CD3-targeting antibodies, bispecific antibodies and uses thereof

Abstract

To provide a CD3 antibody that is capable of binding to primate CD3, has a suitable CD3 binding capacity, and has a stable single-chain scFv structure.SOLUTION: Disclosed are a CD3-targeting antibody, a bispecific antibody and the use thereof. The CD3-targeting antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH). The VL has the amino acid sequence as shown in SEQ ID NO: 56 or a mutation thereof. The VH has mutations on the amino acid sequence as shown in SEQ ID NO: 42, and the mutations occur at one or more of the sites of amino acid residues selected from positions 30, 73, 76, 78, 93 and 94. The bispecific antibody comprises a first protein domain and a second protein domain, wherein the first protein domain comprises the CD3-targeting antibody as described above. The CD3-targeting antibody reduces the toxicity caused by cytokine release syndrome, and the bispecific antibody prepared therefrom is stable and has the ability to bind to T cells, and also reduces the difficulty of producing.SELECTED DRAWING: Figure 11

Description

[Technical Field]

[0001] This application claims priority from Chinese Patent Application No. 2019109413286, filed on September 30, 2019, the entire text of which is incorporated herein by reference.

[0002] The present invention relates to the field of biopharmaceuticals, and in particular to antibodies, bispecific antibodies, and uses thereof that target CD3. [Background technology]

[0003] T cells are an important class of cells involved in adaptive immune responses. They recognize antigens via their T cell receptors (TCRs). TCRs cannot directly recognize epitopes on antigens; instead, they specifically recognize antigen peptide-MHC molecule complexes (pMHC) presented on the surface of antigen-presenting cells (APCs) or target cells. The specificity of T cell responses is mediated by pMHC recognition by the TCR-CD3 molecular complex. TCRs are heterodimers composed of two distinct transmembrane polypeptide chains. There are four types of peptide chains: α, β, γ, and δ. Depending on the combination of peptide chains, TCRs are classified as TCRαβ and TCRγδ. CD3 has different transmembrane polypeptide chains: γ, δ, ε, and ζ. These peptide chains interact to form homodimers or heterodimers, which become part of the TCR-CD3 complex. For example, the TCR-CD3 complex may contain TCRαβ dimers, CD3γε dimers, CD3δε dimers, and CD3ζζ dimers. Because the cytoplasmic region of the TCR peptide chain is very short, it is generally believed that the activation signal generated by antigen recognition by the TCR is transmitted to the T cell by the CD3 peptide chain.

[0004] Given the crucial role of CD3 in initiating immune responses, monoclonal antibodies targeting TCR-CD3 signaling, particularly CD3, are believed to modulate immune processes and be effective drugs for the treatment of inflammatory or autoimmune diseases. Indeed, the anti-CD3 antibody, Orthoclone OKT3, was the first approved therapeutic antibody. OKT3 was initially approved by the US FDA in 1985 for the treatment of acute rejection after organ transplantation. The immunosuppressive potential of OKT3 with repeated administration made it an effective treatment for kidney transplant rejection. However, its use was limited by an early toxic dose-response syndrome, which is thought to be related to OKT3-mediated T cell activation and cytokine release. Subsequently, OKT3 was withdrawn from the market in 2010 due to severe cytokine storms, immunogenicity due to murine antibodies, and other issues.

[0005] Another problem with CD3 antibodies is their species specificity. For example, OKT3 reacts with chimpanzee CD3 but not with CD3 homologs in other primates, such as rhesus macaques, or with mouse CD3 homologs. The species specificity of CD3 monoclonal antibodies poses a major obstacle to their development as antibody drugs for the treatment of human diseases. Rigorous preclinical validation is required for drug candidates to be used in human clinical trials. The goal of preclinical testing is to confirm the desired activity and, most importantly, safety of the candidate drug. In preclinical safety testing, drug candidates are administered to the target species, preferably non-human primates. However, higher primates, especially chimpanzees, are endangered species, and their use in drug safety testing is severely restricted. Rhesus macaques, particularly cynomolgus macaques, are a suitable species for safety evaluation in this field. However, CD3 antibodies that lack species-specific cross-reactivity with primates cannot provide effective preclinical safety evaluation data. Among known antibodies that bind to human CD3, SP34 is one of the leading antibodies that can bind to CD3 from multiple primates (e.g., human and cynomolgus monkey CD3) (see Salmeron, A., et. al, J Immunol 147 (1991) 3047-3052; Conrad ML, et. al, Cytometry A 71 (2007) 925-933).

[0006] While CD3 monoclonal antibodies have already been clinically proven to be effective against certain diseases, in recent years, CD3 antibodies have primarily been used in the development of bispecific antibody drugs. Currently, CD3-based bispecific T-cell engager (BsTCE) projects account for more than half of the bispecific antibody projects currently in the clinical or preclinical stages worldwide. CD3 bispecific antibody BsTCEs not only demonstrate potent efficacy similar to CAR-T cell therapy, but can also be produced and commercialized like traditional monoclonal antibodies. Among the bispecific antibody drugs currently approved worldwide, the first to be marketed, catumaxomab (approved by the European Medicines Agency in 2009 and withdrawn in the United States in 2013) and blinatumomab (approved by the US FDA in 2014), are both BsTCEs. CD3 antibodies are a key component in the construction of BsTCEs. BsTCE bispecific antibodies can simultaneously bind to two targets: one side recognizes tumor-associated antigens (TAAs) on the surface of tumor cells, and the other side binds to CD3 molecules on T cells. In the presence of tumor cells, BsTCE bispecific antibodies bind to the tumor cell surface and then recruit and activate nearby T cells to kill the tumor cells. When designing and constructing various BsTCE bispecific antibody structures, the selection and optimization of CD3 antibodies is particularly important. First, the species specificity of CD3 monoclonal antibodies, especially cross-reactivity in monkeys, is very important. Second, the affinity of CD3 antibodies for the CD3 complex is also important; CD3 antibodies with too high an affinity may restrict the antibodies to sites such as the spleen, making it difficult for them to contact the tumor. Furthermore, too high an affinity can overstimulate T cells, resulting in high levels of cytokine release. Third, the CD3 antibody valency plays a major role, and it has been shown that multivalent CD3 bispecific antibodies activate T cells without binding to tumor-associated antigens, causing side effects. Therefore, most CD3 bispecific antibodies under development are monovalent CD3.

[0007] In addition to CD3 antibodies, the structural design of BsTCE bispecific antibodies is also an important consideration. BsTCE bispecific antibodies have diverse structures and can be divided into two main categories: Fc-containing IgG-like structures and Fc-free antibody fragment structures. For example, blinatumomab is a single polypeptide chain consisting of two single-chain variable fragments (scFv) connected in tandem. However, this structure has a very short half-life and requires continuous intravenous infusion, making it difficult to use. Therefore, many BsTCE bispecific antibodies incorporate an Fc structure to improve molecular stability and pharmacokinetic properties. However, because BsTCE generally requires the CD3-binding domain to be in a monovalent form, Fc-containing structures are often asymmetric. Such asymmetric Fc-containing structures present many technical challenges to overcome, including heavy chain homodimerization in asymmetric structures, light chain mismatching, and Fcγ receptor-induced molecular cross-linking and effector functions such as ADCC or CDC. When constructing a BsTCE bispecific antibody from an anti-TAA IgG antibody and an anti-CD3 IgG antibody (Figure 16(A)), different asymmetric structures can be selected. One structure is an IgG-like structure that retains two independent Fab domains. This structure contains four distinct polypeptide chains (two distinct heavy chains and two distinct light chains, as shown in Figure 16(B)), with a molecular weight similar to that of a conventional monoclonal antibody. However, because this structure contains multiple distinct polypeptide chains, it can potentially produce various combinations of by-products, which poses a major challenge to the antibody expression, purification, and production process. Converting the Fab of the CD3 antibody into an scFv structure converts the "four-chain" structure into a "three-chain" structure (as shown in Figure 16(C)), reducing the number of by-product combinations and thus reducing the complexity of its production. The present inventors have attempted to convert the SP34 mouse antibody IgG into an scFv to construct a BsTCE bispecific antibody. However, no stable scFv was obtained, regardless of the (VH / VL) configuration or the length of the connecting peptide. Therefore, there is a need in the art for a stable anti-CD3 monoclonal antibody, particularly a stable scFv structure.

[0008] As can be seen from the above, there is a need in the art for CD3 antibodies that can bind to primate CD3, have appropriate CD3 binding ability, and have a stable single-chain scFv structure. Summary of the Invention [Problem to be solved by the invention]

[0009] To solve the technical problem in the field of the lack of low-antigenicity, effective, and safe anti-CD3 antibodies and bispecific antibodies with asymmetric structures, the present invention provides antibodies, bispecific antibodies, and uses thereof that target CD3. [Means for solving the problem]

[0010] The technical solution of the first aspect of the present invention to solve the above technical problems is as follows: Provided is an antibody targeting CD3, the CD3-targeting antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL has the amino acid sequence set forth in SEQ ID NO: 56 or a variant thereof, and the VH has mutations in the amino acid sequence set forth in SEQ ID NO: 42, selected from one or more of the amino acid residues at positions 30, 73, 76, 78, 93, and 94 (these positions are numbered using the Chothia numbering system). The mutations are addition, deletion, or substitution of one or more amino acid residues in the initial amino acid sequence. The CD3-targeting antibody of the present invention has altered binding ability to T cells and reduced cytokine release levels, and is therefore expected to reduce toxicity caused by cytokine release syndrome.

[0011] In a particularly preferred embodiment, the mutations in the VH are selected from the following combinations: (a) 30th place, (b) 30th, 73rd and 76th places; (c) 30th, 93rd and 94th places, (d) 30th, 73rd and 93rd places; (e) 30th place, 93rd place, (f) 30th, 76th and 78th places; (g) 73rd, 76th, 93rd and 94th places, (h) 76th, 78th and 93rd places, (i) 30th, 73rd, 76th, 93rd and 94th places; (j) 30th, 76th, 78th and 93rd places.

[0012] In a particularly preferred embodiment, the mutations in the VH are selected from the following combinations: (a) N30S, (b) N30S, D73N and S76N, (c) N30S, V93A and R94K, (d) N30S, D73N and V93A, (e) N30S and V93T, (f) N30S, S76N and L78A, (g) D73N, S76N, V93A and R94K, (h) S76N, L78A and V93T, (i) N30S, D73N, S76N, V93A and R94K, (j) N30S, S76N, L78A and V93T.

[0013] The antibodies of the present invention having the above-mentioned mutations in the VH of the antibody further include those having mutations in the VL of the amino acid sequence shown in SEQ ID NO: 56 or in the VH of the amino acid sequence shown in SEQ ID NO: 42, whereby the amino acid sequence after the mutation has 80%, 85%, 90%, 95%, 98%, 99% or more identity with the initial amino acid sequence, and which maintain or improve the function of the antibody, are also included in the scope of protection of the present invention.

[0014] In a particularly preferred embodiment, the amino acid sequence of the VH is as set forth in any of SEQ ID NOs: 43 to 55, and / or the amino acid sequence of the VL is as set forth in any of SEQ ID NOs: 57 to 60.

[0015] In certain preferred embodiments, the amino acid sequence of the VH is as set forth in SEQ ID NO: 44 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 51 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 44 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 60; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 51 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 60; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 45 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 52 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 43 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 43 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 60; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 50 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 47 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 48 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 49 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 53 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 54 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 43 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 57; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 44 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 57; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 43 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 59; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 44 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 59; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 51 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 57; or the amino acid sequence of the VH is as set forth in SEQ ID NO: 55 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or The amino acid sequence of the VH is as shown in SEQ ID NO:46, and the amino acid sequence of the VL is as shown in SEQ ID NO:58.

[0016] In certain preferred embodiments, the antibody comprises a single-chain variable fragment (scFv) that is VL-Linker-VH or VH-Linker-VL. Preferably, the Linker (i.e., connecting peptide) is (GGGGS). n [Abbreviation (G4S) n or a variant thereof, wherein n is a natural number other than 0, preferably 1 to 20, and more preferably the amino acid sequence set forth in SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67. More preferably, the amino acid sequence of the scFv is as set forth in SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80. Even more preferably, the antibody further comprises a fragment crystallizable (Fc), and the Fc is connected to the scFv by a hinge region.

[0017] In certain preferred embodiments, the antibody further comprises a constant region, preferably a human constant region. Preferably, the human constant region comprises a human light chain constant region and a human heavy chain constant region, and the human light chain constant region is preferably the human κ light chain constant region set forth in SEQ ID NO: 61 or the human λ light chain constant region set forth in SEQ ID NO: 62. More preferably, the human heavy chain constant region is hIgG1, hIgG2, hIgG3, hIgG4 or a variant thereof, preferably the heavy chain constant region set forth in SEQ ID NO: 63 or SEQ ID NO: 64.

[0018] The technical solution of the second aspect of the present invention to solve the above technical problems is as follows: A bispecific antibody is provided. The bispecific antibody of the present invention has a three-chain structure, which reduces the number of by-product combinations and thus reduces the complexity of its production. However, its development is not simply a matter of slightly modifying a conventional antibody. As mentioned in the background art, the present inventors attempted to convert the SP34 mouse antibody IgG into an scFv to construct a BsTCE bispecific antibody, but no stable scFv was obtained, regardless of the (VH / VL) configuration or the length of the connecting peptide. After repeated mutation design and testing, the present inventors found that only certain mutations could stably maintain the scFv structure. The bispecific antibody of the present invention comprises a first protein domain and a second protein domain, wherein the first protein domain comprises an antibody targeting CD3 according to the first aspect of the present invention, and preferably the bispecific antibody comprises three chains: (1) a first protein domain VL1-Linker-VH1-Hinge-CH2-CH3 (knob) or VH1-Linker-VL1-Hinge-CH2-CH3 (knob), (2) a second protein domain VH2-CH1-Hinge-CH2-CH3 (hole), and (3) a second protein domain VL2-CL, wherein the second protein domain is an antibody targeting another target, preferably an antibody targeting B7H4 or an antibody targeting ROR1, and the Linker is (G4S). nwherein n is a natural number other than 0, preferably 1-20, and more preferably the amino acid sequence shown in SEQ ID NO: 65, SEQ ID NO: 66, or SEQ ID NO: 67. More preferably, the bispecific antibody comprises VL1-Linker-VH1-Hinge-CH2-CH3 (knob) shown in SEQ ID NO: 88, VH2-CH1-Hinge-CH2-CH3 (hole) shown in SEQ ID NO: 86, and VL2-CL shown in SEQ ID NO: 83, or VL1-Linker-VH1-Hinge-CH2-CH3 (knob) shown in SEQ ID NO: 88, VH2-CH1-Hinge-CH2-CH3 (hole) shown in SEQ ID NO: 87, and VL2-CL shown in SEQ ID NO: 85. The bispecific antibody of the present invention overcomes the drawback of instability of single-chain antibody arms targeting CD3, and is stable and has T cell binding ability. Bispecific antibodies containing only three chains are easier to manufacture and less difficult to produce.

[0019] The technical solution of the third aspect of the present invention to solve the above technical problem is as follows: An isolated nucleic acid encoding the CD3-targeting antibody according to the first aspect of the present invention or the bispecific antibody according to the second aspect of the present invention is provided.

[0020] To solve the above technical problems, the technical solution of the fourth aspect of the present invention is as follows: There is provided an expression vector comprising the isolated nucleic acid according to the third aspect of the present invention, and preferably, the expression vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

[0021] The technical solution of the fifth aspect of the present invention to solve the above technical problems is as follows: A genetically modified cell transfected with the expression vector according to the fourth aspect of the present invention is provided, and preferably, the genetically modified cell is a eukaryotic cell.

[0022] To solve the above technical problems, the technical solution of the sixth aspect of the present invention is as follows: There is provided a pharmaceutical composition comprising an antibody targeting CD3 according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, a gene-modified cell according to the fifth aspect of the present invention, and a pharmaceutically acceptable carrier, and preferably, the pharmaceutical composition further comprises an immune checkpoint antibody.

[0023] To solve the above technical problems, the technical solution of the seventh aspect of the present invention is as follows: Use of an antibody targeting CD3 according to the first aspect of the present invention, a bispecific antibody according to the second aspect of the present invention, an isolated nucleic acid according to the third aspect of the present invention, an expression vector according to the fourth aspect of the present invention, a genetically modified cell according to the fifth aspect of the present invention, or a pharmaceutical composition according to the sixth aspect of the present invention for the manufacture of a medicament for treating a tumor is provided.

[0024] Furthermore, the technical solution of the eighth aspect of the present invention to solve the above technical problems is as follows: A kit combination is provided, comprising Kit A and Kit B, wherein Kit A comprises an antibody targeting CD3 according to the first aspect of the present invention, a bispecific antibody according to the second aspect, a genetically modified cell according to the fifth aspect, or a pharmaceutical composition according to the sixth aspect, and Kit B comprises another antibody, bispecific antibody, genetically modified cell, or pharmaceutical composition, wherein the other antibody, bispecific antibody, genetically modified cell, or pharmaceutical composition targets CD3, B7H4, ROR1, or another target. The order of use of Kit A and Kit B is not fixed, and Kit A may be used first, followed by Kit B, or Kit B may be used first, followed by Kit A. The drug in Kit A is in an injectable form, for example, an injection, and the drug in Kit B is in an injectable form, for example, an injection, or a swallowable form, for example, a tablet or pill.

[0025] The combination of an antibody targeting CD3 according to the first aspect of the invention, a bispecific antibody according to the second aspect, a genetically modified cell according to the fifth aspect, a pharmaceutical composition according to the sixth aspect or a kit according to the eighth aspect of the invention may be administered to a patient for the treatment of a tumour.

[0026] Unless inconsistent with common knowledge in this field, any combination of the above-mentioned preferred conditions can be used to obtain any preferred embodiment of the present invention.

[0027] All of the reagents and raw materials used in the present invention may be commercially available products.

[0028] The beneficial effects of the present invention are as follows: 1. The monoclonal antibodies of the present invention have altered binding ability to T cells and reduced levels of cytokine release, which is expected to reduce toxicity caused by cytokine release syndrome. 2. The bispecific antibody produced using it overcomes the disadvantage of instability of the single-chain antibody arm targeting CD3, and is stable and has T cell binding ability. 3. Bispecific antibodies containing only three chains are easier to manufacture and less difficult to produce. [Brief explanation of the drawings]

[0029] [Figure 1] Figure 1 shows the HPLC-SEC results after one-step purification of CD3 single-chain antibodies: (A) PR000275, (B) PR000276, (C) PR000307, ​​and (D) PR000308. [Figure 2] FIG. 2 is a comparison of SP34 VH humanized variant sequences. [Figure 3] FIG. 3 is a comparison of SP34 VL humanized variant sequences. [Figure 4] Figure 4 shows the differences in key regions between different VH / VL variant sequences, where (A) is the VH variant sequence and (B) is the VL variant sequence. [Figure 5]FIG. 5 shows (A) the SDS-PAGE results and (B) the HPLC-SEC results of the CD3 single-chain antibody PR000510 after one-step purification. [Figure 6] FIG. 6 shows the binding ability of the CD3 antibody PR000260 to (A) recombinant CHOK1 cells overexpressing human CD3 and (B) recombinant CHOK1 cells overexpressing cynomolgus monkey CD3. [Figure 7-1] FIG. 7 shows the binding ability of CD3 antibodies to human T cells, including binding curves and relative MFI intensities (fluorescence intensity MFI at which an antibody at a specific concentration binds to human T cells, and the ratio to the initial antibody PR000260 (SP34)) or maximum MFI values. (A) shows the binding of PR000511, PR000512, PR000513, PR000514, and PR000260 to human T cells. (B) shows the binding of PR001848, PR001849, and PR000260 to human T cells. (C) shows the binding of PR002467, PR002468, PR002469, PR002470, PR002471, PR002472, (D) Binding of PR001848 and PR000260 to human T cells; (E) Binding of PR002833, PR002834, PR002835, PR002836, PR002837, PR002742, PR001848, PR002469, and PR000260 to human T cells; (F) Binding of PR003886, PR001848, and PR002742 to human T cells; and (G) Binding of PR001848, PR002469, and PR004616 to human T cells. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 7-3] This is a continuation of Figure 7-2. [Figure 8]Figure 8 shows the binding ability of CD3 single-chain antibodies to human T cells, including binding curves and relative MFI intensities (fluorescence intensity MFI at which a specific antibody concentration binds to human T cells, and the ratio to the initial antibody PR000260 (SP34)). (A) shows the binding of PR000510, PR000624, PR000627, and PR000260 to human T cells, and (B) shows the binding of PR001850 and PR000260 to human T cells. [Figure 9] FIG. 9 shows the binding ability of CD3 antibodies to cynomolgus monkey T cells. [Figure 10-1] Figure 10 shows the ability of CD3 antibodies to activate human T cells to produce the cytokine IFN-γ. (A) T cell activation by PR000511, PR000512, PR000513, PR000514, and PR000260; (B) T cell activation by PR001848, PR001849, and PR000260; (C) T cell activation by PR002468, PR002469, PR002471, and PR001848; (D) T cell activation by PR002742, PR001848, and PR000260; (E) T cell activation by PR002833, PR002834, PR002835, PR002836, PR002837, and PR000260; (F) T cell activation by PR003886, PR001848, and PR002742; (G) T cell activation by PR001848, PR002469, and PR004616. [Figure 10-2] This is a continuation of Figure 10-1. [Figure 10-3] This is a continuation of Figure 10-2. [Figure 11] Figure 11 shows the ability of CD3 single chain antibodies (PR000510, PR000623, PR000624, PR000627 and PR000260) to activate human T cells to produce the cytokine IFN-γ. [Figure 12] FIG. 12 shows the results of SDS-PAGE of samples after one-step purification of bispecific antibodies (A) PR002883 and (B) PR002885. [Figure 13]FIG. 13 shows the binding ability of monoclonal and bispecific antibodies to (A) SK-BR-3 cells and (B) human T cells. [Figure 14] FIG. 14 shows the in vitro target cell killing mediated by bispecific antibody PR002883: (A) SK-BR-3 cell killing, and (B) IFN-γ release levels. [Figure 15] FIG. 15. Binding ability of monoclonal and bispecific antibodies to (A) Panc-1 cells and (B) human T cells. [Figure 16] Figure 16 shows monoclonal or bispecific antibody structures: (A) an IgG structure, (B) an asymmetric "four-chain" structure, and (C) an asymmetric "three-chain" structure containing a single-chain antibody. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention will be further explained by the following examples, but the present invention is not limited to these examples. In the following examples, experimental methods for which conditions are not specifically described are carried out according to conventional methods and conditions, or are selected based on the product instructions.

[0031] As used herein, the term "antibody" generally refers to a protein comprising an antigen-binding portion and, optionally, a scaffold or framework portion that may adopt a conformation that promotes binding of the antibody to the antigen. Typically, the antibody may comprise an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both. The VH and VL regions may be further divided into hypervariable regions called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL may be composed of three CDRs and four FR regions, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with antigens. Examples of antibodies include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv and / or dAb), immune complexes, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, as long as they exhibit the desired antigen-binding activity.

[0032] As used herein, the term "variable" generally refers to the fact that certain portions of the antibody variable domain sequence vary significantly and contribute to the binding and specificity of various specific antibodies to specific antigens. However, variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three segments of the light and heavy chain variable regions, called complementarity-determining regions (CDRs) or hypervariable regions (HVRs). The highly conserved portions of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions, most of which form a β-sheet structure connected by three CDRs to form loops, although in some cases they form part of a β-sheet structure. In each chain, the CDRs are tightly connected by the FR regions and, together with the CDRs from other chains, form the antigen-binding site of the antibody. The constant regions are not directly involved in antibody-antigen binding but exhibit various effector functions, such as antibody-dependent cellular cytotoxicity. In the art, antibody CDRs can be defined by various methods, such as the Kabat definition based on sequence variability (see Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda, Md (1991)) and the Chothia definition based on the location of structural loop regions (see Al-Lazikani, et al., J Mol Biol 273:927-48, 1997). In addition, in the present application, a combined definition method of the Kabat and Chothia definitions is used to determine the amino acid residues of variable domain sequences and full-length antibody sequences (Table 1). [Table 1]

[0033] Here, Laa-Lbb may refer to the amino acid sequence from positions aa (Chothia numbering system) to bb (Chothia numbering system) from the N-terminus of the antibody light chain, and Haa-Hbb may refer to the amino acid sequence from positions aa (Chothia numbering system) to bb (Chothia numbering system) from the N-terminus of the antibody heavy chain. For example, L24-L34 may refer to the amino acid sequence from positions 24 to 34 in the Chothia numbering system from the N-terminus of the antibody light chain, and H26-H32 may refer to the amino acid sequence from positions 26 to 32 in the Chothia numbering system from the N-terminus of the antibody heavy chain.

[0034] Effector functions mediated by the Fc domain of antibodies, such as ADCC and CDC, also have very important biological functions. Different IgG subtypes have different ADCC or CDC functions; for example, IgG1 and IgG3 have strong ADCC and CDC effects, while IgG2 and IgG4 have weaker effects. Furthermore, the inherent effector functions of Fc can be regulated by altering the binding ability of Fc to Fc receptors through amino acid mutation or modification. For example, the "LALA" double mutant (L234A / L235A) of IgG1 significantly reduces affinity for FcγRIIIA (CD16A) and reduces ADCC. Furthermore, the P329G mutation significantly reduces binding to various Fcγ receptors (see Schlothauer T, Herter S, Koller CF, et al., Protein Eng Des Sel. 2016 Oct;29(10):457-466). In the present application, a "LALA" double mutation (L234A / L235A) or a "LALAPG" triple mutation (L234A / L235A / P329G) was introduced into the Fc of these CD3 antibodies to reduce binding to Fcγ receptors.

[0035] Example 1: Recombinant antibody production and characterization analysis 1.1 Production of IgG recombinant antibodies After obtaining sequences encoding the light and heavy chain variable domains of an antibody molecule, conventional recombinant DNA techniques can be used to fusion-express the light and heavy chain variable domain sequences with the corresponding human antibody light and heavy chain constant domain sequences to obtain a recombinant antibody molecule. In this example, an antibody heavy chain variable domain sequence (VH) is genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding a human IgG1 antibody heavy chain constant domain sequence to encode a full-length heavy chain for generating an IgG1 antibody. Furthermore, the IgG1 heavy chain constant region is modified with a "LALA" double mutant (L234A / L235A) (SEQ ID NO: 63) or a "LALAPG" triple mutant (L234A / L235A / P329G) (SEQ ID NO: 64) to reduce antibody Fcγ receptor binding. Encode a full-length kappa light chain for generating antibodies by genetically synthesizing an antibody light chain variable domain sequence (VL) and cloning it into a mammalian cell expression plasmid vector encoding a human antibody kappa light chain constant domain sequence (SEQ ID NO: 61), or encode a full-length lambda light chain for generating antibodies by genetically synthesizing a VL and cloning it into a mammalian cell expression plasmid vector encoding a human antibody lambda light chain constant domain sequence (SEQ ID NO: 62).

[0036] Mammalian host cells (e.g., human embryonic kidney cells HEK293) are transfected with both the antibody heavy chain-encoding plasmid and the antibody light chain-encoding plasmid, and purified recombinant antibodies with correctly paired and assembled light and heavy chains can be obtained using standard recombinant protein expression and purification techniques. Specifically, HEK293 cells were expanded in FreeStyle™ F17 Expression Medium (Thermo #A1383504). Prior to transient transfection, the cell concentration was adjusted to 6–8 × 10 5 The cells were cultured in a shaker at 37°C and 8% CO2 for 24 hours, with a cell concentration of 1.2 × 10 6The cell density was 30 mL. The antibody heavy chain-encoding plasmid and the antibody light chain-encoding plasmid were mixed at a 2:3 ratio, resulting in a total of 30 μg of plasmid. The resulting mixture was dissolved in 1.5 mL of Opti-MEM reduced serum medium (Thermo #31985088) and sterilized by filtration through a 0.22 μm membrane filter. 1.5 mL of Opti-MEM was then dissolved in 120 μL of 1 mg / mL PEI (Polysciences #23966-2) and allowed to stand for 5 minutes. The PEI was slowly added to the plasmid and incubated at room temperature for 10 minutes. The plasmid and PEI mixture was then slowly added dropwise while shaking the culture flask. The culture was then cultured for 5 days at 37°C and 8% CO2 in a shaker. Cell viability was measured after 5 days. The culture was harvested and centrifuged at 3300 g for 10 minutes to obtain the supernatant. The supernatant was then removed by high-speed centrifugation. A gravity-flow column (Bio-Rad #7311550) containing MabSelect™ (GE Healthcare Life Science #71-5020-91 AE) was equilibrated with PBS (pH 7.4) and washed with 2–5 column volumes. The supernatant sample was passed through the column, washed with 5–10 column volumes of PBS, and the target protein was eluted with 0.1 M glycine, pH 3.5. The solution was then neutralized with Tris-HCl, pH 8.0, and finally concentrated and exchanged into PBS buffer using an ultrafiltration centrifuge tube (Millipore #UFC901024) to obtain the purified recombinant antibody solution. Finally, the concentration was measured using a NanoDrop™ (Thermo Scientific™ NanoDrop™ One), aliquoted, and stored.

[0037] 1.2 Production of monovalent scFv-his recombinant antibodies The VH and VL sequences of an antibody are connected by a flexible peptide segment (Linker) to create a single polypeptide chain encoding both VH and VL, i.e., a single-chain variable fragment (scFv). By selecting a connecting peptide of appropriate length, such as (G4S)3 (SEQ ID NO: 65) or (G4S)4 (SEQ ID NO: 66), the VH and VL can be correctly folded and assembled into a functional antibody. Depending on the arrangement of VH and VL and the connecting peptide, different scFv structures (VH-Linker-VL or VL-Linker-VH) can be constructed. A single scFv contains an antigen-binding region consisting of a VH and VL pair and is generally capable of binding only one antigen molecule, and is therefore called a monovalent binding molecule.

[0038] In this example, a His tag consisting of six histidine residues was fused to the C-terminus of the scFv to facilitate purification. A polypeptide sequence encoding the scFv and His tag was genetically synthesized and cloned into a mammalian cell expression plasmid vector to obtain a plasmid encoding scFv-His. This plasmid was then transfected into mammalian host cells (e.g., human embryonic kidney cells HEK293). Purified recombinant protein was obtained using standard recombinant protein expression and purification techniques. Specifically, HEK293 cells were expanded in FreeStyle™ F17 Expression Medium (Thermo #A1383504). Prior to transient transfection, the cell concentration was adjusted to 6–8 × 10 5 The cells were cultured in a shaker at 37°C and 8% CO2 for 24 hours, with a cell concentration of 1.2 × 10 6The concentration was 30 cells / mL. A 30 mL culture was prepared. 30 μg of the plasmid was dissolved in 1.5 mL of Opti-MEM reduced serum medium (Thermo #31985088) and sterilized by filtration through a 0.22 μm membrane filter. Further, 1.5 mL of Opti-MEM was dissolved in 120 μL of 1 mg / mL PEI (Polysciences #23966-2) and allowed to stand for 5 minutes. The PEI was slowly added to the plasmid and incubated at room temperature for 10 minutes. The plasmid and PEI mixture was then slowly added dropwise while shaking the culture flask. The culture was then cultured for 5 days in a shaker at 37°C and 8% CO2. Cell viability was measured after 5 days. The culture was harvested and centrifuged at 3300 g for 10 minutes to obtain the supernatant. Impurities were then removed from the supernatant by high-speed centrifugation. A gravity-flow column (Bio-Rad #7311550) containing Ni Sepharose Excel (GE Healthcare Life Science #17-3712-01) was equilibrated with PBS buffer (pH 7.4) and washed with 2–5 column volumes. The supernatant sample was passed through the column and washed with 5–10 column volumes of PBS. Nonspecifically adsorbed impurity proteins were eluted with Buffer A (20 mM imidazole, 150 mM phosphate, pH 8.0), followed by elution of the target protein with Buffer B (500 mM imidazole, 150 mM phosphate, pH 8.0). Finally, the purified recombinant antibody solution was concentrated and exchanged into PBS buffer using an ultrafiltration centrifuge tube (Millipore #UFC901024). Finally, the concentration was measured using a NanoDrop (Thermo Scientific™ NanoDrop™ One), aliquoted, and stored.

[0039] 1.3 Production of bivalent scFv-Fc recombinant antibodies In this example, a human IgG1 constant region Fc sequence (Glu216-Lys447, hinge region, CH2 domain, and CH3 domain) was fused to the C-terminus of the scFv to construct an scFv-Fc recombinant molecule. Fc homodimerization was utilized to generate a bivalent scFv-Fc dimer molecule capable of simultaneously binding two antigen molecules. Furthermore, the "LALA" double mutant (L234A / L235A) or the "LALAPG" triple mutant (L234A / L235A / P329G) was introduced into the Fc to reduce antibody Fcγ receptor binding. The scFv-Fc-encoding polypeptide sequence was genetically synthesized and cloned into a mammalian cell expression plasmid vector to obtain a plasmid encoding the scFv-Fc. Mammalian host cells (e.g., human embryonic kidney cells HEK293) were then transfected with the resulting plasmid. Purified recombinant protein was then obtained using the protein expression and purification method described in Example 1.1.

[0040] 1.4 Protein Purity Analysis by HPLC-SEC Protein samples were analyzed for purity and multimeric form using size-exclusion chromatography (SEC). A TSKgel G3000SWxl analytical column (Tosoh Bioscience #08541, 5 μm, 7.8 mm × 30 cm) was connected to a high-performance liquid chromatograph (HPLC) (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS buffer at room temperature for at least 1 hour. An appropriate amount of protein sample (at least 10 μg) was filtered through a 0.22 μm membrane filter and injected into the system. The HPLC program was set as follows: the sample was passed through the column using PBS buffer at a flow rate of 1.0 mL / min for a maximum time of 20 minutes. The HPLC generated an analytical report reporting the retention times of components of different molecular sizes within the sample.

[0041] Example 2: Recombinant expression of a mouse-human chimeric antibody of the CD3 antibody SP34 SP34 is a mouse-derived anti-human CD3e antibody that can bind to various primate CD3s and activate T cells. The variable region sequences (VH and VL) of SP34 are disclosed in WO2016071004A1. In this application, the amino acid sequence of the VH of SP34 is SEQ ID NO: 42, and its corresponding mouse germline V gene is IGHV10-1. The amino acid sequence of the VL of SP34 is SEQ ID NO: 56, and its corresponding mouse germline V gene is IGLV1. In this example, the full-length heavy chain of the SP34 mouse-human chimeric IgG1 antibody was generated by fusing the VH sequence of SP34 with a human IgG1 antibody heavy chain constant domain sequence (SEQ ID NO: 63) containing the "LALA" double mutant (L234A / L235A). The full-length λ light chain of the SP34 mouse-human chimeric antibody was generated by fusing the amino acid sequence of the VL of SP34 with a human antibody λ light chain constant domain sequence (SEQ ID NO: 62).

[0042] SP34 mouse-human chimeric recombinant antibody PR000260 was prepared according to the method in Example 1.1. Table 2 shows the recombinant expression data of PR000260. [Table 2]

[0043] Example 3: Conversion of the CD3 antibody SP34 murine antibody into a recombinant scFv antibody The VH sequence (SEQ ID NO: 42) and VL sequence (SEQ ID NO: 56) of SP34 were connected by a flexible peptide segment (Linker) to obtain a single polypeptide chain encoding both VH and VL, i.e., a single-chain variable fragment (scFv). Different scFv structures can be constructed by varying the VH and VL arrangement and connecting peptides of different lengths (SEQ ID NO: 65, SEQ ID NO: 66). Furthermore, a His tag consisting of six histidines is fused to the C-terminus of the scFv for easy purification. The connecting peptide shown in SEQ ID NO: 67 can also be used to construct the scFv of the present application.

[0044] In this example, four recombinant scFv antibody molecules (PR000275, PR000276, PR000307, ​​and PR000308) were produced based on the method described in Example 1.2. Table 3 lists the sequence numbers of the four recombinant scFv antibody molecules, and Table 4 lists the recombinant expression data for the four molecules. Figure 1 shows the HPLC-SEC results of the four molecules after one-step purification: (A) PR000275, (B) PR000276, (C) PR000307, ​​and (D) PR000308. As shown in the figure, constructing an scFv using the VH and VL sequences of SP34 failed to produce a stable scFv, regardless of the (VH / VL) configuration or the length of the connecting peptide. [Table 3] [Table 4]

[0045] Example 4: Sequence optimization of SP34 4.1 Humanization of variable region sequences and framework region mutations In this example, humanization of sequences is performed using the "CDR grafting" method. The CDRs of a mouse antibody VH are grafted onto the framework regions of a human antibody VH, and the CDRs of a mouse antibody VL are grafted onto the framework regions of a human antibody VL. The sequences of the framework regions of a human antibody VH or VL may be derived from human germline gene sequences, antibody sequences after V(D)J rearrangement, or consensus sequences of a specific human antibody VH or VL gene family. In this example, framework region sequences provided by human germline gene sequences are used as humanization template sequences. That is, a human germline V gene fragment provides the sequences of framework regions FR1, FR2, and FR3, and a human germline J gene fragment provides the sequence of framework region FR4. The final humanized variable region (VH or VL) sequence is constructed in the following configuration: (human)FR1-(mouse)CDR1-(human)FR2-(mouse)CDR2-(human)FR3-(mouse)CDR3-(human)FR4.

[0046] In this example, the human germline V gene fragment IGHV3-73*01 or the human germline V gene fragment IGHV3-23*01 combined with the human germline J gene fragment IGHJ1*01 was used as a humanization template to provide framework region sequences, and amino acid mutations were introduced at one or more positions 30, 73, 76, 78, 93, or 94 (Chothia numbering system) to obtain multiple different VH variant sequences.

[0047] In this example, the human germline V gene fragment IGLV7-46*02 linked to the human germline J gene fragment IGLJ2*01 or the human germline V gene fragment IGKV1-39*01 linked to the human germline J gene fragment IGKJ4*01 was used as a humanization template to provide framework region sequences, and zero or multiple amino acid mutations were introduced at positions 2, 36, 46, 49, 66, 69, 71, or 87 (Chothia numbering system) to generate multiple different VL variant sequences.

[0048] Table 5 shows the sequence numbers of the CDR and FR region sequences defined by Chothia for the antibody variable regions and optimized variant sequences (FV). [Table 5]

[0049] Figure 2 shows a comparison of VH variant sequences. Figure 3 shows a comparison of VL variant sequences. Figures 4(A) and (B) show the differences at key positions between the VH and VL variant sequences, respectively. As can be seen from Figures 2 to 4, the mutations in the VH of the antibody of the present invention are at one or more of the amino acid residues at positions 30, 73, 76, 78, 93, and 94 of the amino acid sequence set forth in SEQ ID NO: 42. The mutations in the VL are at the amino acid residues at positions 2, 36, 46, 49, 66, 69, 71, and / or 87 of the sequence set forth in SEQ ID NO: 56. For more detailed information on the mutations, see Table 5 for details of the VH3730, VH3731, VH3732, VH3733, VH3734, VH3735, VH3230, VH3231, VH3232, VH3233, VH3234, VH3235, VH3236, VL7460, VL7461, VK1392 and VK1393 sequences.

[0050] 4.2 Recombinant antibody molecules with sequence-optimized variants The VH variant sequences and VL variant sequences obtained in Example 4.1 were paired and combined to construct IgG recombinant antibodies according to the method of Example 1.1, and the "LALA" double mutant or "LALAPG" triple mutant was introduced into the IgG1 heavy chain constant region to reduce Fc effector function. Table 6 shows the sequence of the recombinant antibody molecules that underwent sequence optimization. Table 7 shows the expression data of the recombinant antibodies. The expression yields of the three IgG molecules constructed with VH variant VH3230 were very low, but the expression yields of all the other IgG molecules were reasonable. [Table 6] [Table 7]

[0051] 4.3 Recombinant scFv molecules with sequence-optimized variants The VH and VL variant sequences obtained in Example 4.1 were paired and combined to produce multiple recombinant bivalent scFv antibody molecules according to the method described in Example 1.3. Tables 8 and 9 show the scFv sequence information and protein expression status, respectively. As can be seen from Table 9, PR000510 and PR000627 in particular showed good expression status and yielded stable molecules. Figure 5 shows the results of (A) SDS-PAGE and (B) HPLC-SEC of PR000510, demonstrating good monomer purity and no obvious multimers. [Table 8] [Table 9]

[0052] Example 5: Measurement of the binding ability of CD3 antibodies to CD3-expressing cells by FACS Flow cytometry (FACS) was used to analyze the binding of CD3 antibodies to CD3-expressing cells. The CD3-expressing cells may be CHOK1 cells or HEK293 cells overexpressing human CD3 (host cells CHOK1 (ATCC, CCL-61) or HEK293 (ATCC, CRL-1573) were transfected with both a plasmid encoding the gamma, delta, epsilon, and zeta chains of human CD3 and a plasmid encoding the alpha and beta chains of human TCR to construct a stable cell line expressing the human TCR / CD3 complex structure), CHOK1 or HEK293 cells overexpressing cynomolgus monkey CD3, human pan-T cells (isolated from PBMCs using a human pan-T cell isolation kit (Miltenyi #130-096-535)), or cynomolgus monkey pan-T cells. Specifically, the collected cells were washed twice with PBS containing 2% FBS (FACS buffer), resuspended in FACS buffer, and then plated at 1 × 10 cells per well. 5Cells were dispensed into a 96-well plate and centrifuged at 500 g for 5 minutes. The supernatant was discarded, and 100 μL of pre-gradiently diluted CD3 antibody was added. The cells were incubated at room temperature for 1 hour, washed twice with FACS buffer, and resuspended in the secondary antibody Alexa Fluor 488 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch #109-545-098) diluted in FACS buffer. The cells were incubated at room temperature in the dark for 30 minutes, washed twice with FACS buffer, and resuspended in 200 μL of FACS buffer. Fluorescence signal readings were read using a flow cytometer (BD FACS CANTO II or ACEA NovoCyte). Data were processed and analyzed using FlowJo v10 software (FlowJo, LLC). Data processing and graphing analysis were performed using GraphPad Prism 8 software. Binding curves and EC2 binding curves were calculated using a four-parameter nonlinear fitting method. 50 The parameters were obtained as follows.

[0053] Figure 6 shows the binding ability of the CD3 antibodies obtained in Example 2 to recombinant CHOK1 cells overexpressing human CD3 (Figure 6(A)) and recombinant CHOK1 cells overexpressing cynomolgus monkey CD3 (Figure 6(B)). The results show that the SP34 chimeric antibody PR000260 has high binding ability to both human CD3 and cynomolgus monkey CD3.

[0054] Figure 7 (A)-(G) show the binding ability of the CD3 antibodies (including PR000260 and its variants) obtained in Example 4.2 to human pan-T cells, and also show the calculated fluorescence intensity (MFI) and ratio to the initial antibody PR000260 when the CD3 antibodies bind to human pan-T cells at antibody concentrations of 7.4 or 10 μg / mL. Specifically, after optimizing the SP34 IgG antibody sequence, PR000512, PR000513, PR001849, and PR002837 had binding abilities equivalent to PR000260 (i.e., SP34 chimeric antibody), PR000514 had slightly higher binding ability than PR000260, PR000511, PR001848, PR002469, PR002472, PR002742, PR002833, PR002834, PR002835, PR002836, PR003886, and PR004616 had low T cell binding ability, and PR002467, PR002468, PR002470, PR002471, and PR002743 hardly bound to T cells (no signal was detected at the current antibody concentration). The above results demonstrate that the present invention has yielded several novel antibodies through sequence optimization of CD3 antibody, which have different binding abilities to human T cells and can be used in different application scenarios.

[0055] Figures 8(A) and (B) show the human pan-T cell binding ability of the anti-CD3 scFv-Fc single-chain antibodies obtained in Example 4.3. The fluorescence intensity (MFI) and ratio of the CD3 antibody to human pan-T cells at antibody concentrations of 7.4 and 10 μg / mL were calculated. Specifically, after humanization optimization of the SP34 scFv antibody, PR000624 had the same or slightly higher binding ability as PR000260, PR000510 and PR000627 had the same or slightly lower binding ability as PR000260, and the T cell binding ability of PR001850 was significantly lower than PR000260. These results demonstrate that the present invention has yielded several stable scFv-format single-chain antibodies by sequence optimization of the CD3 antibody, which can bind to human T cells and can be used in applications such as the construction of bispecific antibodies.

[0056] Figure 9 shows the binding ability of several CD3 antibodies obtained in Example 4.2 to cynomolgus monkey pan-T cells. As shown in the figure, the binding ability of each molecule to cynomolgus monkey pan-T cells varies, and there is a positive correlation with the binding ability to human pan-T cells. In other words, molecules that bind strongly to human pan-T cells also have a strong binding ability to cynomolgus monkey pan-T cells, and vice versa.

[0057] Example 6: Measurement of the activation effect of CD3 antibodies on human T cells A gradient dilution of CD3 antibody (e.g., 50, 10, 5, 1, 0.5, or 0.05 μg / mL) was applied to each well of a 96-well cell culture plate in triplicate at 50 μL each, and the plate was coated overnight at 4°C. Human PBMCs (Myotsu Seizo) or human pan-T cells (isolated from PBMCs using a human pan-T cell isolation kit (Miltenyi #130-096-535)) were incubated at a cell density of 7.5 × 10 5 The solution was adjusted to 1 μg / mL, and human CD28 antibody was added to the plate at a concentration of 1 μg / mL. 200 μL of the solution was added to each well of the cell culture plate and the plate was placed in a CO2 incubator. After 72 hours of culture, the supernatant was collected and the IFN-γ content of the supernatant was measured using an IFN-γ ELISA kit (Thermo #88-7316-77). Data analysis and graphing were performed using GraphPad Prism software.

[0058] Figure 10 (A)-(G) show the human T cell activation ability of each CD3 antibody (including the SP34 chimeric antibody) obtained in Example 4.2. At an antibody concentration of 1 μg / mL, PR000511, PR000512, PR000513, and PR000514 activated T cells to produce significantly lower IFN-γ levels than PR000260. At an antibody concentration of 10 μg / mL, PR000512, PR000513, and PR000514 produced slightly lower IFN-γ levels than PR000260 (Figure 10(A)). At antibody concentrations of 0.5 μg / mL and 5 μg / mL, PR001848 produced significantly lower IFN-γ levels than PR000260 (Figure 10(B)). Furthermore, the T cell activation effects of antibodies PR002468, PR002469, PR002471, PR002742, PR002833, PR002834, PR002835, PR002836, PR002837, PR001848, PR003886, and PR004616 at concentrations of 0.5 μg / mL, 5 μg / mL, and 50 μg / mL (FIG. 10 (C)-(G)) were detected, and the results showed that these antibodies were able to activate T cells and induce the production of IFN. The IFN-γ levels were found to be much lower than those of PR000260, no IFN-γ was detected for PR002468 and PR002471, slight IFN-γ levels were detected only at 50μg / mL for PR002469 and PR002835, comparable levels were slightly weaker than those of PR001848 for PR002742 and PR003886, and comparable levels were significantly weaker than those of PR001848 for PR002469 and PR004616. The above results demonstrate that the present invention has yielded several novel antibodies by optimizing the CD3 antibody sequence, which have different human T cell activation abilities and can achieve different cytokine release levels, making them suitable for use in different application scenarios.

[0059] Figure 11 shows the human T cell activation ability of the anti-CD3 scFv-Fc antibodies obtained in Example 4.3. At concentrations of 1 μg / mL and 10 μg / mL, PR000510, PR000623, PR000624, and PR000627 all induced lower IFN-γ levels than PR000260 and higher levels than the isotype control antibody, indicating that these four molecules limit cytokine release by regulating T cell activation levels. These results demonstrate that the present invention has yielded several stable scFv-format single-chain antibodies through sequence optimization of CD3 antibodies. These antibodies have weaker human T cell activation ability and lower cytokine release levels, making them suitable for applications such as the construction of bispecific antibodies.

[0060] Example 7: Bispecific antibodies comprising anti-CD3 scFv antibodies targeting B7H4 B7H4 is a member of the B7 family of transmembrane proteins and is highly expressed in various solid tumor tissues, including breast cancer, ovarian cancer, and endometrial cancer, but is not or only weakly expressed in normal tissues, making it a highly specific tumor-associated target antigen. Constructing a bispecific antibody molecule targeting both B7H4 and CD3 can selectively activate T cells near tumor cells by targeting and binding to B7H4 on the surface of tumor cells, leading to specific killing of tumor cells.

[0061] 7.1 Production of B7H4 antibodies The variable region sequences of the B7H4 antibody were determined in accordance with WO2016040724, and a recombinant IgG antibody PR000014 against B7H4 was constructed according to the method described in Example 1.1. Table 10 shows the sequence information of the B7H4 antibody PR000014. [Table 10]

[0062] 7.2 Production of bispecific antibodies containing anti-CD3 scFv antibodies targeting B7H4 A bispecific antibody molecule PR002883 targeting B7H4×CD3 was constructed from the sequence of B7H4 antibody PR000014 obtained in Example 7.1 and the sequence of CD3 single-chain antibody PR000627 obtained in Example 4.3. It contains three polypeptide chains: a heavy chain containing a CD3 single-chain antibody scFv (SEQ ID NO: 88), a heavy chain containing a B7H4 antibody VH (SEQ ID NO: 86), and a light chain containing a B7H4 antibody VL (SEQ ID NO: 83). Its structure is shown in Figure 16(C). Because this molecule has a unique asymmetric structure, different amino acid mutations were introduced into the constant regions of the two heavy chains to reduce the formation of homologous heavy chain dimers. Furthermore, a "LALAPG" triple mutation (L234A / L235A / P329G) was introduced into the heavy chain constant region to prevent cross-linking caused by Fcγ receptor binding and reduce effector function.

[0063] The recombinant protein of bispecific antibody PR002883 was produced by one-step affinity purification using the method described in Example 1.1 in combination with a plasmid blend (e.g., 1:1:1 or other ratio). Table 11 shows the sequence of bispecific antibody PR002883, and Table 12 shows the expression status of the bispecific antibody. [Table 11] [Table 12]

[0064] Figure 12(A) shows the results of SDS-PAGE analysis of bispecific antibody PR002883 after one-step purification. The main by-products were incompletely assembled molecules with low polymer content, and optimization of the purification step or the transfection ratio of the plasmids could reduce the by-products.

[0065] 7.3 Binding to B7H4-expressing tumor cells In this example, we examine the ability of bispecific antibodies to bind to SK-BR-3 (ATCC, HTB-30) tumor cells expressing human B7H4. Specifically, SK-BR-3 cell suspensions were harvested and the cell density was adjusted to 1 × 10 6 The antibody was adjusted to 1 mL / mL and seeded at 100 μL / well in a V-bottom 96-well plate (Corning #3894). A 3-fold gradient dilution of the test antibody (2x the final concentration) was then added at 100 μL / well. The plate was incubated at 4°C in the dark for 2 hours. Cells were then rinsed twice with 100 μL / well of pre-chilled PBS, centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was discarded. 100 μL / well of fluorescent secondary antibody Alexa Fluor 488 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch #109-545-098) was added and incubated at 4°C in the dark for 1 hour. Cells were then washed twice with 100 μL / well of pre-chilled PBS, centrifuged at 500×g for 5 minutes, and the supernatant was discarded. Finally, 200 μL / well of pre-chilled PBS was added to resuspend the cells. Fluorescence signal values ​​were read using a flow cytometer (BD FACS CANTO II or ACEA NovoCyte), and data were processed and analyzed using FlowJo v10 software (FlowJo, LLC). Data processing and graphing analysis were performed using GraphPad Prism 8 software, and binding curves, EC 50 The parameters were obtained as follows.

[0066] Figure 13(A) shows the binding ability of the monoclonal antibody obtained in Example 7.1 and the bispecific antibody obtained in Example 7.2 to SK-BR-3 cells. Bispecific antibody PR002883 was found to have binding ability equivalent to or greater than that of monoclonal antibody PR000014.

[0067] 7.4 Binding to human T cells The binding ability of bispecific antibody PR002883 to human pan-T cells was detected by the method described in Example 5. As shown in Figure 13 (B), PR002883 can bind to human pan-T cells.

[0068] 7.5 In vitro killing and cytokine release of bispecific antibodies against the B7H4-highly expressing cell line SK-BR-3 To study the in vitro target cell killing ability of the B7H4xCD3 bispecific antibody, we performed an in vitro killing experiment using human PBMCs as effector cells and the B7H4-highly expressing cell line SK-BR-3 (ATCC, HTB-30) as target cells to detect cytokine release. Specifically, 50 μL of RPMI 1640 / 10% FBS medium was added to each well of an E-plate (ACEA Biosciences Inc. #05232368001), and the plate was placed in a 37°C, 5% CO2 incubator for 30 minutes to equilibrate. The E-plate was then placed in an xCELLigence RTCA (ACEA Biosciences) device to confirm normal cell death. SK-BR-3 cells were cultured at a density of 0.4 x 10 in RPMI 1640 / 10% FBS medium. 6 The PBMCs were cultured at a density of 4 × 10 cells / mL in RPMI1640 / 10% FBS medium and seeded onto E-plates at 50 μL cells / well. The E-plates were then subjected to xCELLigence RTCA overnight to detect the cell index. 6The antibody was adjusted to 0.2 nM and seeded onto an E-plate at 50 μL / well. A 5-fold gradient dilution of the test antibody (4x the final concentration) was then added at 50 μL / well. The highest final antibody concentration was 0.2 nM, with seven concentrations of each antibody. The final effector cell to target cell ratio was 10:1. This was repeated twice. Additionally, a blank control was added to the plate, containing SKBR3+PBMC+RPMI 1640 / 10% FBS medium. The E-plate was then incubated at 37°C and 5% CO2 for 24 hours. After incubation, the E-plate was placed in the xCELLigence RTCA instrument to detect the cell index.

[0069] The specific killing of the antibody against the cells is calculated from the detected cell index using the following formula. % cell killing = (1 - detection sample / blank control) x 100%.

[0070] The cell culture supernatant was collected to detect the release of the cytokine IFN-γ. The ELISA detection method was described in accordance with the instructions for the IFN-γ kit (IFN gamma Human Uncoated ELISA Kit, Thermo #88-7316-77).

[0071] As shown in Figure 14 (A) and (B), the bispecific antibody PR002883 can activate T cells to release cytokines (e.g., IFN-γ) and effectively kill tumor cells SK-BR-3. Nearly 100% of tumor cells were killed when the bispecific antibody concentration was 0.01 μg / mL (Figure 14 (A)).

[0072] Example 8: Bispecific antibodies comprising anti-CD3 scFv antibodies targeting ROR1 ROR1 is an inactive tyrosine protein kinase transmembrane protein that is overexpressed in many tumors but is barely expressed in normal tissues. When ROR1 interacts with Wnt5a as a receptor, it transduces the Wnt signaling pathway, contributing to cell proliferation and migration in chronic lymphocytic leukemia and epithelial-mesenchymal transition (EMT) in solid tumors. Its tumor-specific expression makes ROR1 a suitable tumor-associated antigen target for therapeutic drug development. Constructing a bispecific antibody molecule targeting both ROR1 and CD3 can selectively activate T cells in the vicinity of tumor cells by targeting and binding to ROR1 on the surface of tumor cells, resulting in specific tumor cell killing.

[0073] 8.1 Production of ROR1 antibodies The variable region sequences of the ROR1 antibody are based on WO2016094873, and a recombinant IgG antibody PR000374 against ROR1 is constructed according to the method in Example 1.1. Table 13 shows the sequence listing of the ROR1 antibody PR000374. [Table 13]

[0074] 8.2 Production of bispecific antibodies containing anti-CD3 scFv antibodies targeting ROR1 A bispecific antibody molecule PR002885 targeting ROR1 × CD3 was constructed from the sequence of the ROR1 antibody PR000374 obtained in Example 8.1 and the sequence of the CD3 single-chain antibody PR000627 obtained in Example 4.3. It contains three polypeptide chains: a heavy chain containing a CD3 single-chain antibody scFv (SEQ ID NO: 88), a heavy chain containing a ROR1 antibody VH (SEQ ID NO: 87), and a light chain containing a ROR1 antibody VL (SEQ ID NO: 85). Its structure is shown in Figure 16(C). Because the molecule has a unique asymmetric structure, different amino acid mutations were introduced into the constant regions of the two heavy chains to reduce the formation of homologous heavy chain dimers. Furthermore, a "LALAPG" triple mutation (L234A / L235A / P329G) was introduced into the heavy chain constant region to prevent cross-linking caused by Fcγ receptor binding and reduce effector function.

[0075] The recombinant protein of bispecific antibody PR002885 was produced by one-step affinity purification using the method described in Example 1.1 in combination with a plasmid blend (e.g., 1:1:1 or other ratio). Table 14 shows the sequence information of bispecific antibody PR002885, and Table 15 shows the expression status of the bispecific antibody. [Table 14] [Table 15]

[0076] Figure 12 (B) shows the SDS-PAGE analysis of bispecific antibody PR002885 after one-step purification. The main by-products were incompletely assembled molecules with low polymer content, and optimization of the purification step or the transfection ratio of the plasmids could reduce the by-products.

[0077] 8.3 Binding to ROR1-expressing tumor cells In this example, we investigated the ability of bispecific antibodies to bind to tumor cells Panc-1 (ATCC, CRL-1469) expressing human ROR1. Specifically, Panc-1 cell suspensions were harvested and the cell density was adjusted to 1 × 10 6The antibody was adjusted to 1 mL / mL and seeded at 100 μL / well in a V-bottom 96-well plate (Corning #3894). A 3-fold gradient dilution of the test antibody (2x the final concentration) was then added at 100 μL / well. The plate was incubated at 4°C in the dark for 2 hours. Cells were then rinsed twice with 100 μL / well of pre-chilled PBS, centrifuged at 500 g for 5 minutes, and the supernatant was discarded. 100 μL / well of fluorescent secondary antibody Alexa Fluor 488 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch #109-545-098) was added and incubated at 4°C in the dark for 1 hour. Cells were then washed twice with 100 μL / well of pre-chilled PBS, centrifuged at 500 g for 5 minutes, and the supernatant was discarded. Finally, 200 μL / well of pre-chilled PBS was added to resuspend the cells. Fluorescence signal values ​​were read using a flow cytometer (BD FACS CANTO II or ACEA NovoCyte), and data were processed and analyzed using FlowJo v10 software (FlowJo, LLC). Data processing and graphing analysis were performed using GraphPad Prism 8 software, and binding curves, EC 50 The parameters were obtained as follows.

[0078] Figure 15(A) shows the binding ability of the monoclonal antibody obtained in Example 8.1 and the bispecific antibody obtained in Example 8.2 to Panc-1 cells. Both the bispecific antibody PR002885 and the monoclonal antibody PR000374 can bind to Panc-1 cells.

[0079] 8.4 Binding to human T cells The binding ability of bispecific antibody PR002885 to human pan-T cells was detected by the method described in Example 5. As shown in Figure 15(B), PR002885 can bind to human pan-T cells.

Claims

1. 1. A CD3-targeting antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), the amino acid sequence of the VH is as set forth in SEQ ID NO: 55 and the amino acid sequence of the VL is as set forth in SEQ ID NO: 58; or the amino acid sequence of the VH is as set forth in SEQ ID NO:45 and the amino acid sequence of the VL is as set forth in SEQ ID NO:58; or the amino acid sequence of the VH is as set forth in SEQ ID NO:43 and the amino acid sequence of the VL is as set forth in SEQ ID NO:58; or the amino acid sequence of the VH is as set forth in SEQ ID NO:50 and the amino acid sequence of the VL is as set forth in SEQ ID NO:58; or the amino acid sequence of the VH is as set forth in SEQ ID NO:47 and the amino acid sequence of the VL is as set forth in SEQ ID NO:58; or the amino acid sequence of the VH is as set forth in SEQ ID NO:48 and the amino acid sequence of the VL is as set forth in SEQ ID NO:58; or the amino acid sequence of the VH is as set forth in SEQ ID NO:49 and the amino acid sequence of the VL is as set forth in SEQ ID NO:58; or The amino acid sequence of the VH is as shown in SEQ ID NO: 53, and the amino acid sequence of the VL is as shown in SEQ ID NO:

58. An antibody that targets CD3, characterized in that:

2. An antibody targeting CD3, characterized in that the antibody comprises a single chain antibody (scFv), and the amino acid sequence of the scFv is as set forth in SEQ ID NO: 73 or SEQ ID NO:

78.

3. The antibody of claim 2, characterized in that the antibody comprises an Fc connected to the scFv by a hinge region (Hinge).

4. the antibody further comprises a human constant region; the human constant region comprises a human light chain constant region and a human heavy chain constant region; The antibody of claim 1, wherein the human heavy chain constant region is hIgG1, hIgG2, hIgG3, hIgG4, or a mutant thereof.

5. The antibody according to claim 4, characterized in that the human heavy chain constant region is the heavy chain constant region shown in SEQ ID NO: 63 or SEQ ID NO: 64, and the human light chain constant region is the human κ light chain constant region shown in SEQ ID NO: 61 or the human λ light chain constant region shown in SEQ ID NO:

62.

6. A bispecific antibody comprising a first protein domain comprising an antibody that targets CD3 according to any one of claims 1 to 5, and a second protein domain.

7. 7. The bispecific antibody of claim 6, wherein the bispecific antibody comprises three chains: (1) the first protein domain VL1-Linker-VH1-Hinge-CH2-CH3 (knob) or VH1-Linker-VL1-Hinge-CH2-CH3 (knob), (2) the second protein domain VH2-CH1-Hinge-CH2-CH3 (hole), and (3) the second protein domain VL2-CL, wherein the second protein domain is an antibody that targets a target other than CD3.

8. The second protein domain is an antibody targeting B7H4 or an antibody targeting ROR1, and the Linker is ４ S) ｎ and n is 1 to 20.

9. The bispecific antibody according to claim 7 or 8, characterized in that the amino acid sequence of the Linker is as set forth in SEQ ID NO: 65, SEQ ID NO: 66 or SEQ ID NO:

67.

10. A bispecific antibody comprising VL1-Linker-VH1-Hinge-CH2-CH3 (knob) shown in SEQ ID NO: 88, VH2-CH1-Hinge-CH2-CH3 (hole) shown in SEQ ID NO: 86, and VL2-CL shown in SEQ ID NO: 83, or VL1-Linker-VH1-Hinge-CH2-CH3 (knob) shown in SEQ ID NO: 88, VH2-CH1-Hinge-CH2-CH3 (hole) shown in SEQ ID NO: 87, and VL2-CL shown in SEQ ID NO:

85.

11. An isolated nucleic acid encoding the CD3-targeting antibody of any one of claims 1 to 5 or the bispecific antibody of any one of claims 6 to 10.

12. An expression vector comprising the isolated nucleic acid of claim 11.

13. A genetically modified cell transfected with the expression vector of claim 12.

14. 14. A pharmaceutical composition comprising an antibody targeting CD3 according to any one of claims 1 to 5, a bispecific antibody according to any one of claims 6 to 10, a genetically modified cell according to claim 13, and a pharmaceutically acceptable carrier.

15. 15. Use of an antibody targeting CD3 according to any one of claims 1 to 5, a bispecific antibody according to any one of claims 6 to 10, an isolated nucleic acid according to claim 11, an expression vector according to claim 12, a genetically modified cell according to claim 13, or a pharmaceutical composition according to claim 14 for the manufacture of a medicament for treating a tumor.

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