compared with the control group.

[0378] Specifically, about 5 mL of blood was collected from the jugular vein or central ear artery of a male New Zealand rabbit, and red blood cells were extracted and prepared into a 2% (v / v) suspension with 0.9% sodium chloride injection. 2.5 mL of 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 system was adjusted to 5 mL with 0.9% sodium chloride injection. The test group and the control group were incubated at 37°C ± 1°C for 3 hours, observed every 15 minutes for the first hour, and then every 1 hour. The changes in the color of the upper solution in the test tube and the red blood cell sedimentation, as well as the formation of flocculent precipitation, were photographed and recorded.

[0379] The results showed that LMOAME at a final concentration of 0.0028-0.014 mg / mL did not cause hemolysis in rabbit erythrocytes. This was manifested by varying degrees of erythrocyte sedimentation in the test tubes with increasing incubation time, and the supernatant solution remained colorless and transparent. Furthermore, the results showed no reddish-brown or brownish-red flocculent precipitate, indicating no drug-induced agglutination.

[0380] Example 10. Anti-tumor effect of LMOAME in the Raji-luc / human PBMC co-transplanted subcutaneous tumor model in NPG mice

[0381] This example examined the anti-tumor effect of the trispecific antibody LMOAME in the Raji-luc / human PBMC co-transplanted subcutaneous tumor model in NPG mice.

[0382] Each female immunodeficient NPG mouse (Beijing Weitongda Animal Breeding Co., Ltd.) weighing about 21 grams was subcutaneously inoculated with 5×10 6 Raji-luc cells and 2.5×10 6 Human PBMC cells. Raji-luc cell line used in this experiment was cultured in RPMI-1640 medium supplemented with 10% FBS in a 37°C incubator containing 5% CO2. 5×10 6 The cells were suspended in 50 μL PBS and mixed with an equal volume of 2.5×10 6PBMCs were mixed with an equal volume of Matrigel and inoculated subcutaneously on the right side of the back of each mouse in a volume of 200 μL. Animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Pengli Biotechnology.

[0383] On the 6th day after inoculation, 32 mice were selected and randomly divided into 4 groups (8 mice / group) according to body weight and tumor volume, including a vehicle control group (G1) and 20, 80, and 150 μg / kg dose groups (G2-G4) of the LMOAME purified in Example 2.

[0384] Each group of mice was intravenously administered twice a week (day 1 and day 4) for 3 weeks (defined as the administration period D1-D21), for a total of 6 doses. The body weight and tumor volume of the mice were measured and recorded twice a week, and the following indicators were calculated: tumor volume (V), tumor growth inhibition rate (TGI), relative tumor volume (RTV) and tumor weight inhibition rate (IR TW %). The calculation methods of each index are as follows: V = (length × width 2 ) / 2

[0385] RTV=V t / V0,V t is the tumor volume measured at time point t, and V0 is the tumor volume at the beginning of treatment.

[0386] TGI=(1-T / C)×100%, where T and C are the RTV of the experimental group and the vehicle control group at a specific time point, respectively.

[0387] IR TW % = (average tumor weight of vehicle control group - average tumor weight of test group) / average tumor weight of vehicle control group × 100%.

[0388] The results are shown in Figures 12A, 12B, and 13.

[0389] Mouse weight

[0390] 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 dose groups (G2-G4) of LMOAME from D1 to D21 (P>0.05).

[0391] Tumor volume (V), relative tumor volume (RTV) and tumor growth inhibition rate (TGI)

[0392] On days 8-21, LMOAME at doses of 20, 80, and 150 μg / kg (G2-G4) exhibited significant tumor inhibition compared to the vehicle control (G1) (P < 0.001), with tumor volume reduction occurring in a dose-dependent manner with increasing LMOAME concentration. Complete tumor clearance was observed in all eight mice in the 150 μg / kg LMOAME group (G4) at the end of dosing.

[0393] At the end of the study, the tumors were removed, weighed, and the inhibition rate of tumor weight (IRTW%) was calculated. The results showed that the IRTW% values ​​of the G2-G4 groups at the end of the administration were 87.21%, 95.35%, and 100%, respectively.

[0394] Example 11. Tumor Suppressant Effect and Toxicity Evaluation of the Trispecific Antibody LMOAME in the Raji-luc Subcutaneous Tumor Model of huHSC-NCG Mice

[0395] This example examines the tumor suppressor effect and toxicity of the trispecific antibody LMOAME in the Raji-luc subcutaneous tumor model of huHSC-NCG mice.

[0396] huHSC-NCG mice are designed to reconstruct the human immune system in severely immunodeficient NCG mice by transplanting human hematopoietic stem cells (HSCs). 5×10 6 When the average tumor volume reached 59.99 mm 3 At the time of the study, 54 mice were randomly divided into the following 9 groups (6 mice / group) based on the flow cytometry data of hCD45%×hCD3% in the mouse peripheral blood, mouse body weight, and tumor volume: vehicle control group G1; blinatumomab control groups G2-G3, administered at a dose of 100 μg / kg; and LMOAME-administered groups G4-G9, of which the doses of groups G4-G5 were 50 μg / kg, the doses of groups G6-G7 were 150 μg / kg, and the doses of groups G8-G9 were 500 μg / kg. The molar concentrations of the blinatumomab group (100 μg / kg) and the LMOAME group (150 μg / kg) were the same.

[0397] Mice in groups G1, G2, G4, G6, and G8 were intravenously administered twice a week (day 1 and day 4) for 4 weeks, for a total of 8 doses. Mice in groups G3, G5, G7, and G9 were intravenously administered once a week for 4 weeks, for a total of 4 doses.

[0398] The date of grouping was defined as D1, and the administration period was D1-D28. The body weight and tumor volume of 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 removed, weighed, and the tumor weight inhibition rate (TGI) was calculated. TW The drug effect is expressed as tumor growth inhibition rate (TGI), including tumor volume inhibition rate (TGI TV ) and tumor weight inhibition rate (TGI TW ).

[0399] TGI TV The calculation formula is as follows: Vnt: tumor volume of mouse numbered n on day T; Vn0: tumor volume of mouse numbered n on day 0; RTVn: relative tumor volume of mouse numbered n on day T; meanRTVtreat: mean RTV of treatment group; meanRTVvehicle: mean RTV of vehicle control group.

[0400] TGI TW The calculation formula is as follows: meanTWtreat: the mean tumor weight of the drug-treated group at the end of the study; meanTWvehicle: the mean tumor weight of the vehicle-controlled group at the end of the study.

[0401] The pharmacodynamic and toxicological evaluations of LMOAME were conducted based on clinical observations such as tumor growth inhibition rate, mouse body weight, and 24-hour food and water intake. Furthermore, the results of HE staining of major tissues and organs, IHC staining of human CD3 antigen (tumor tissue, spleen, and brain tissue), and flow cytometric analysis of mouse peripheral blood were used. The animal operation procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Jicui Pharmaceutical Kang Biotechnology.

[0402] Tumor volume inhibition rate (TGI) of each group during the administration period TV ) are shown in Table 1. As shown in Table 1, LMOAME can inhibit tumor growth in a dose-dependent manner, and mice given LMOAME twice a week showed a higher tumor inhibition rate than mice given LMOAME once a week. + and hCD20 + The cell numbers showed that different doses of LMOAME (50μg / kg, 150μg / kg and 500μg / kg) and 100μg / kg Blinatumomab were able to inhibit the hCD19 in this mouse tumor model. + and hCD20+ cell proliferation, thereby exerting a tumor suppressor effect.

[0403] Table 1. Changes in tumor volume and tumor inhibition rate in different groups during the administration period (D1-D28) (TGI TV )

[0404] At the end of the study, the tumors were removed, weighed, and the tumor weight inhibition rate (TGI) was calculated. TW ), the results are shown in Table 2 and Figure 14. When the dosing frequency was changed from twice a week (BIW) to once a week (QW), the tumor inhibition rate of LMOAME in the G7 group was higher than that in the blinatumomab (G3) group with the same molar concentration and dosing frequency.

[0405] Table 2. End point tumor weight and tumor weight inhibition rate (TGI) of each drug group TW ) Note: Tumor weight is expressed as mean ± SEM. The independent sample T test was used to analyze the tumor inhibition rate between different groups. G2 vs G1 (*: P < 0.05, **: P < 0.01, ***: P < 0.001); G2 vs G6, and G3 vs G7 ( ^ :P<0.05).

[0406] During the dosing period, no abnormalities were found in the ear temperature or food and water intake of mice in each dosing group. Compared with the vehicle control group, there was no significant difference in the body weight of mice in each dosing group. The spleen weight of mice in groups G2, G4, and G8 was significantly reduced. Since the spleen is rich in lymphocytes, and Blinatumomab and LMOAME have a significant B lymphocyte clearance effect, the reduction in spleen weight of mice in the dosing group is believed to be related to the pharmacological effect of the test product. Histopathological studies did not find any drug-related pathological reactions. IHC staining of hCD3 found that hCD3 positivity was mainly concentrated in the spleen and tumor tissue, and there was no hCD3 positive staining in brain tissue.

[0407] Example 12. Tumor inhibitory effect of the trispecific antibody LMOAME in the huHSC-NCG mouse Nalm6-luc tail vein tumor model

[0408] Female huHSC-NCG mice were inoculated via the tail vein with 1×10 6 Nalm6-luc cells. When the average photon count reaches 5.25×10 4 p / sec / cm 2 / sr, based on the flow cytometry data of hCD45% × hCD3% cells in peripheral blood, mouse body weight, and tumor photon count, 32 mice were randomly divided into 4 groups (8 mice / group), including a vehicle control group (G1) and 50, 120, and 300 μg / kg dose groups (G2-G4) of the LMOAME purified in Example 2.

[0409] Each group of mice received intravenous administration twice weekly (Days 1 and 4) for 4 weeks (defined as the administration period D1-D28), for a total of 8 doses. Mouse body weight and tumor photon counts were measured and recorded twice weekly. Tumor growth inhibition (TGI) was calculated based on the measured photon counts. The TGI was calculated as follows: Rnt: the tumor photon number of mouse numbered n on day t; Rn0: the tumor photon number of mouse numbered n on day 0; RRn: the relative tumor photon number of mouse numbered n on day t; meanRRtreat: the mean RR of the drug-treated group; meanRTVvehicle: the mean RR of the vehicle control group.

[0410] The animal operation procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Jicui Yaokang Biotechnology.

[0411] 12.1 Mouse Weight

[0412] As shown in Figure 15A , the body weight of mice in each group decreased, and tumor growth was considered to be the main cause of the weight loss.

[0413] 12.2 Survival curve and mortality statistics

[0414] No mice were euthanized during the study. The survival of mice in different groups is shown in Table 3 and Figure 16. In the G1 group, mice began to die from D15, and only one mouse survived on D20. By D21, all mice in the G1 group had died. For the G2-G4 groups, mice began to die from D20. By the end of the study, the number of surviving mice in the G2-G4 groups was 4, 3, and 7, respectively. Combined with tumor fluorescence imaging data, the death of mice was considered to be caused by tumor burden. It can be seen that compared with the vehicle control, different doses of LMOAME can significantly prolong the average survival of mice (P < 0.001***).

[0415] Table 3. Survival statistics of mice in different groups (days) Note: Data are presented as mean ± SEM. Log-rank (Mantel-Cox) test. ***: P < 0.001. N / A: Median survival could not be determined because more than half of the mice survived the observation period.

[0416] 12.3 Tumor Photon Count and Tumor Growth Inhibition (TGI)

[0417] The average tumor photon counts of mice in each group are shown in Table 4 and FIG15B , and the tumor growth inhibition rate TGI values ​​are shown in Table 5 .

[0418] Table 4. Average tumor photon counts (p / sec / cm2 / sr) of mice in different groups during the study period Note: Data are presented as mean ± SEM. “-” indicates no statistical data due to mouse death.

[0419] Table 5. Tumor inhibition rate (TGI) of mice in different groups

[0420] As shown in Tables 4 and 5, LMOAME at doses of 50, 120, and 300 μg / kg (G2-G4) all exhibited tumor suppression effects on days 5, 10, and 15, significantly prolonging the survival of mice. The TGI values ​​for the G2-G4 groups on day 5 were 67.12%, 76.18%, and 86.08%, respectively; on day 10, they were 83.86%, 86.11%, and 94.38%, respectively; and on day 15, they were 66.77%, 79.12%, and 92.86%, respectively.

[0421] 12.4 Flow Cytometry Data Analysis of Human Immune Cells in Mouse Peripheral Blood

[0422] Peripheral blood of mice was collected on D3 and D28 days for flow cytometry analysis to detect the proportion and number of mCD45, hCD45, hCD19, hCD20, hCD3, hCD4 and hCD8 cells. The data showed that in the 50, 120, and 300 μg / kg dose groups (G2-G4) of LMOAME, the hCD19 in the peripheral blood of mice was significantly increased. + and hCD20 + The cell numbers were significantly lower than the levels on D3, proving that LMOAME can target and eliminate human B cells.

[0423] Example 13. Phase I clinical study of LMOAME

[0424] This example describes a Phase I clinical study involving a trispecific antibody of the present invention, in which the LMOAME trispecific antibody was prepared as a lyophilized powder injection and tested for safety, pharmacokinetics (PK), and immunogenicity in subjects with relapsed / refractory (r / r) B-cell-associated tumors. Specifically, the B-cell-associated tumors include CD19-expressing B-cell non-Hodgkin's lymphoma (NHL) and B-cell acute lymphoblastic leukemia (B-ALL). Patients must have experienced disease progression after receiving currently available, approved, or established therapies known to produce clinical benefit.

[0425] 13.1 Enrolled Population

[0426] Relapsed / refractory B-ALL and B-cell lymphoma.

[0427] 13.2 Study Objectives

[0428] Main research objectives:

[0429] To evaluate the safety of LMOAME and determine the recommended Phase II dose (RP2D) of LMOAME in adult patients based on dose-limiting toxicity (DLT) assessment.

[0430] Secondary study objectives:

[0431] - To evaluate LMOAME drug-related toxicities and their relationship to LMOAME dose and PK.

[0432] - To evaluate the pharmacokinetic (PK) parameters and immunogenicity of LMOAME in patients with CD19-expressing B-cell malignancies.

[0433] - Identify a safe, biologically active dosing dose and treatment regimen for Phase 2 clinical trials.

[0434] - To evaluate any objective tumor response rate with LMOAME in B-cell related tumors.

[0435] Exploratory research objectives:

[0436] -To explore the relationship between PK parameters and B cell depletion, serum cytokine levels, CRS, and their correlation with clinical efficacy.

[0437] -Perform immunophenotyping of peripheral blood, including activation of CD4+ and CD8+ T cells, percentages of CD19 and CD20, etc.

[0438] -Assess the MRD status of ALL patients who achieved CR / CRi using multiparameter flow cytometry.

[0439] 13.3 Scheme Design

[0440] This Phase I, multicenter, open-label, dose-escalation study was divided into two phases. The first phase enrolled patients and sample size followed the protocol of Simon R, et al., while the second phase followed a traditional 3+3 dose-escalation design.

[0441] Phase I: Accelerated Titration Design

[0442] The primary objective of Phase I is to determine the priming dose level. Other objectives include safety, PK, and immunogenicity.

[0443] A total of five dose groups were set as follows: 0.3mcg, 0.6mcg, 1.2mcg, 2.4mcg, 4.8mcg; each dose group had one patient.

[0444] The dose-limiting toxicity (DLT) assessment time was 21 days.

[0445] In the first dosing cycle, the drug was administered once a week in week 1 and twice a week in weeks 2 and 3, with a DLT observation period of 21 days.

[0446] From the second dosing cycle onwards, the drug is administered twice a week for 3 weeks, continuing until disease progression (PD) or intolerance.

[0447] All five dose groups were accelerated dose escalation; if a DLT or grade ≥2 adverse event (AE) occurred during the 21-day period, the dose group was expanded to 3 patients and the dose escalation was changed to a 3+3 design.

[0448] If a single grade ≥3 cytokine release syndrome (CRS) or two grade 2 CRS occurs at a given dose level, a second lead-in dose, referred to as the “intermediate” lead-in dose, will be added to the first cycle of each dose level. The intermediate lead-in dose will be 1 dose level higher than the determined initial lead-in dose level. The DLT period will be extended to 28 days to include the lead-in dose schedule (one week each for the initial lead-in dose and the intermediate lead-in dose) and 2 weeks of escalating dose levels. The determination and optimization of the lead-in dose schedule (initial and intermediate doses) will be based on all available data, including safety, efficacy, PK, PD (including cytokine data), and other data that will be reviewed by the SMC. The pharmacodynamic (PD) analysis used to determine the active biologic dose will include B cell clearance, first grade 2 CRS, and clinical response.

[0449] If the first patient treated with 0.3 mcg develops grade 3 or higher cytokine release syndrome (CRS) or neurotoxicity, a cohort of patients will be treated with 0.15 mcg using a 3+3 approach. If no grade 3 or higher CRS is seen at 0.15 mcg or the maximum tolerated dose (MTD) is reached, the dose will be escalated to 0.3 mcg.

[0450] If no DLT or grade ≥2 AE is reported (excluding exogenous causes or disease progression), patients at the next dose level will be enrolled after Day 21 of 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.

[0451] Phase II: Traditional 3+3 dose escalation design

[0452] The primary objectives of Phase II are to study safety, PK, immunogenicity, preliminary efficacy, and to determine the recommended Phase II dose (RP2D).

[0453] A total of five dose groups were set as follows: 9.6mcg, 16mcg, 24mcg, 34mcg, and 45mcg; each dose group had three patients.

[0454] In the first dosing cycle, the drug was administered once a week in week 1 and twice a week in weeks 2 and 3, with a DLT observation period of 21 days.

[0455] From the second dosing cycle onwards, the drug was administered twice a week for 3 weeks, continuing until PD or intolerance.

[0456] Patients will be admitted for observation within the first 72 hours after enrollment to administer the initial dose, intermediate doses (if applicable), and escalating doses. The first dose (Cycle 1 Day 1, C1D1) will be infused slowly over 3 hours. If there are no ≥Grade 2 infusion-related reactions (IRRs), the next dose (Cycle 1 Day 8, C1D8) can be administered within 2 hours. If there are no IRRs, the third and subsequent doses can be administered within 1 hour.

[0457] Premedication with an antihistamine and acetaminophen is required 1 hour before the first three doses of LMOAME (e.g., before the initial dose on C1D1 and before the first two BIW doses on C1D8 and C1D11). Dexamethasone may be added for subsequent infusions as clinically indicated. Subsequent premedication doses of dexamethasone may be reduced or eliminated at the investigator's discretion. Patients will be monitored after the infusion for a period equivalent to twice the duration of the infusion. If CRS occurs after the first three doses, premedication with 20 mg of dexamethasone, an antihistamine, and acetaminophen should be given within 1 hour before the subsequent dose. If two events of grade 2 or greater CRS or one event of grade 3 or greater CRS occur during the study, all patients will be given prophylactic acetaminophen 650 mg orally, diphenhydramine 50 mg (orally or intravenously), and tocilizumab 8 mg / kg (intravenously over 1 hour) prior to the first escalating dose.

[0458] On day 8 of cycle 1, patients will receive the first dose at the escalating dose level according to the BIW schedule for 2 weeks. The DLT period is 3 weeks (1 week of induction dose and 2 weeks of escalating dose). If a single ≥ grade 3 CRS or two grade 2 CRS events occur at a given dose level, a second intermediate induction dose will be added to the first cycle of each dose level, the same as Part I (see above). After all patients at the previous dose level have passed the DLT evaluation period and have not exceeded the MTD, the escalating dose will be increased. For cycle 2 and beyond, patients will be dosed BIW starting on day 1. These cycles are 3 weeks in length.

[0459] Patients will continue treatment until disease progression, DLT or intolerable toxicity, withdrawal from treatment or withdrawal of informed consent, or at the discretion of the investigator if it is in the best interest of the patient.

[0460] After reviewing all safety, PK, PD, and preliminary efficacy data, the RP2D was determined. This dose level was then expanded to 6-10 patients with NHL and 6-10 patients with B-ALL at the estimated RP2D.

[0461] 13.4 Number of people to be included

[0462] Phase I

[0463] Phase II

[0464] Once the maximum tolerated dose (MTD) is determined, an additional 6-10 studies will be included in the NHL and B-ALL populations as extension phase studies to further determine the safety and efficacy at this MTD dose.

[0465] 13.5 Administration

[0466] After reconstitution, the lyophilized powder of LMOAME is administered by intravenous infusion at the dose and frequency described in Section 13.3 above.

[0467] 13.6 Inclusion Criteria

[0468] Male or female patients aged 18 years and above who meet the following criteria will be included in this study:

[0469] - Histologically confirmed disease from one of the following groups of B-cell malignancies. Lymphocytes or lymphoma cells must be positive for CD19 antigen expression by immunohistochemistry and / or flow cytometry. If the patient has previously received CD19-targeted therapy, CD19 expression must be determined after treatment.

[0470] Non-Hodgkin lymphoma (NHL):

[0471] Aggressive B-cell NHLs include:

[0472] Diffuse large B-cell lymphoma (DLBCL), NOS

[0473] Primary mediastinal large B-cell lymphoma

[0474] High-grade B-cell lymphoma with rearrangements of MYC and BCL2 and / or BCL6

[0475] High-grade B-cell lymphoma, NOS

[0476] High-grade B-cell lymphoma arising from indolent NHL

[0477] Mantle cell lymphoma (can present as indolent or aggressive disease)

[0478] Follicular lymphoma, grade 3B

[0479] Indolent B-cell NHLs include:

[0480] Follicular lymphoma (grade 1-3A)

[0481] Splenic marginal zone lymphoma

[0482] Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma, excluding gastric MALT lymphoma)

[0483] Additional requirements for inclusion of NHL patients included:

[0484] NHL patients entering the Phase II study must have ≥1 measurable target lesion defined by the Lugano criteria (SAD for nodal lesions should be ≥15 mm; LD for extranodal lesions should be ≥10 mm).

[0485] Patients with indolent lymphomas will be considered for treatment only if they meet criteria for systemic therapy.

[0486] Patients with rapidly progressive, high-grade NHL will be enrolled only after reaching a biologically active dose level.

[0487] 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), received ≥2 lines of systemic therapy, and have received or been intolerant to all other standard therapies considered to provide clinical benefit.

[0488] Indolent NHL: Patients who have received ≥2 prior systemic therapies, including rituximab or other anti-CD20 monoclonal antibodies alone or in combination with chemotherapy (e.g., bendamustine, CHOP, CVP), are refractory or relapsed, and have received or are intolerant to all other standard therapies deemed clinically beneficial.

[0489] B-cell acute lymphoblastic leukemia (B-ALL):

[0490] - Philadelphia chromosome-positive or -negative B-cell leukemia refractory or relapsed after first-line therapy (induction / consolidation / maintenance), salvage therapy (e.g., blinatumomab, inotuzumab bozogamycin, chemotherapy), and all available Bcr-Abl tyrosine kinase inhibitors.

[0491] - Have received or are intolerant to all other standard therapies known to provide clinical benefit.

[0492] - Relapsed or refractory CD19+ B-cell ALL who have failed all available therapies with known clinical efficacy.

[0493] - No active acute or chronic graft-versus-host disease in the 2 months prior to enrollment and no immunosuppressive therapy at enrollment. Peripheral blood counts must be <10,000 / mm³ at the time of the first dose. This value can be maintained with prednisone and / or hydroxyurea during the screening period. B-ALL patients will be enrolled in the dose-escalation portion of the study only after reaching a biologically active dose level. The biologically active dose level is determined based on the first dose level at which ≥Grade 2 CRS and peripheral B-cell depletion are achieved.

[0494] Both NHL and ALL patients should meet the following criteria:

[0495] - ECOG performance status 0-2, life expectancy greater than 3 months.

[0496] - During the screening period, clinical and biochemical indicators meet the following requirements:

[0497] Total bilirubin must be <1.5 times the upper limit of the normal range (ULN). If the elevation in total bilirubin can be reasonably attributed to the presence of metastatic disease in the liver or in patients with documented Gilbert's syndrome (unconjugated hyperbilirubinemia), total bilirubin may be increased to 3x ULN.

[0498] Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) must be < 3×ULN. AST and ALT elevations up to 5×ULN are permitted if the elevations can be reasonably attributed to the presence of metastatic disease in the liver.

[0499] Calculated creatinine clearance > 50 mL / min (Cockcroft-Gault formula)

[0500] Hemoglobin must be ≥8 g / dL.

[0501] Neutrophil count must be >1,500 / mm 3 In ALL, if neutropenia is thought to be due to leukemic cell involvement of the BM, there is no standard for neutrophil count.

[0502] Platelet count must be greater than 75,000 / mm 3 For patients with ALL, if thrombocytopenia was thought to be due to leukemic cell involvement of the BM, there was no platelet count criterion; platelet transfusions were administered as needed at the investigator's discretion.

[0503] All patients must have a peripheral blood cell count ≤ 10,000 / mm3 before the first dose of LMOAME 3 This can be achieved by taking dexamethasone 20-40 mg / m2 for 4 consecutive days. Hydroxyurea can also be used to control the white blood cell count. If these measures do not reduce the white blood cell count to ≤10,000 / mm 3 , the patient will not be admitted.

[0504] The prothrombin time-international normalized ratio (PT-INR) must be ≤1.5.

[0505] - If female, negative urine pregnancy test or postmenopausal status for at least 1 year prior to screening.

[0506] - Female patients of childbearing potential or male patients with reproductive potential partners must use one or more contraceptive methods starting from screening and continuing during study treatment until 3 months after the last dose. Contraceptive methods include:

[0507] a. Complete abstinence (if consistent with the patient's usual lifestyle preferences). Cyclic abstinence (e.g., calendar, ovulation, sympathetic, postovulation) and withdrawal are unacceptable methods of contraception.

[0508] b. Female sterilization (bilateral salpingectomy with or without hysterectomy) or tubal ligation performed at least 12 weeks before study treatment.

[0509] c. Male sterilization (at least 6 months before screening). For female patients participating in this study, their male partner who has undergone vasectomy should be their only partner.

[0510] d. Use of oral, injectable, or implantable hormonal contraceptive methods; implantable intrauterine device (IUD) or intrauterine contraceptive system (IUS); use of other equally effective hormonal contraceptive methods (failure rate <1%), such as hormonal vaginal rings or transdermal hormonal contraceptives.

[0511] e. If female patients are taking oral contraceptives, they should take the same medication stably for at least 3 months before taking study treatment.

[0512] - Able to understand and willing to provide written informed consent and comply with scheduled visits and study procedures.

[0513] 13.7 Exclusion Criteria

[0514] - Systemic anticancer treatment (including I / O therapy) within 4 weeks before starting LMOAME treatment.

[0515] - Received radiotherapy treatment within 2 weeks prior to study entry.

[0516] - Received CAR-T or transplantation therapy within 3 months prior to study entry.

[0517] - Severe infection requiring treatment with antibiotics within 14 days prior to study entry.

[0518] - Patients who have previously received anti-CD19 targeted therapy can only join this study if their tumor cells are proven to still express CD19 after completing CD19 targeted therapy.

[0519] - Patients with brain metastases or other significant neurological disease, except those with brain metastases who were asymptomatic and radiologically stable within 2 weeks prior to the first dose of LMOAME and did not require steroids. Patients with ALL must not have significant CNS disease.

[0520] -Treatment with corticosteroids (>10 mg of prednisone or equivalent daily) or immunosuppressive medications ≤7 days before the first dose of LMOAME, except for the following:

[0521] Topical, ophthalmic, intra-articular, intranasal, or inhaled corticosteroids

[0522] Dexamethasone to lower peripheral blood cell counts in ALL

[0523] - Previous treatment with any investigational product (including cell or gene therapy) within 5 half-lives of the agent or within 4 weeks prior to starting LMOAME, whichever is shorter.

[0524] - Current autoimmune disease or history of autoimmune disease with potential central nervous system involvement.

[0525] - Hypersensitivity to any ingredient contained in the drug product formulation, including polysorbate 80.

[0526] - Use or have evidence of illegal drugs, drug abuse, or alcohol abuse.

[0527] - Any cerebrovascular accident (CVA), transient ischemic attack (TIA), myocardial infarction (MI), unstable angina, or New York Heart Association (NYHA) class III or IV heart failure within 6 months prior to enrollment; uncontrolled arrhythmia within < 3 months after enrollment.

[0528] - Major surgery within 4 weeks or minor surgery within 2 weeks prior to screening; the wound must be completely healed (minor surgery such as catheter placement is not an exclusion criterion).

[0529] - History of Grade 3-4 hypersensitivity reactions to other monoclonal antibody treatments, or known hypersensitivity to protein drugs or recombinant proteins or excipients in LMOAME drug formulations.

[0530] - Presence of congenital long QT syndrome, QTcF > 480 milliseconds, pacemaker use, left ventricular ejection fraction (LVEF) < 50%, clinically significant arrhythmias 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).

[0531] - Any other serious underlying medical condition (e.g., active gastric ulcer, uncontrolled seizures, cerebrovascular events, gastrointestinal bleeding, severe signs and symptoms of coagulation and coagulopathy, cardiac conditions), psychiatric, psychological, familial, or geographic condition that, in the investigator's judgment, may interfere with planned staging, treatment, and follow-up, affect patient compliance, or place the patient at high risk for treatment-related complications.

[0532] - Patients infected with COVID-19, who have been isolated and tested negative for the virus, can participate.

[0533] - Patients with other concurrent malignancies within 5 years before enrollment, except for carcinoma in situ of the cervix, local cutaneous squamous cell carcinoma, basal cell carcinoma, local prostate cancer, ductal carcinoma in situ of the breast, or <T1 urothelial carcinoma that have been adequately treated.

[0534] - Those who have previously undergone organ or allogeneic stem cell / bone marrow transplantation. Patients who received autologous stem cell transplantation >6 months before study screening are eligible if their hematological parameters meet the enrollment requirements.

[0535] - Those with a history of grade 3-4 immune-related adverse events (irAEs) or irAEs that required discontinuation of previous treatment, except for grade 3 endocrinopathies managed with hormone replacement therapy.

[0536] - Pleural effusion, pericardial effusion, or ascites that require frequent drainage or medical intervention

[0537] - Those who received live virus vaccines within 4 weeks before study enrollment.

[0538] - Active hepatitis B or C; HBV carriers without active disease (HBV DNA titer <1000 cps / mL or 200 IU / mL), or those with cured hepatitis C (negative HCV RNA test) can be enrolled.

[0539] - Known HIV infection.

[0540] 13.8 Study endpoints

[0541] Safety study endpoints

[0542] - The proportion of patients with serious adverse events (SAEs) and adverse events (AEs) at each dose level;

[0543] - The type, frequency, and dose correlation of DLTs;

[0544] - Unexpected clinical or laboratory findings.

[0545] PK study endpoints:

[0546] PK parameters, including AUC, C max , C min , initial clearance rate, volume of distribution, and elimination half-life

[0547] PD study endpoints:

[0548] B cell depletion, cytokine release (including IL-2, IL-6, IFNg, TNFa)

[0549] Immunogenicity study endpoints:

[0550] Proportion of patients with anti-drug antibodies (ADA), neutralizing antibodies.

[0551] Efficacy endpoints:

[0552] Objective response rate (ORR)

[0553] Sequence information

[0554] Table 6. Linker sequences

Claims

1. A trispecific antibody that specifically binds to CD19, CD3, and CD28, which is a single-chain fusion protein and comprises: (1) The 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) The third binding domain specifically binds to CD28.

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

3. The trispecific antibody according to 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 trispecific antibody according to any one of claims 1 to 3, 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), (a) the VH comprises a heavy chain CDR 1 as set forth in SEQ ID NO: 11, a heavy chain CDR2 as set forth in SEQ ID NO: 12, and a heavy chain CDR3 as set forth in SEQ ID NO: 13, and the VL comprises a light chain CDR 1 as set forth in SEQ ID NO: 14, a light chain CDR2 as set forth in SEQ ID NO: 15, and a light chain CDR3 as set forth in SEQ ID NO: 16; or (b) the VH comprises a heavy chain CDR 1 as shown in SEQ ID NO: 46, a heavy chain CDR2 as shown in SEQ ID NO: 47, and a heavy chain CDR3 as shown in SEQ ID NO: 48, and the VL comprises a light chain CDR 1 as shown in SEQ ID NO: 49, a light chain CDR2 as shown in SEQ ID NO: 50, and a light chain CDR3 as shown in SEQ ID NO:

51.

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

6. The trispecific antibody according to claim 5, wherein the first binding domain comprises the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 56, or an amino acid sequence that is at least 80% homologous (such as at least 85% homologous, preferably at least 90% homologous, more preferably at least 95% homologous, even more preferably at least 96%, at least 97%, at least 98% or at least 99% homologous) to the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO: 56 and all the amino acid differences are in non-CDR regions; or consists of such an amino acid sequence.

7. The trispecific antibody according to any one of claims 1 to 6, wherein the second binding domain is a single-chain antibody specifically binding 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 as set forth in SEQ ID NO: 22, a heavy chain CDR2 as set forth in SEQ ID NO: 23, and a heavy chain CDR3 as set forth in SEQ ID NO: 24, and the VL comprises a light chain CDR1 as set forth in SEQ ID NO: 25, a light chain CDR2 as set forth in SEQ ID NO: 26, and a light chain CDR3 as set forth in SEQ ID NO: 27; or (b) the VH comprises a heavy chain CDR 1 as shown in SEQ ID NO: 57, a heavy chain CDR2 as shown in SEQ ID NO: 58, and a heavy chain CDR3 as shown in SEQ ID NO: 59, and the VL comprises a light chain CDR 1 as shown in SEQ ID NO: 60, a light chain CDR2 as shown in SEQ ID NO: 61, and a light chain CDR3 as shown in SEQ ID NO:

62.

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

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

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 comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a heavy chain CDR 1 as set forth in SEQ ID NO: 33, a heavy chain CDR2 as set forth in SEQ ID NO: 34, and a heavy chain CDR3 as set forth in SEQ ID NO: 35, and the VL comprises a light chain CDR 1 as set forth in SEQ ID NO: 36, a light chain CDR2 as set forth in SEQ ID NO: 37, and a light chain CDR3 as set forth in SEQ ID NO:

38.

11. The trispecific antibody according to claim 10, wherein The VH comprises the amino acid sequence shown in SEQ ID NO: 39, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 39 and all the amino acid differences are in non-CDR regions, or consists of such an amino acid sequence; and The VL comprises the amino acid sequence shown in SEQ ID NO: 41, or an amino acid sequence that has at least 80% homology (such as 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 shown in SEQ ID NO: 41 and all amino acid differences are in non-CDR regions, or consists of such an amino acid sequence.

12. The trispecific antibody according to claim 11, wherein the third binding domain comprises the amino acid sequence as shown in SEQ ID NO: 43, or an amino acid sequence that has at least 80% homology (such as 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 as shown in SEQ ID NO: 43 and all amino acid differences are in non-CDR regions; or consists of such an amino acid sequence.

13. The trispecific antibody according to any one of claims 1 to 12, comprising or consisting of the amino acid sequence of SEQ ID NO: 44 or SEQ ID NO: 68; or comprising or consisting of an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous to the amino acid sequence of 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. The isolated nucleic acid molecule of claim 14, comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 45 or SEQ ID NO:

69.

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

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

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

21. The use according to claim 20, wherein the medicament is used to treat B cell-related tumors, such as B cell lymphoma, B cell leukemia, refractory or relapsed B cell tumors, aggressive or indolent non-Hodgkin's lymphoma, and B cell acute lymphoblastic leukemia.

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

23. A pharmaceutical combination comprising the trispecific antibody of any one of claims 1 to 13 and another therapeutic agent, such as a therapeutic agent for treating a B cell-associated tumor.

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

Images