CD3-targeting antibodies, bispecific antibodies, and their use

Abstract

One objective is to provide a CD3 antibody that can bind to primate CD3, possesses appropriate CD3-binding ability, and has a stable single-chain scFv structure. [Solution] The present invention discloses an antibody targeting CD3, a bispecific antibody, and its use. The CD3-targeting antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL is the amino acid sequence shown in SEQ ID NO: 56 or a variant thereof, and the VH is a mutation in the amino acid sequence shown in SEQ ID NO: 42, wherein the mutation is selected from one or more amino acid residues at 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 contains the CD3-targeting antibody. The CD3-targeting antibody of the present invention reduces the toxicity caused by cytokine release syndrome, and the bispecific antibody produced using it is stable, has T cell binding ability, and is easier to produce.

Description

[Technical Field]

[0001] This application claims priority from Chinese Patent Application No. 2019109413286, filed on September 30, 2019, and the entire text of the said Chinese Patent Application is cited.

[0002] This invention relates to the field of biopharmaceuticals, and more particularly to antibodies targeting CD3, bispecific antibodies, and their use. [Background technology]

[0003] T cells are a crucial cell class involved in adaptive immune responses, and they recognize antigens via the T cell receptor (TCR). TCRs cannot directly recognize epitopes on the antigen surface; instead, they specifically recognize antigen peptide-MHC molecular complexes (pMHCs) presented on the surface of antigen-presenting cells (APCs) or target cells. The specificity of the T cell response is mediated by the recognition of pMHCs by the TCR-CD3 molecular complex. TCRs are heterodimers composed of two distinct transmembrane polypeptide chains, each with four peptide chain types: α, β, γ, and δ. Depending on the combination of peptide chains, TCRs are classified as TCRαβ or TCRγδ. CD3 has different transmembrane polypeptide chains: γ, δ, ε, and ζ. Homodimers or heterodimers formed by the interaction of these peptide chains become part of the TCR-CD3 complex. For example, the TCR-CD3 complex includes TCRαβ dimer, CD3γε dimer, CD3δε dimer, and CD3ζζ dimer. Because the cytoplasmic region of the TCR peptide chain is very short, it is generally believed that the activation signal generated by TCR antigen recognition is transmitted to T cells by the CD3 peptide chain.

[0004] Due to the crucial role of CD3 in initiating the immune response, monoclonal antibodies targeting TCR-CD3 signaling, particularly CD3, are considered effective drugs that can modulate immune processes and be used to treat inflammatory or autoimmune diseases. In fact, the anti-CD3 antibody, orthoclone OKT3, was the first therapeutic antibody approved. OKT3 was initially approved by the US FDA in 1985 as a treatment for acute rejection after organ transplantation. While the immunosuppressive capacity of repeated doses of OKT3 was recognized as an effective treatment for rejection after kidney transplantation, 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, due to severe cytokine storms, immunogenicity caused by mouse antibodies, and other problems, OKT3 was withdrawn from the market in 2010.

[0005] Another problem with CD3 antibodies is that many CD3 antibodies are species-specific. For example, OKT3 reacts with chimpanzee CD3 but not with CD3 homologs of other primates such as rhesus macaques, or mouse CD3 homologs. The species specificity of CD3 monoclonal antibodies is a major obstacle to their development as antibody drugs to treat human diseases. Before a candidate drug can be used in clinical trials involving humans, rigorous preclinical validation must be performed. The purpose of preclinical trials is to confirm that the candidate drug has the required activity and, most importantly, its safety. In preclinical safety studies, the candidate drug is 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, especially cynomolgus macaques, are suitable species for safety evaluation studies in this field. However, CD3 antibodies that do not exhibit primate species-specific cross-reactivity cannot provide effective preclinical safety evaluation data. Among known antibodies that bind to human CD3, SP34 is one of the few antibodies that can bind to multiple primate CD3 species (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 effective for certain diseases, in recent years they 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 in the clinical or preclinical stages worldwide. CD3 bispecific antibody BsTCEs not only exhibit potent effects similar to CAR-T cell therapy, but can also be produced and commercialized like conventional monoclonal antibodies. Of the bispecific antibody drugs approved worldwide to date, catumaxomab (approved by the European EMA in 2009 and withdrawn in the US in 2013) and blinatumomab (approved by the US FDA in 2014) are both BsTCEs. CD3 antibodies are a crucial component in constructing BsTCEs. BsTCE bispecific antibodies can bind to two targets simultaneously: one side can recognize tumor-associated antigens (TAAs) on the surface of tumor cells, and the other side can bind to CD3 molecules on T cells. In the presence of tumor cells, after binding to the tumor cell surface, BsTCE bispecific antibodies can recruit and activate T cells near the tumor cells, thereby killing the tumor cells. When designing and constructing various structures of BsTCE bispecific antibodies, the selection and optimization of the CD3 antibody is particularly important. Firstly, species specificity of the CD3 monoclonal antibody, especially cross-reactivity with monkeys, is very important. Secondly, the affinity of the CD3 antibody to the CD3 complex is also important; a CD3 antibody with too high an affinity may restrict the antibody to sites such as the spleen, making contact with the tumor difficult. Also, if the affinity is too high, T cells may be excessively stimulated, resulting in high levels of cytokine release. Thirdly, the CD3 antibody binding titer has a significant impact, and it has been found that polyvalent CD3 bispecific antibodies activate T cells without binding to tumor-associated antigens, causing side effects. Therefore, most CD3 bispecific antibodies currently under development are monovalent CD3.

[0007] Structural design of BsTCE bispecific antibodies, as well as CD3 antibodies, is a crucial consideration. BsTCE bispecific antibody structures vary and can be broadly categorized into two types: IgG-like structures containing Fc and antibody fragment structures without Fc. For example, blinatumomab is a single polypeptide chain structure in which two single-chain variable region fragments (scFv) are linked in series. However, such structures have a very short half-life and require continuous intravenous injection, making them difficult to use. Therefore, many BsTCE bispecific antibodies employ structures containing Fc to improve molecular stability and pharmacokinetic properties. However, since BsTCE generally requires the CD3 binding domain to be in a monovalent form, structures containing Fc are often asymmetric. Such asymmetric structures containing Fc present many technical challenges to overcome, such as the problem of heavy chain homodimerization in asymmetric structures, light chain mismatch, and molecular crosslinking caused by the Fcγ receptor and effector functions such as ADCC or CDC. When constructing a BsTCE bispecific antibody from anti-TAA IgG antibody and anti-CD3 IgG antibody (Figure 16 (A)), different asymmetric structures can be selected. One such structure is an IgG-like structure retaining two independent Fab domains. This structure contains four different polypeptide chains (two different heavy chains and two different light chains, as shown in Figure 16 (B)) and has a molecular weight close to that of conventional monoclonal antibodies. However, because such a structure contains multiple different polypeptide chains, it can result in various combinations of byproducts, which poses a significant challenge to the antibody expression purification and production process. By changing the Fab of the CD3 antibody to an scFv structure, the "quadruple-chained" structure becomes a "triple-chained" structure (as shown in Figure 16 (C)), reducing the number of byproduct combinations and thus lowering the complexity of its production. The inventors attempted to construct a BsTCE bispecific antibody by converting SP34 mouse antibody IgG into scFv, but were unable to obtain a stable scFv regardless of the (VH / VL) configuration or the length of the connecting peptide. Therefore, a stable anti-CD3 monoclonal antibody, particularly a stable scFv structure, is desired in this field.

[0008] As can be seen from the above, in this field, there is a demand for CD3 antibodies that can bind to primate CD3, have appropriate CD3 binding ability, and possess a stable single-chain scFv structure. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] To address the technical challenges in this field, namely the lack of asymmetric structures for low-antigenic, effective, and safe anti-CD3 antibodies and bispecific antibodies, the present invention provides a CD3-targeting antibody, a bispecific antibody, and its use. [Means for solving the problem]

[0010] The first aspect of the present invention provides a technical solution to the above technical problems. The CD3-targeting antibody comprises a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL is the amino acid sequence shown in SEQ ID NO: 56 or a variant thereof, and the VH is a mutation in the amino acid sequence shown in SEQ ID NO: 42, wherein the mutation is selected from one or more amino acid residues at positions 30, 73, 76, 78, 93, and 94 (the Chothia numbering system position numbers are used for the said sites). The mutation is an 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 reduces cytokine release levels, thus reducing toxicity caused by cytokine release syndrome.

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

[0012] In certain preferred embodiments, the mutations occurring 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, [[ID=]) [[ID=))(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] In the antibodies of the present invention having the above-described mutations in the VH of the antibody, there are further mutations in the VL having the amino acid sequence shown in SEQ ID NO: 56 or in the VH having 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 the amino acid sequences that maintain or improve the function of the antibody are also included in the protection scope of the present invention.

[0014] In certain preferred embodiments, the amino acid sequence of the VH is as shown in any of the sequences of SEQ ID NOs: 43 to 55, and / or the amino acid sequence of the VL is as shown in any of the sequences of SEQ ID NOs: 57 to 60.

[0015] In certain preferred embodiments, [[ID=4)) the amino acid sequence of the VH is as shown in SEQ ID NO: 44, the amino acid sequence of the VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of the VH is as shown in SEQ ID NO: 51, and the amino acid sequence of the VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of the VH is as shown in SEQ ID NO: 44, and the amino acid sequence of the VL is as shown in SEQ ID NO: 60, or, The amino acid sequence of the VH is as shown in SEQ ID NO: 51, and the amino acid sequence of the VL is as shown in SEQ ID NO: 60, or, The amino acid sequence of the VH is as shown in SEQ ID NO: 45, and the amino acid sequence of the VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of the VH is as shown in SEQ ID NO: 52, and the amino acid sequence of the VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of the VH is as shown in SEQ ID NO: 43, and the amino acid sequence of the VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of the VH is as shown in SEQ ID NO: 43, and the amino acid sequence of the VL is as shown in SEQ ID NO: 60, or, The amino acid sequence of the VH is as shown in SEQ ID NO: 50, and the amino acid sequence of the VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of the VH is as shown in SEQ ID NO: 47, and the amino acid sequence of the VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of the VH is as shown in SEQ ID NO: 48, and the amino acid sequence of the VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of the VH is as shown in SEQ ID NO: 49, and the amino acid sequence of the VL is as shown 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, or, The amino acid sequence of the VH is as shown in SEQ ID NO: 54, and the amino acid sequence of the VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of VH is as shown in SEQ ID NO: 43, and the amino acid sequence of VL is as shown in SEQ ID NO: 57, or, The amino acid sequence of VH is as shown in SEQ ID NO: 44, and the amino acid sequence of VL is as shown in SEQ ID NO: 57, or, The amino acid sequence of VH is as shown in SEQ ID NO: 43, and the amino acid sequence of VL is as shown in SEQ ID NO: 59, or, The amino acid sequence of VH is as shown in SEQ ID NO: 44, and the amino acid sequence of VL is as shown in SEQ ID NO: 59, or, The amino acid sequence of VH is as shown in SEQ ID NO: 51, and the amino acid sequence of VL is as shown in SEQ ID NO: 57, or, The amino acid sequence of VH is as shown in SEQ ID NO: 55, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of VH is shown in SEQ ID NO: 46, and the amino acid sequence of VL is shown in SEQ ID NO: 58.

[0016] In certain preferred embodiments, the antibody comprises a single-chain variable region fragment (scFv) which is VL-Linker-VH or VH-Linker-VL. Preferably, the Linker (i.e., the linking peptide) is (GGGGS) n [Abbreviation (G4S)] n The antibody is either ] or a variant thereof, where 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, SEQ ID NO: 67. More preferably, the amino acid sequence of scFv is as shown 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 crystallizable fragment (Fc), the Fc being 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, wherein the human light chain constant region is preferably 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. More preferably, the human heavy chain constant region is hIgG1, hIgG2, hIgG3, hIgG4 or a variant thereof, preferably the heavy chain constant region shown in SEQ ID NO: 63 or SEQ ID NO: 64.

[0018] The following is a technical solution according to a second aspect of the present invention to solve the above technical problems: A bispecific antibody is provided. The bispecific antibody of the present invention has a triple-chain structure, and the complexity of its production is reduced because the number of by-product combinations is reduced. On the other hand, its development is not just a matter of slightly modifying conventional antibodies. As described in the background art, the inventors attempted to construct a BsTCE bispecific antibody by changing the SP34 mouse antibody IgG to scFv, but a stable scFv could not be obtained regardless of the (VH / VL) configuration or the length of the connecting peptide. After repeated mutation design and verification by the inventors, it was discovered that only some mutations could stably maintain the structure of scFv. 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 as described in the first aspect of the present invention, and preferably the bispecific antibody comprises three chains: (1) VL1-Linker-VH1-Hinge-CH2-CH3(knob) or VH1-Linker-VL1-Hinge-CH2-CH3(knob) of the first protein domain, (2) VH2-CH1-Hinge-CH2-CH3(hole) of the second protein domain, and (3) VL2-CL of the second protein domain, 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) nPreferably, n is a natural number other than 0, preferably between 1 and 20, and more preferably the amino acid sequence shown in SEQ ID NO: 65, SEQ ID NO: 66, and 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 instability of single-chain antibody arms targeting CD3, is stable, and has T cell binding ability. Bispecific antibodies containing only three chains are easy to manufacture and the difficulty of production is reduced.

[0019] The following is a technical solution according to a third aspect of the present invention for solving the above technical problems: A separated nucleic acid is provided that encodes a CD3-targeting antibody as described in the first aspect of the present invention or a bispecific antibody as described in the second aspect of the present invention.

[0020] The following is a technical solution according to a fourth aspect of the present invention for solving the above technical problems. An expression vector containing the isolated nucleic acid described in the third aspect of the present invention is provided, preferably the expression vector being selected from a retroviral vector, a lentiviral vector, an adenovirus vector, and an adeno-associated virus vector.

[0021] The following is a technical solution according to a fifth aspect of the present invention for solving the above technical problems: A gene-modified cell transfected with the expression vector described in the fourth aspect of the present invention is provided, preferably the gene-modified cell is a eukaryotic cell.

[0022] The sixth aspect of the present invention provides a technical solution to the above technical problems: a pharmaceutical composition comprising a CD3-targeting antibody as described in the first aspect of the present invention, a bispecific antibody as described in the second aspect of the present invention, gene-modified cells as described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier, preferably the pharmaceutical composition further comprising an immune checkpoint antibody.

[0023] The following is a technical solution according to a seventh aspect of the present invention to solve the above technical problems: the use of a CD3-targeting antibody 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 gene-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 producing a drug to treat tumors.

[0024] Furthermore, the eighth aspect of the present invention provides a technical solution to the above technical problems: a combination of kits comprising Kit A and Kit B, wherein Kit A comprises a CD3-targeting antibody as described in the first aspect of the present invention, a bispecific antibody as described in the second aspect, a gene-modified cell as described in the fifth aspect, or a pharmaceutical composition as described in the sixth aspect; Kit B comprises another antibody, a bispecific antibody, a gene-modified cell, or a pharmaceutical composition, wherein the other antibody, bispecific antibody, gene-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; Kit A may be used first, then Kit B, or Kit B may be used first, then Kit A. The drug in Kit A exists in an injectable form, for example, as an injection, and the drug in Kit B exists in an injectable form, for example, as an injection, or in a swallowable form, for example, as a tablet or pill.

[0025] A combination of the CD3-targeting antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect, the gene-modified cells described in the fifth aspect, the pharmaceutical composition described in the sixth aspect, or the kit described in the eighth aspect can be administered to a patient for the treatment of a tumor.

[0026] As long as it does not contradict common sense in this field, any preferred combination of the above preferred conditions can be used to obtain any preferred embodiment of the present invention.

[0027] The reagents and raw materials used in this invention may all be commercially available products.

[0028] The beneficial effects of this invention are as follows: 1. The monoclonal antibody of the present invention has altered binding ability to T cells and reduces cytokine release levels, thus reducing toxicity caused by cytokine release syndrome. 2. The bispecific antibodies produced using this method overcome the instability drawback of single-chain antibody arms targeting CD3, and are stable and possess T-cell binding ability. 3. Bispecific antibodies containing only three chains are easier to manufacture, reducing the difficulty of production. [Brief explanation of the drawing]

[0029] [Figure 1] Figure 1 shows the HPLC-SEC results after one-step purification of CD3 single-chain antibodies, where (A) is PR000275, (B) is PR000276, (C) is PR000307, ​​and (D) is PR000308. [Figure 2] Figure 2 shows a comparison of SP34 VH humanized variant sequences. [Figure 3] Figure 3 shows a comparison of SP34 VL humanized variant sequences. [Figure 4] Figure 4 shows the differences in key regions of different VH / VL variant sequences, where (A) is the VH variant sequence and (B) is the VL variant sequence. [Figure 5]Figure 5 shows (A) SDS-PAGE results and (B) HPLC-SEC results after one-step purification of the CD3 single-chain antibody PR000510. [Figure 6] Figure 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] Figure 7 shows the binding ability of CD3 antibodies to human T cells, including binding curves and MFI relative intensity (the fluorescence intensity MFI at which a specific concentration of antibody binds to human T cells, and the ratio to the initial antibody PR000260 (SP34)) or maximum MFI value. (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, and (C) shows the binding of PR002467, PR002468, PR002469, PR002470, PR002471, PR002472, (D) shows the binding of PR001848 and PR000260 to human T cells, (E) shows the binding of PR002833, PR002834, PR002835, PR002836, PR002837, PR002742, PR001848, PR002469 and PR000260 to human T cells, (F) shows the binding of PR003886, PR001848 and PR002742 to human T cells, and (G) shows the 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 MFI relative intensities (the fluorescence intensity MFI at which a specific concentration of antibody binds to human T cells, and its 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] Figure 9 shows the binding ability of the CD3 antibody to cynomolgus monkey T cells. [Figure 10-1] Figure 10 shows the ability of CD3 antibodies to activate human T cells and generate the cytokine IFN-γ, with (A) T cell activation by PR000511, PR000512, PR000513, PR000514 and PR000260, (B) T cell activation by PR001848, PR001849 and PR000260, and (C) T cell activation by PR002468, PR002469, PR002471 and PR001848. (D) represents T cell activation by PR002742, PR001848, and PR000260; (E) represents T cell activation by PR002833, PR002834, PR002835, PR002836, PR002837, and PR000260; (F) represents T cell activation by PR003886, PR001848, and PR002742; and (G) represents 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 and generate the cytokine IFN-γ. [Figure 12] Figure 12 shows the SDS-PAGE results of samples after one-step purification of bispecific antibodies (A)PR002883 and (B)PR002885. [Figure 13]Figure 13 shows the binding ability of monoclonal antibodies and bispecific antibodies to (A) SK-BR-3 cells and (B) human T cells. [Figure 14] Figure 14 shows the in vitro target cell killing ability mediated by the bispecific antibody PR002883, where (A) is SK-BR-3 cell killing and (B) is IFN-γ release level. [Figure 15] Figure 15 shows the binding ability of monoclonal antibodies and bispecific antibodies to (A) Panc-1 cells and (B) human T cells. [Figure 16] Figure 16 shows monoclonal or bispecific antibody structures, where (A) is an IgG structure, (B) is an asymmetric "quadruple-chain" structure, and (C) is an asymmetric "triple-chain" structure containing a single-chain antibody. [Modes for carrying out the invention]

[0030] The present invention will be further explained below with reference to examples, and the present invention is not limited to the scope of these examples. Experimental methods in the following examples that do not specify the conditions in detail will be carried out using conventional methods and conditions, or selected based on the product's instruction manual.

[0031] In this application, the term "antibody" generally refers to a protein containing a portion that binds to an antigen, and optionally, a scaffold or skeletal portion that may employ conformations to promote antibody-antigen binding in the antigen-binding portion. Typically, it may include an antibody light chain variable region (VL), an antibody heavy chain variable region (VH), or both. The VH and VL regions may further be divided into hypervariable regions called complementarity-determining regions (CDRs) scattered within a more conserved region called a framework region (FR). Each of the VH and VL may consist of three CDR and four FR regions arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. Examples of antibodies include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragment, 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] In this application, the term "variable" generally refers to the significant change in a specific portion of the sequence of an antibody's variable domain, contributing to the binding and specificity of various antibodies to a particular antigen. However, variability is not evenly distributed throughout the entire variable region of an antibody. 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 portion of the variable domain is called the framework (FR). The natural heavy and light chain variable domains each contain four FR regions, mostly forming a β-sheet structure connected by three CDRs to form loops, although in some cases they are formed as part of a β-sheet structure. In each chain, the CDRs are tightly bound by FR regions and, together with CDRs from other chains, form the antigen-binding site of the antibody. The constant region does not directly participate in antibody-antigen binding but exhibits various effector functions, such as participating in antibody-dependent cell-mediated cytotoxicity. In this field, the CDR of antibodies can be defined using various methods, such as the Kabat definition method 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 method based on the position of the structural loop region (see Al-Lazikani, et.al, JMol Biol 273:927-48, 1997). In this application, a combined definition method combining the Kabat and Chothia definitions is adopted to determine the amino acid residues of the variable domain sequence and the full-length antibody sequence (Table 1). [Table 1]

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

[0034] Effector functions mediated by the Fc domain of antibodies such as ADCC and CDC also have very important biological functions, and the ADCC or CDC function differs depending on the IgG subtype; for example, IgG1 and IgG3 have strong ADCC and CDC effects, while IgG2 and IgG4 have weaker effects. Furthermore, the intrinsic effector function of Fc can be regulated by altering the binding ability of Fc to Fc receptors through amino acid mutations or modifications. For example, the "LALA" double mutant of IgG1 (L234A / L235A) can significantly reduce its affinity for FcγRIIIA (CD16A) and thus reduce its ADCC effect. Also, the P329G mutation can significantly reduce 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 this invention, in order to reduce the binding of CD3 antibodies to the Fcγ receptor, the Fc of these CD3 antibodies is modified to either the "LALA" double mutant (L234A / L235A) or the "LALAPG" triple mutant (L234A / L235A / P329G).

[0035] Example 1: Production and characterization analysis of recombinant antibodies 1.1 Production of recombinant IgG antibodies After obtaining sequences encoding the variable light and heavy chain domains of an antibody molecule, recombinant antibody molecules can be obtained by fusion expression of the corresponding light and heavy chain constant domain sequences of a human antibody using conventional recombinant DNA technology. In this example, the antibody heavy chain variable domain sequence (VH) is genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human IgG1 antibody heavy chain constant domain sequence to generate a full-length heavy chain that generates an IgG1 antibody. Furthermore, the binding of the antibody to the Fcγ receptor is reduced by introducing either the "LALA" double mutant (L234A / L235A) (SEQ ID NO: 63) or the "LALAPG" triple mutant (L234A / L235A / P329G) (SEQ ID NO: 64) into the IgG1 heavy chain constant region. The antibody light chain variable domain sequence (VL) is genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody κ light chain constant domain sequence (SEQ ID NO: 61) to generate an antibody, thereby encoding a full-length λ light chain, or the VL is genetically synthesized and cloned into a mammalian cell expression plasmid vector encoding the human antibody λ light chain constant domain sequence (SEQ ID NO: 62) to generate an antibody.

[0036] Mammalian host cells (e.g., human embryonic kidney cells HEK293) can be transfected with both plasmids encoding the antibody heavy chain and plasmids encoding the antibody light chain. Purified recombinant antibodies with correctly paired light and heavy chains can then be obtained using standard recombinant protein expression and purification techniques. Specifically, HEK293 cells were cultured in FreeStyle® F17 Expression Medium (Thermo#A1383504). Before initiating transient transfection, the cell concentration was increased to 6–8 × 10⁶. 5 The solution was adjusted to cells / mL and cultured in a shaker at 37°C and 8% CO2 for 24 hours, resulting in a cell concentration of 1.2 × 10⁶. 6The cell count was 4 / mL. 30 mL of cultured cells was prepared. The plasmid encoding the antibody heavy chain and the plasmid encoding the antibody light chain were mixed in a ratio of 2:3, and a total of 30 μg of plasmid was dissolved in 1.5 mL of Opti-MEM low-serum medium (Thermo#31985088). The mixture was filtered through a 0.22 μm membrane filter to sterilize it. 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-PEI mixture was slowly added dropwise while shaking the culture flask, and the culture was incubated at 37°C and 8% CO2 in a shaker for 5 days. Cell viability was measured after 5 days. The culture was harvested, centrifuged at 3300 g for 10 minutes to obtain the supernatant, and then impurities were removed from the supernatant 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 a volume equivalent to 2-5 columns. The supernatant sample was passed through the column, washed with a volume equivalent to 5-10 columns of PBS, and the target protein was eluted with 0.1 M glycine at pH 3.5. The solution was then neutralized with Tris-HCl at pH 8.0, and finally concentrated in an ultrafiltration centrifuge tube (Millipore#UFC901024) and converted to PBS buffer to obtain a purified recombinant antibody solution. Finally, the concentration was measured using NanoDrop (Thermo Scientific® NanoDrop® One), aliquoted, and stored.

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

[0038] In this example, a His tag consisting of six histidines is fused to the C-terminus of scFv for easier purification. By genetically synthesizing the polypeptide sequences encoding scFv and the His tag and cloning them into a mammalian cell expression plasmid vector, a plasmid encoding scFv-his is obtained. Mammalian host cells (e.g., human embryonic kidney cells HEK293) are transfected, and purified recombinant protein can be obtained using standard recombinant protein expression and purification techniques. Specifically, HEK293 cells were cultured in FreeStyle® F17 Expression Medium (Thermo#A1383504). Before initiating transient transfection, the cell concentration was increased to 6-8 × 10⁶. 5 The solution was adjusted to cells / mL and cultured in a shaker at 37°C and 8% CO2 for 24 hours, resulting in a cell concentration of 1.2 × 10⁶. 6The cell count was 4 / mL. 30 mL of cultured cells was prepared. 30 μg of the plasmid was dissolved in 1.5 mL of Opti-MEM low-serum medium (Thermo#31985088), filtered through a 0.22 μm membrane filter, and sterilized. 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-PEI mixture was slowly added dropwise while shaking the culture flask, and the culture was incubated at 37°C and 8% CO2 in a shaker for 5 days. Cell viability was measured after 5 days. The culture was harvested, centrifuged at 3300 g for 10 minutes to obtain the supernatant, and then impurities were 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 a volume equivalent to 2-5 columns. The supernatant sample was passed through the column, and the column was washed with a volume equivalent to 5-10 columns of PBS. First, nonspecifically adsorbed impurity proteins were eluted with buffer A (containing 20 mM imidazole and 150 mM phosphate, pH 8.0), then the target protein was eluted with buffer B (containing 500 mM imidazole and 150 mM phosphate, pH 8.0), and finally the solution was concentrated in an ultrafiltration centrifuge tube (Millipore#UFC901024) and replaced with PBS buffer to obtain a purified recombinant antibody solution. Finally, the concentration was measured using NanoDrop (Thermo Scientific® NanoDrop® One), dispensed, and stored.

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

[0040] 1.4 Protein purity analysis by HPLC-SEC Size exclusion chromatography (SEC) was used to analyze the purity and polymer morphology of protein samples. An analytical column, TSKgel G3000SWxl (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, and the HPLC program was set as follows. The sample was passed through the column at a flow rate of 1.0 mL / min using PBS buffer for a maximum time of 20 minutes. HPLC generated an analytical report reporting the retention times of components of different molecular sizes within the sample.

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

[0042] The SP34 mouse-human chimeric recombinant antibody PR000260 was prepared using the method described in Example 1.1. Table 2 shows the recombinant expression data for PR000260. [Table 2]

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

[0044] In this example, four recombinant scFv antibody molecules (PR000275, PR000276, PR000307, ​​PR000308) were prepared based on the method of Example 1.2. Table 3 shows the sequence numbers of the four recombinant scFv antibody molecules, and Table 4 shows the recombinant expression data for the four molecules. Figure 1 shows the HPLC-SEC results after one-step purification of the four molecules, with (A) being PR000275, (B) being PR000276, (C) being PR000307, ​​and (D) being PR000308. As shown, when constructing scFv using the VH and VL sequences of SP34, a stable scFv cannot be obtained regardless of the (VH / VL) configuration or the length of the connecting peptide. [Table 3] [Table 4]

[0045] Example 4: Array optimization of SP34 4.1 Humanization of Variable Region Sequences and Framework Region Variation In this example, humanization of the sequence is performed using the "CDR grafting" method. The CDR of mouse antibody VH is grafted into the framework region of human antibody VH, and the CDR of mouse antibody VL is grafted into the framework region of human antibody VL. The sequence of the framework region of human antibody VH or VL may be derived from a human germline gene sequence, a rearranged antibody sequence of V(D)J, or a consensus sequence of a specific VH or VL gene family of human antibodies. In this example, the framework region sequence provided from the human germline gene sequence is used as the humanization template sequence; that is, the human germline V gene fragment provides the sequences of framework regions FR1, FR2, and FR3, and the human germline J gene fragment provides the sequence of framework region FR4. Finally, the humanized variable region (VH or VL) sequence is constructed in the configuration of (human)FR1-(mouse)CDR1-(human)FR2-(mouse)CDR2-(human)FR3-(mouse)CDR3-(human)FR4.

[0046] In this example, a humanization template is provided for a framework region sequence in which the human germline V gene fragment IGHV3-73*01 or the human germline V gene fragment IGHV3-23*01 is bound to the human germline J gene fragment IGHJ1*01. Furthermore, by introducing one or more amino acid mutations at positions 30, 73, 76, 78, 93, or 94 (Chothia numbering method), multiple different VH variant sequences were obtained.

[0047] In this example, a framework region sequence is provided as a humanization template, consisting of a sequence in which the human germline V gene fragment IGLV7-46*02 is bound to the human germline J gene fragment IGLJ2*01, or a sequence in which the human germline V gene fragment IGKV1-39*01 is bound to the human germline J gene fragment IGKJ4*01. Furthermore, by introducing zero or multiple amino acid mutations at positions 2, 36, 46, 49, 66, 69, 71, or 87 (Chothia numbering scheme), multiple different VL variant sequences were obtained.

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

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

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

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

[0052] Example 5: Measurement of the binding ability of a CD3 antibody to CD3-expressing cells by FACS Flow cytometry-FACS was used to analyze the binding status of CD3 antibodies to CD3-expressing cells. CD3-expressing cells may include human CD3-overexpressing CHOK1 cells or HEK293 cells (host cells CHOK1 (ATCC, CCL-61) or HEK293 (ATCC, CRL-1573) transfected with both plasmids encoding the γ, δ, ε, and ζ chain ORFs of human CD3 and plasmids encoding the α and β chain ORFs of human TCR to construct a stable cell line expressing the human TCR / CD3 complex structure), cynomolgus monkey CD3-overexpressing CHOK1 or HEK293 cells, human pan-T cells (isolated from PBMCs using the human pan-T cell isolation kit (Miltenyi#130-096-535)), or cynomolgus monkey pan-T cells. Specifically, the harvested cells were washed twice with PBS (FACS buffer) containing 2% FBS, then resuspended in FACS buffer, and 1 × 10⁶ cells were placed in each well. 5Dispense into 96-well plates with cells, centrifuge at 500 g for 5 minutes, discard the supernatant, add 100 μL of pre-diluted CD3 antibody, incubate at room temperature for 1 hour, wash twice with FACS buffer, add secondary antibody Alexa Fluor 488 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch #109-545-098) diluted with FACS buffer to resuspend the cells, incubate at room temperature in the dark for 30 minutes, wash twice with FACS buffer, and resuspend with 200 μL of FACS buffer. Read the fluorescence signal values using a flow cytometer (BD FACS CANTOII or ACEA NovoCyte), and process and analyze the data with software FlowJo v10 (FlowJo, LLC). Perform data processing and graph creation analysis using software GraphPad Prism 8, and obtain parameters such as binding curves, EC 50 and other parameters by four-parameter non-linear fitting.

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

[0054] Figures 7(A)-(G) show the binding ability of the CD3 antibodies obtained in Example 4.2 (including PR000260 and its variants) to human pan-T cells, and the fluorescence intensity MFI and the ratio to the initial antibody PR000260 when the antibody concentration is 7.4 or 10 μg / mL are calculated. Specifically, after optimizing the SP34 IgG antibody sequences, PR000512, PR000513, PR001849, and PR002837 had binding ability equivalent to PR000260 (i.e., the 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 current antibody concentrations). The results above indicate that, in this invention, multiple novel antibodies can be obtained by optimizing the sequence of CD3 antibodies, and that these antibodies have different binding capabilities to human T cells and can be used in different application scenarios.

[0055] Figures 8(A) and (B) show the binding ability of the anti-CD3 scFv-Fc single-chain antibodies obtained in Example 4.3 to human pan-T cells. The fluorescence intensity MFI and the ratio to the initial antibody PR000260 are calculated when the CD3 antibody binds to human pan-T cells at antibody concentrations of 7.4 or 10 μg / mL. Specifically, after optimizing the SP34 scFv antibody through humanization, PR000624 had binding ability equivalent to or slightly higher than PR000260, PR000510 and PR000627 had binding ability equivalent to or slightly lower than PR000260, and PR001850's T cell binding ability was clearly lower than PR000260. From these results, it was found that the present invention allows for the acquisition of several stable scFv-form single-chain antibodies by optimizing the sequence of CD3 antibodies, and since these can bind to human T cells, they can be used in application scenarios 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, different molecules exhibited different binding abilities to cynomolgus monkey pan-T cells, and these abilities were positively correlated with their binding ability to human pan-T cells. In other words, molecules that strongly bind to human pan-T cells also showed high binding ability to cynomolgus monkey pan-T cells, and vice versa.

[0057] Example 6: Measurement of the activation effect of CD3 antibody on human T cells Gradient-diluted CD3 antibodies (e.g., 50, 10, 5, 1, 0.5, 0.05 μg / mL) were applied to each well of a 96-well cell culture plate in 50 μL doses, repeating the process in three wells, and then coated overnight at 4°C. The cell density of human PBMCs (Myotsū Bio) or human pan-T cells (isolated from PBMCs using the human pan-T cell isolation kit (Miltenyi #130-096-535)) was set to 7.5 × 10⁻⁶. 5 The solution was adjusted to 1 μg / mL, human CD28 antibody was added to bring the concentration to 1 μg / mL, and 200 μL was added to each well of a cell culture plate. The cells were then cultured in a CO2 incubator. After 72 hours of incubation, the supernatant was obtained, and the IFN-γ content of the supernatant was measured using an IFN-γ ELISA kit (Thermo#88-7316-77). Data analysis and graph creation were performed using GraphPad Prism software.

[0058] Figures 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, the levels of IFN-γ produced by T cell activation by PR000511, PR000512, PR000513, and PR000514 were significantly lower than those of PR000260. At an antibody concentration of 10 μg / mL, the IFN-γ levels produced by activation of PR000512, PR000513, and PR000514 were slightly lower than those of PR000260 (Figure 10(A)). At antibody concentrations of 0.5 μg / mL and 5 μg / mL, the IFN-γ levels produced by activation of PR001848 were significantly lower than those of PR000260 (Figure 10(B)). Furthermore, the T-cell activation effects (Figure 10 (C)-(G)) of antibodies such as 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 were detected, and the IFN production that these antibodies induce in T cells was detected from the results. The levels of -γ were found to be much lower than those of PR000260, no IFN-γ release was detected for PR002468 and PR002471, only slight IFN-γ levels were detected for PR002469 and PR002835 at 50 μg / mL, PR002742 and PR003886 were comparable and slightly weaker than PR001848, and PR002469 and PR004616 were comparable and clearly weaker than PR001848. From the above results, it was found that the present invention allows for the generation of multiple novel antibodies by optimizing the sequence of CD3 antibodies, and that these antibodies have different human T cell activation capabilities and yield different cytokine release levels, thus being usable 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. PR000510, PR000623, PR000624, and PR000627 at concentrations of 1 μg / mL and 10 μg / mL all showed lower IFN-γ levels than PR000260 and higher IFN-γ levels than the isotype control antibody, indicating that these four molecules restrict cytokine release by regulating the T cell activation level. From the above results, it was found that in the present invention, several stable scFv-form single-chain antibodies can be obtained by sequence optimization of the CD3 antibody, and because they have weak human T cell activation ability and low cytokine release levels, they can be used in application scenarios such as the construction of bispecific antibodies.

[0060] Example 7: Bispecific antibody containing anti-CD3 scFv antibody targeting B7H4 B7H4 is a member of the B7 family of transmembrane proteins and is highly expressed in various solid tumor tissues such as breast cancer, ovarian cancer, and endometrial cancer. However, it is not expressed or is only expressed to a small extent in normal tissues, making B7H4 a highly specific tumor-associated target antigen. Constructing a bispecific antibody molecule that targets both B7H4 and CD3 allows for the selective activation of T cells near tumor cells by targeting and binding to B7H4 on the surface of tumor cells, thereby specifically killing tumor cells.

[0061] 7.1 Production of B7H4 Antibody The variable region sequence of the B7H4 antibody conforms to WO2016040724, and recombinant IgG antibody PR000014 against B7H4 is constructed according to the method of Example 1.1. Table 10 shows the sequence information of the B7H4 antibody PR000014. [Table 10]

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

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

[0064] Figure 12(A) shows the SDS-PAGE analysis results after one-step purification of the bispecific antibody PR002883. The main by-products were found to be incompletely assembled molecules with a small amount of polymer content, suggesting that by-products can be reduced by optimizing the purification step or the plasmid transfection ratio.

[0065] 7.3 Binding to tumor cells expressing B7H4 In this example, we investigate the ability of the bispecific antibody human B7H4 to bind to tumor cells SK-BR-3 (ATCC, HTB-30) expressing this antibody. Specifically, we collect the SK-BR-3 cell suspension and increase the cell density to 1 × 10⁶. 6 The solution was adjusted to 1 / mL and seeded at 100 μL / well in a 96-well V-bottom plate (Corning #3894). Then, the test antibody, diluted 3-fold at twice the final concentration, was added at 100 μL / well. The cells were incubated at 4°C in the dark for 2 hours. Subsequently, the cells were rinsed twice with 100 μL / well of pre-cooled PBS, centrifuged at 500 g at 4°C for 5 minutes, and the supernatant was discarded. Next, 100 μL / well of the fluorescent secondary antibody Alexa Fluor 488 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch #109-545-098) was added, and the cells were incubated at 4°C in the dark for 1 hour. Finally, the cells were washed twice with 100 μL / well of pre-cooled PBS, centrifuged at 500 g for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended in 200 μL / well of pre-cooled PBS. Fluorescence emission signal values ​​were read using a flow cytometer (BD FACS CANTO II or ACEA NovoCyte), and the data were processed and analyzed using FlowJo v10 software (FlowJo, LLC). Data processing and graphing analysis were performed using GraphPad Prism 8 software, and nonlinear fitting of four parameters was used to obtain bond curves and EC. 50 We obtained parameters such as these.

[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. It was found that the bispecific antibody PR002883 has binding ability equivalent to or better than that of the monoclonal antibody PR000014.

[0067] 7.4 Binding with human T cells The binding ability of the bispecific antibody PR002883 to human pan-T cells is 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 toxicity and cytokine release of bispecific antibodies against the B7H4-overexpressing cell line SK-BR-3. To study the in vitro target cell-killing ability of the B7H4×CD3 bispecific antibody, human PBMCs were used as effector cells, and in vitro cell-mediated chemotherapeutic experiments were performed using the B7H4-highly expressing cell line SK-BR-3 (ATCC, HTB-30) as the target cell to detect cytokine release. Specifically, 50 μL of RPMI1640 / 10% FBS medium was added to each well of an E-plate (ACEA Biosciences Inc. #05232368001), and the plate was equilibrated for 30 minutes at 37°C in a 5% CO2 incubator. Then, the E-plate was placed in the xCELLigence RTCA (ACEA Biosciences) instrument to check for normality. The density of SK-BR-3 in RPMI1640 / 10% FBS medium was 0.4 × 10⁶. 6 The cells were adjusted to the required concentration per mL and seeded into E-plates at 50 μL cells / well. The E-plates were then subjected to xCELLigence RTCA overnight to detect the cell index. The density of PBMCs was measured in RPMI1640 / 10% FBS medium at 4 × 10⁻¹⁶. 6The cells were adjusted to the required concentration per mL and seeded into E-plates at 50 μL per well. Subsequently, 50 μL per well of the test antibody, diluted 5-fold to four times the final concentration, was added. The maximum final antibody concentration was 0.2 nM, with a total of seven concentrations for each antibody. The final ratio of effector cells to target cells was 10:1, and this process was repeated twice. Furthermore, SKBR3+PBMC+RPMI1640 / 10% FBS medium was placed in the plate as a blank control, and the E-plates were incubated in a 37°C, 5% CO2 incubator for 24 hours. After incubation was complete, the E-plates were placed in an xCELLigence RTCA instrument to detect the cell index.

[0069] The specificity of antibody-mediated toxicity against cells is calculated using the following formula based on the detected cell index. Cell killing percentage = (1 - detected sample / blank control) × 100%.

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

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

[0072] Example 8: Bispecific antibody containing anti-CD3 scFv antibody targeting ROR1 ROR1 is an inactive tyrosine protein kinase transmembrane protein that is overexpressed in many tumors but is hardly expressed in normal tissues. When ROR1 interacts with Wnt5a as a receptor, it transmits the Wnt signaling pathway, contributing to cell proliferation and migration in chronic lymphocytic leukemia and to epithelial-mesenchymal transition (EMT) in solid tumors. Due to its tumor-specific expression, ROR1 is a suitable tumor-associated antigen target for therapeutic drug development. Constructing a bispecific antibody molecule that targets both ROR1 and CD3 allows for the selective activation of T cells near tumor cells by targeting and binding to ROR1 on the surface of tumor cells, thereby specifically killing tumor cells.

[0073] 8.1 Production of ROR1 Antibodies The variable region sequence of the ROR1 antibody conforms to WO2016094873, and the recombinant IgG antibody PR000374 against ROR1 is constructed according to the method of Example 1.1. Table 13 shows the sequence listing of the ROR1 antibody PR000374. [Table 13]

[0074] 8.2 Production of a bispecific antibody containing an anti-CD3 scFv antibody that targets 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. This molecule contains three polypeptide chains: a heavy chain containing the CD3 single-chain antibody scFv (SEQ ID NO: 88), a heavy chain containing the ROR1 antibody VH (SEQ ID NO: 87), and a light chain containing the ROR1 antibody VL (SEQ ID NO: 85). Its structure is shown in Figure 16(C). Because this molecule has a special asymmetric structure, different amino acid mutations were introduced into the constant regions of the two heavy chains to reduce the generation of homologous heavy chain dimers. In addition, a "LALAPG" triple mutant (L234A / L235A / P329G) was introduced into the constant region of the heavy chain to prevent crosslinking caused by Fcγ receptor binding and reduce effector function.

[0075] The recombinant protein of the bispecific antibody PR002885 is produced by combining the method described in Example 1.1 with a plasmid formulation (e.g., 1:1:1 or other ratios) and performing one-step affinity purification. Table 14 shows the sequence information of the 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 results after one-step purification of the bispecific antibody PR002885. The main by-products were found to be incompletely assembled molecules with a small amount of polymer content, suggesting that by-products can be reduced by optimizing the purification step or the plasmid transfection ratio.

[0077] 8.3 Binding to tumor cells expressing ROR1 In this example, we investigate the ability of the bispecific antibody human ROR1 to bind to Panc-1 (ATCC, CRL-1469) tumor cells expressing ROR1. Specifically, Panc-1 cell suspensions are collected, and the cell density is set to 1 × 10⁶. 6The solution was adjusted to 1 / mL and seeded at 100 μL / well in a V-bottom 96-well plate (Corning #3894). Then, the test antibody, diluted 3-fold at twice the final concentration, was added at 100 μL / well. The cells were incubated at 4°C in the dark for 2 hours. After that, the cells were rinsed twice with 100 μL / well of pre-cooled PBS, centrifuged at 500 g for 5 minutes, and the supernatant was discarded. Next, 100 μL / well of the fluorescent secondary antibody Alexa Fluor 488 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch #109-545-098) was added, and the cells were incubated at 4°C in the dark for 1 hour. Finally, the cells were washed twice with 100 μL / well of pre-cooled PBS, centrifuged at 500 g for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended in 200 μL / well of pre-cooled PBS. Fluorescence emission signal values ​​were read using a flow cytometer (BD FACS CANTO II or ACEA NovoCyte), and the data were processed and analyzed using FlowJo v10 software (FlowJo, LLC). Data processing and graphing analysis were performed using GraphPad Prism 8 software, and nonlinear fitting of four parameters was used to obtain bond curves and EC. 50 We obtained parameters such as these.

[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.

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

Claims

1. A CD3-targeting antibody comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL is the amino acid sequence shown in SEQ ID NO: 56 or a variant thereof, and the VH is a mutation in the amino acid sequence shown in SEQ ID NO: 42, wherein the mutation is selected from one or more amino acid residues at positions 30, 73, 76, 78, 93, and 94, and the positional numbers of the Chothia numbering system are used for the said sites.

2. The antibody targeting CD3 according to claim 1, characterized in that the mutation occurring in the aforementioned VH is selected from the following combinations. (a) 30th place, (b) 30th, 73rd and 76th, (c) 30th, 93rd and 94th, (d) 30th, 73rd and 93rd, (e) 30th place, 93rd place, (f) 30th, 76th and 78th, (g) 73rd, 76th, 93rd and 94th, (h) 76th, 78th and 93rd, (i) 30th, 73rd, 76th, 93rd and 94th, (j) 30th, 76th, 78th, and 93rd.

3. The antibody targeting CD3 according to claim 1, characterized in that the mutation occurring in the aforementioned VH is 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.

4. The antibody targeting CD3 according to any one of claims 1 to 3, characterized in that the amino acid sequence of VH is as shown in any one of sequence numbers 43 to 55, and / or the amino acid sequence of VL is as shown in any one of sequence numbers 57 to 60.

5. The amino acid sequence of VH is as shown in SEQ ID NO: 44, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or The amino acid sequence of VH is as shown in SEQ ID NO: 51, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of VH is as shown in SEQ ID NO: 44, and the amino acid sequence of VL is as shown in SEQ ID NO: 60, or The amino acid sequence of VH is as shown in SEQ ID NO: 51, and the amino acid sequence of VL is as shown in SEQ ID NO: 60, or The amino acid sequence of VH is as shown in SEQ ID NO: 45, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of VH is as shown in SEQ ID NO: 52, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of VH is as shown in SEQ ID NO: 43, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or The amino acid sequence of VH is as shown in SEQ ID NO: 43, and the amino acid sequence of VL is as shown in SEQ ID NO: 60, or The amino acid sequence of VH is as shown in SEQ ID NO: 50, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of VH is as shown in SEQ ID NO: 47, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of VH is as shown in SEQ ID NO: 48, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of VH is as shown in SEQ ID NO: 49, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of VH is as shown in SEQ ID NO: 53, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or, The amino acid sequence of VH is as shown in SEQ ID NO: 54, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or The amino acid sequence of VH is as shown in SEQ ID NO: 43, and the amino acid sequence of VL is as shown in SEQ ID NO: 57, or, The amino acid sequence of VH is as shown in SEQ ID NO: 44, and the amino acid sequence of VL is as shown in SEQ ID NO: 57, or The amino acid sequence of VH is as shown in SEQ ID NO: 43, and the amino acid sequence of VL is as shown in SEQ ID NO: 59, or, The amino acid sequence of VH is as shown in SEQ ID NO: 44, and the amino acid sequence of VL is as shown in SEQ ID NO: 59, or, The amino acid sequence of VH is as shown in SEQ ID NO: 51, and the amino acid sequence of VL is as shown in SEQ ID NO: 57, or, The amino acid sequence of VH is as shown in SEQ ID NO: 55, and the amino acid sequence of VL is as shown in SEQ ID NO: 58, or, The antibody targeting CD3 according to claim 4, characterized in that the amino acid sequence of VH is as shown in SEQ ID NO: 46, and the amino acid sequence of VL is as shown in SEQ ID NO:

58.

6. It contains a single-chain antibody (scFv) which is VL-Linker-VH or VH-Linker-VL. Preferably, the Linker is (G ４ S) ｎ or a variant thereof, where n is a natural number other than 0, preferably 1 to 20, and more preferably the amino acid sequence shown in SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO:

67. More preferably, the amino acid sequence of the scFv is as shown 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. More preferably, the antibody according to any one of claims 1 to 5, further comprising an Fc connected to the scFv by a hinge region (Hinge).

7. The steady-state region, preferably a human steady-state region, further comprises Preferably, the human steady-state region includes a human light chain steady-state region and a human heavy chain steady-state region, and the human light chain steady-state region is preferably the human κ light chain steady-state region shown in Sequence ID No. 61 or the human λ light chain steady-state region shown in Sequence ID No.

62. The antibody according to any one of claims 1 to 6, wherein the human heavy chain constant region is hIgG1, hIgG2, hIgG3, hIgG4 or a mutation thereof, and preferably the heavy chain constant region shown in SEQ ID NO: 63 or SEQ ID NO:

64.

8. A bispecific antibody comprising a first protein domain containing an antibody targeting CD3 as described in any one of claims 1 to 7, and a second protein domain.

9. (1) The first protein domain is VL1-Linker-VH1-Hinge-CH2-CH3(knob) or VH1-Linker-VL1-Hinge-CH2-CH3(knob), (2) the second protein domain is VH2-CH1-Hinge-CH2-CH3(hole), and (3) the second protein domain is VL2-CL, the second protein domain is an antibody that targets something other than CD3, preferably an antibody that targets B7H4 or an antibody that targets ROR1, and the Linker is (G ４ S) ｎ The bispecific antibody according to claim 8, wherein n is preferably a natural number other than 0, preferably 1 to 20, and more preferably the amino acid sequence shown in SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO:

67.

10. The bispecific antibody according to claim 9, characterized by 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. Isolated nucleic acids encoding a CD3-targeting antibody according to any one of claims 1 to 7 or a bispecific antibody according to any one of claims 8 to 10.

12. An expression vector comprising the isolated nucleic acid described in claim 11, preferably selected from a retroviral vector, a lentiviral vector, an adenovirus vector, and an adeno-associated virus vector.

13. A gene-modified cell characterized by being transfected with the expression vector described in claim 12, and preferably being a eukaryotic cell.

14. A pharmaceutical composition comprising a CD3-targeting antibody according to any one of claims 1 to 7, a bispecific antibody according to any one of claims 8 to 10, a gene-modified cell according to claim 13, and a pharmaceutically acceptable carrier, preferably further comprising an immune checkpoint antibody.

15. Use of a CD3-targeting antibody according to any one of claims 1 to 7, a bispecific antibody according to any one of claims 8 to 10, an isolated nucleic acid according to claim 11, an expression vector according to claim 12, a gene-modified cell according to claim 13, or a pharmaceutical composition according to claim 14 for manufacturing a drug to treat a tumor.

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