Multispecific antibody, cancer growth inhibitor, polynucleotide, and expression vector

Abstract

The present invention improves an effect of killing or an effect of inhibiting the growth of cancer cells. The present invention comprises: a binding domain that includes a first antigen-binding site that binds to a cancer cell and a second antigen-binding site that binds to a T cell; a cleavable sequence that is linked to the binding domain; and a masking moiety that is linked to the N-terminus of a single-chain antibody via the cleavable sequence when the binding domain is composed of the single-chain antibody, and that is linked to the C-terminus of each light chain via the cleavable sequence when the binding domain is composed of heavy chains and light chains. In a state in which the cleavable sequence is cleaved, the masking moiety functions as an agonist of a co-stimulatory molecule for the T cell or an antagonist of a co-inhibitory molecule for the T cell.

Description

Multispecific antibodies, cancer growth inhibitors, polynucleotides and expression vectors

[0001] The present disclosure relates to multispecific antibodies, cancer growth inhibitors, polynucleotides, and expression vectors.

[0002] Immunotherapy using antibodies or modified immune cells for cancer has attracted attention. Among these, the use of multispecific antibodies makes it possible to bridge immune cells and cancer cells, induce apoptosis in cancer cells, activate multiple costimulatory molecules, and so on, thereby expanding the possibilities of immunotherapy for cancer. For example, a bispecific antibody (Ex3) is known that combines the variable region of an antibody that binds to EGFR, which is overexpressed on the surface of cancer cells, with the variable region of an antibody that binds to CD3 on T cell-derived activated lymphocytes.

[0003] Maejima, A. et al., N. Biotechnol., 2023, 77, 80-89 discloses fusing a masking moiety, PAS, to the N-terminus of Ex3-Fc, which is Ex3 fused with the Fc region of human IgG1. PAS is a polymer containing proline, alanine, and serine, and is known as a molecule that inhibits the binding of antibody-like proteins. In Maejima, A. et al., N. Biotechnol., 2023, 77, 80-89, an antibody molecule was produced in which PAS was fused to the N-terminus, which is the immune cell binding domain of Ex3-Fc, via a recognition sequence for matrix metalloproteinase, a protease secreted by cancer cells. Maejima, A. et al., N. Biotechnol. , 2023, 77, 80-89, it has been shown that the antibody molecule produced has a long half-life in the body due to its high molecular weight, and does not exhibit cytotoxic activity in the vicinity of normal tissue due to the masking effect of PAS, while in the vicinity of tumors, the PAS is removed, thereby restoring the cytotoxic activity to cancer cells.

[0004] Furthermore, Japanese Patent No. 6529952 discloses a binding polypeptide comprising a masking moiety, a cleavable sequence moiety, and a target-binding moiety (antibody or antibody fragment) arranged in this order. In the binding polypeptide disclosed in Japanese Patent No. 6529952, the masking moiety inhibits binding between the target and the target, and when the cleavable sequence moiety is cleaved and the masking moiety is removed, binding between the target and the target becomes possible. In particular, in the binding polypeptide disclosed in Japanese Patent No. 6529952, the masking moiety not only inhibits binding between the target and the target, but can also function as another target-binding moiety.

[0005] Furthermore, Japanese Patent Application Laid-Open No. 2015-516813 discloses an activatable antibody comprising an antibody or antibody fragment that specifically binds to EGFR, a cleavable sequence portion, and a masking portion, arranged in this order. Japanese Patent Application Laid-Open No. 2015-516813 discloses a specific amino acid sequence consisting of 15 amino acid residues as the masking portion. Furthermore, U.S. Patent Application No. 2022 / 0251169 discloses an activatable antibody comprising, from the N-terminus to the C-terminus, albumin, a matrix metalloproteinase recognition sequence, an immunoadhesin portion comprising CTLA4 having an N-terminal extracellular domain, and an IgG Fc region. In the activatable antibody disclosed in U.S. Patent Application No. 2022 / 0251169, albumin functions as the masking portion. Furthermore, I-Ju Chen et al. Scientific Reports, volume 7, Article number: 11587 (2017) discloses an anti-EGFR antibody or anti-TNF-α antibody having a masking portion called an inhibitory domain. In I-Ju Chen et al. Scientific Reports, volume 7, Article number: 11587 (2017), the inhibitory domain uses the latency-associated peptide (LAP) of TGF-β, C2b in complement factor 2, and CBa in complement factor B.

[0006] As described above, the activatable antibodies containing a masking moiety disclosed in the prior art documents are so-called prodrug antibodies that minimize the effects on normal tissues and are expected to be therapeutic agents with fewer side effects.

[0007] However, even if a multispecific antibody such as a bispecific antibody as described above is used to promote cross-linking between T cells and cancer cells and attempt to kill or inhibit the proliferation of cancer cells, the problem of not being able to achieve the effect of killing or inhibiting the proliferation of cancer cells due to T cell exhaustion remains unresolved. Therefore, an object of the present disclosure is to provide a multispecific antibody, a cancer growth inhibitor, a polynucleotide, and an expression vector that are excellent in the effect of killing or inhibiting the proliferation of cancer cells.

[0008] As a result of extensive research to achieve the above-mentioned object, the present inventors have found that in a multispecific antibody having specific binding sites for cancer cells and T cells, an agonist of a costimulatory molecule for T cells or an antagonist of a co-inhibitory molecule for T cells is used as a masking moiety, and the masking moiety is cleavably positioned at the C-terminus of the light chain or the N-terminus of the single-chain antibody or single-chain antibody fragment, so that the masking moiety after cleavage exhibits an activating effect on T cells, and have completed the present disclosure.

[0009] The present disclosure includes the following: <1> A multispecific antibody comprising: a binding region including a first antigen-binding site that binds to a cancer cell and a second antigen-binding site that binds to a T cell; a cleavable sequence linked to the binding region; and a masking moiety that, when the binding region is composed of a single-chain antibody, is linked to the N-terminus of the single-chain antibody via the cleavable sequence, or, when the binding region is composed of a heavy chain and a light chain, is linked to the C-terminus of the light chain via the cleavable sequence, wherein the masking moiety functions as an agonist of a costimulatory molecule for the T cell or as an antagonist of a co-inhibitory molecule for the T cell when the cleavable sequence is cleaved. <2> The multispecific antibody according to <1>, wherein the masking moiety is a low-molecular-weight antibody having agonistic function corresponding to one ligand selected from the group consisting of 4-1BBL, B7-H2, B7-1, B7-2, CD70, LIGHT, CD40L, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, and CD112. <3> The multispecific antibody according to <2>, wherein the low-molecular-weight antibody is a deletion type in which 1 to 5 amino acid residues are deleted from the C-terminus. <4> The multispecific antibody according to any one of <1> to <3>, wherein the first antigen-binding site comprises a first heavy chain variable region and a first light chain variable region, the second antigen-binding site comprises a second heavy chain variable region and a second light chain variable region, and the binding region comprises, from N-terminus to C-terminus, the second light chain variable region, the first heavy chain variable region, the first light chain variable region, and the second heavy chain variable region. <5> The multispecific antibody according to any one of <1> to <4>, wherein the first antigen-binding site binds to a cancer cell surface receptor.<6> The cancer cell surface receptor is EGFR, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, FGF-2, FGFR1, FGFR2, FGFR3, FGFR4, HER2 / neu, DLL4, NOTCHR1, IL1B, IL1R, IL2, IL2R, IL4, IL6, IL12, IL13, IL15, IL18, IL23, IGF, IGF1R, ERBB3, VCAM-1, CXCR4, CD3, CD11a, CD19, CD20, CD22, CD25, CD28, CD30, The multispecific antibody according to <5>, wherein the first antigen-binding site targets one receptor selected from the group consisting of CD33, CD40, CD40L, CD41, CD44, CD52, CD80, CD86, CTLA-4, TNFα, TNFR, TRAIL-R1, TRAIL-R2, IgE, IgE receptor, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, GPIIB / IIIA, CLAUDIN-3, CLAUDIN-4, C5 complement, RSV F protein, glycoprotein IIb / IIIa receptor, α4β1 integrin, and α4β7 integrin. <7> The multispecific antibody according to any one of <1> to <6>, wherein the first antigen-binding site targets EGFR. <8> The multispecific antibody according to any one of <1> to <7>, wherein the second antigen-binding site targets a T cell surface molecule. <9> The multispecific antibody according to <8>, wherein the T cell surface molecule is a molecule selected from the group consisting of CD2, CD3, CD5, CD7, CD43, CD4, and CD8. <10> The multispecific antibody according to any one of <1> to <9>, wherein the second antigen-binding site targets CD3. <11> The multispecific antibody according to any one of <1> to <10>, wherein the cleavable sequence comprises a recognition sequence for a protease secreted by cancer cells. <12> The multispecific antibody according to <11>, wherein the protease is a matrix metalloprotease. <13> The multispecific antibody according to any one of <1> to <12>, wherein the Fc region comprises heavy chain constant region 2 and heavy chain constant region 3. <14> A cancer growth inhibitor comprising the multispecific antibody according to any one of <1> to <13>.<15> A polynucleotide encoding the multispecific antibody according to any one of <1> to <13>. <16> An expression vector comprising the polynucleotide according to <15>.

[0010] The multispecific antibodies and cancer growth inhibitors of the present disclosure exhibit excellent cancer cell-killing or growth-inhibiting effects. Furthermore, the polynucleotides and expression vectors of the present disclosure can provide multispecific antibodies and cancer growth inhibitors that exhibit excellent cancer cell-killing or growth-inhibiting effects.

[0011] FIG. 1 is a schematic diagram showing an example of a multispecific antibody of the present disclosure. FIG. 2 is a schematic diagram showing another example of a multispecific antibody of the present disclosure. FIG. 3 is a schematic diagram showing a state in which a multispecific antibody of the present disclosure acts on cancer cells and T cells. FIG. 4 is a diagram showing the amino acid sequences constituting a multispecific antibody of the present disclosure. FIG. 5 is a characteristic diagram showing the results of evaluating the binding activity of multispecific antibodies prepared in Examples and Comparative Examples against cancer cells by flow cytometry. FIG. 6 is a characteristic diagram showing the results of evaluating the binding activity of multispecific antibodies prepared in Examples and Comparative Examples against T cells by flow cytometry. FIG. 7 is a characteristic diagram showing the results of evaluating the binding activity of multispecific antibodies prepared in Examples and Comparative Examples against cancer cells by flow cytometry before and after freeze-thawing. FIG. 8 is a characteristic diagram showing the results of evaluating the binding activity of multispecific antibodies prepared in Examples and Comparative Examples against T cells by flow cytometry before and after freeze-thawing. FIG. 9 is a characteristic diagram showing the results of testing the effect of multispecific antibodies prepared in Examples and Comparative Examples on T cell proliferation. FIG. 10 is a characteristic diagram showing the results of testing the cytotoxic activity of multispecific antibodies prepared in Examples and Comparative Examples against cancer cells by MTS assay. FIG. 1 is a characteristic diagram showing the results of a cancer cytotoxicity test on HT1080 cells, A431 cells, and TFK-1 cells for the multispecific antibodies prepared in the Examples.

[0012] Below, an embodiment that is an example of the present disclosure will be described. These descriptions and examples are intended to illustrate the embodiment and do not limit the scope of the invention. In the following embodiments, the components (including element steps, etc.) are not essential unless otherwise specified. The same applies to numerical values ​​and their ranges, and do not limit the present disclosure. For example, the present disclosure allows addition, omission, substitution, modification, etc. of the number, amount, position, ratio, material, configuration, type, order, etc., within the scope that does not deviate from the spirit of the present disclosure.

[0013] In the present disclosure, numerical ranges indicated using "to" include the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In numerical ranges described in stages in the present disclosure, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another numerical range described in stages. Furthermore, in numerical ranges described in the present disclosure, the upper or lower limit value of that numerical range may be replaced with a value shown in the examples. In the present disclosure, each component may contain multiple corresponding substances. When multiple substances corresponding to each component are present in a composition, etc., the content or amount of each component means the total content or amount of the multiple substances present in the composition, etc., unless otherwise specified.

[0014] In the present disclosure, the positions of amino acid residues in the amino acid sequences constituting antibodies or low-molecular-weight antibodies are indicated by numerical values ​​based on Kabat numbering. Therefore, please note that the positions of amino acid residues described in the present disclosure differ from the numerical values ​​based on the amino acid sequences described in the sequence listing attached hereto. The Kabat numbering is based on the Kabat numbering system described in "Sequences of proteins of immunological interest," Elvin A. Kabat et al., NIH publication, no. 91-3242 (National Institutes of Health, 1991, 5 ｔｈ ed.) is adopted.

[0015] In the present disclosure, the term "antibody" is used in the broadest sense to refer to an antibody molecule that exhibits desired antigen-binding activity, and encompasses a variety of antibody structures, including monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and small molecule antibodies, which will be described later.

[0016] <Multispecific antibody> A multispecific antibody according to one embodiment of the present disclosure comprises a binding region including a first antigen-binding site that binds to cancer cells and a second antigen-binding site that binds to T cells, a cleavable sequence linked to the binding region, and a masking moiety that, when the binding region is composed of a single-chain antibody, is linked to the N-terminus of the single-chain antibody via the cleavable sequence, or, when the binding region is composed of a heavy chain and a light chain, is linked to the C-terminus of the light chain via the cleavable sequence, wherein the masking moiety functions as an agonist of a costimulatory molecule for the T cells or an antagonist of a co-inhibitory molecule for the T cells when the cleavable sequence is cleaved. The multispecific antibody of the present disclosure can exhibit a cancer-killing effect or a cancer-proliferation-inhibitory effect, in other words, an antitumor effect, by virtue of the masking moiety released upon cleavage of the cleavable sequence functioning as the agonist or antagonist.

[0017] The multispecific antibody of the present disclosure may be a single-chain antibody (single chain Fv) as shown in Figure 1, or an antibody composed of a heavy chain variable region (VH) and a light chain variable region (VL) as shown in Figure 2. That is, the multispecific antibody shown in Figures 1 and 2 comprises a binding region 3 including a first antigen-binding site 1 that binds to cancer cells and a second antigen-binding site 2 that binds to T cells. The multispecific antibody shown in Figures 1 and 2 also comprises a cleavable sequence 4 linked to the binding region 3. The multispecific antibody shown in Figures 1 and 2 also comprises a masking moiety 5 linked to the binding region 3 via the cleavable sequence 4.

[0018] The multispecific antibody shown in Figure 1 is a single-chain antibody, and therefore the masking moiety 5 is linked to the N-terminus of the binding region 3 via the cleavable sequence 4. The multispecific antibody shown in Figure 2 is composed of a heavy chain and a light chain, and the masking moiety 5 is linked to the C-terminus of the light chain via the cleavable sequence 4. In both Figures 1 and 2, the masking moiety 5 in the multispecific antibody is in close proximity to the second antigen-binding site 2 and can inhibit the binding of the second antigen-binding site 2 to T cells.

[0019] Furthermore, in the multispecific antibody shown in Figures 1 and 2, when the first antigen-binding site 1 is bound to a cancer cell 6, the cleavable sequence 4 is cleaved by a protease 7 produced by the cancer cell 6, as shown in Figure 3. This releases the masking moiety 5 from the multispecific antibody, allowing the second antigen-binding site 2 to bind to a T cell 8 and activating the T cell 8 via the released masking moiety 5. In other words, the released masking moiety 5 functions as an agonist of a costimulatory molecule for the T cell 8 or as an antagonist of a co-inhibitory molecule for the T cell 8, thereby activating the T cell 8.

[0020] Here, "activation of T cells" means observing at least one improvement selected from the group consisting of improved proliferation ability of T cells, improved cytokine production ability of T cells, improved differentiation ability of T cells, improved cytocidal function of T cells, improved memory formation ability of T cells, and improved survival ability of T cells. "Activation of T cells" also means suppression of at least one effect caused by co-inhibitory molecules selected from the group consisting of suppression of cell cycle, suppression of effector function, immune tolerance, exhaustion, and apoptosis induction.

[0021] The activation of the above-mentioned T cells can be evaluated, for example, by measuring the proliferation of cancer cells in the presence of a multispecific antibody using T-LAK cells as T cells and TFK-1 cells, which are extrahepatic cholangiocarcinoma cells, as cancer cells. The proliferation of cancer cells can be measured by measuring the number of viable cancer cells using an MTS assay using a so-called MTS reagent. When the number of viable cancer cells is significantly reduced in the presence of a multispecific antibody, it can be determined that the T cells have been activated.

[0022] <Masking Moiety> The masking moiety according to the present disclosure inhibits binding of T cells to the second antigen-binding site when linked as part of the multispecific antibody, and functions as an agonist of costimulatory molecules for T cells or an antagonist of co-inhibitory molecules for T cells when released from the multispecific antibody. Here, the masking moiety can be released from the multispecific antibody by cleavage of the cleavable sequence in the multispecific antibody, as will be described in detail below.

[0023] Costimulatory molecules for T cells are essential components of the costimulatory signal that is involved in T cell activation along with the primary stimulatory signal mediated by the binding of MHC on antigen-presenting cells to T cell receptors (TCRs). The costimulatory signal is generated when the costimulatory molecule binds to a receptor on a T cell. Examples of costimulatory molecules include a ligand selected from the group consisting of 4-1BBL, B7-H2, B7-1, B7-2, CD70, LIGHT, CD40L, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, and CD112.

[0024] Here, of the costimulatory molecules mentioned above, the T cell receptor for 4-1BBL is 4-1BB. The T cell receptor for B7-H2 is ICOS, and the T cell receptor for B7-H2, B7-1, and B7-2 is CD28. The T cell receptor for CD70 is CD27. The T cell receptor for LIGHT is HVEM. The T cell receptor for CD40L is CD40. The T cell receptor for OX40L is OX40. The T cell receptor for TL1A is DR3. The T cell receptor for GITRL is GITR. The T cell receptor for CD30L is CD30. The T cell receptor for TIM4 is TIM1. The T cell receptor for SLAM is SLAM. The T cell receptor for CD48 and CD58 is CD2. The receptor on T cells for CD155 and CD112 is CD226.

[0025] Thus, the masking moiety according to the present disclosure can be a low-molecular-weight antibody that has agonistic function corresponding to one ligand selected from the group consisting of 4-1BBL, B7-H2, B7-1, B7-2, CD70, LIGHT, CD40L, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, and CD112. In other words, the masking moiety according to the present disclosure can be a low-molecular-weight antibody that specifically binds to one receptor selected from the group consisting of T cell receptors 4-1BB, ICOS, CD28, CD27, HVEM, CD40, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, and CD226, and has agonistic function equivalent to the aforementioned ligand.

[0026] Here, a low molecular weight antibody refers to an antibody molecule that contains a portion of a full-length antibody molecule and retains the ability to bind to an antigen to which the full-length antibody molecule binds. Low molecular weight antibodies are also sometimes called antibody fragments. Low molecular weight antibodies are not particularly limited, but examples include Fv, Fab, Fab', Fab'-SH, and F(ab'). ２Examples of such a masking moiety include: diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and variable domains of heavy chain antibodies (VHH). In particular, the masking moiety according to the present disclosure is preferably an scFv. An scFv is a single-chain polypeptide in which two variable regions are linked, optionally via a linker or the like. The two variable regions contained in an scFv are typically one VH and one VL, but may also be two VHs or two VLs. Generally, an scFv polypeptide contains a linker between the VH and VL domains, thereby forming a pair of VH and VL required for antigen binding. Typically, to form a pair between VH and VL within the same molecule, the linker connecting VH and VL is a peptide linker having a length of 10 amino acids or more. However, the linker of the scFv in the present disclosure is not limited to such peptide linkers, as long as it does not prevent the formation of the scFv.

[0027] In the present disclosure, the class of the antibody is not particularly limited, and may be any of the five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. In the present disclosure, the subclass (isotype) of the antibody is not particularly limited, and may be, for example, IgG １ , IgG ２ , IgG ３ , IgG ４ , IgA １ and IgA ２ It may be any of the above.

[0028] As an example, the masking moiety can be an antibody against 4-1BB: an anti-4-1BB antibody. A known anti-human 4-1BB antibody scFv can be used as an example of an anti-4-1BB antibody. An example of a known anti-human 4-1BB antibody scFv is a single-chain antibody having known sequence information (clone: ​​94G1) (U.S. Patent No. 10,774,151B2). This anti-human 4-1BB antibody scFv has been confirmed to have anti-cancer activity equivalent to that of an anti-PD-1 antibody that targets PD-1 on the surface of T cells. The amino acid sequence of the VH constituting this anti-human 4-1BB antibody scFv is shown in SEQ ID NO: 1, and the amino acid sequence of the VL is shown in SEQ ID NO: 2.

[0029] That is, in the present disclosure, the masking moiety can be a known anti-human 4-1BB antibody scFv having a VH comprising the amino acid sequence shown in SEQ ID NO: 1 and a VL comprising the amino acid sequence shown in SEQ ID NO: 2. Note that the anti-4-1BB antibody used as the masking moiety according to the present disclosure can be an antibody having a VH comprising an amino acid sequence that is 90% or more identical, preferably 93% or more identical, more preferably 95% or more identical, and even more preferably 98% or more identical to the amino acid sequence of SEQ ID NO: 1, which VH has the ability to activate T cells together with the VL, and a VL comprising an amino acid sequence that is 90% or more identical, preferably 93% or more identical, more preferably 95% or more identical, and even more preferably 98% or more identical to the amino acid sequence of SEQ ID NO: 2, which VL has the ability to activate T cells together with the VH.

[0030] Furthermore, the anti-human-4-1BB antibody scFv can have a desired linker sequence between the above-mentioned VH and VL. The linker sequence is not particularly limited, and a GGGS sequence can be used. Specifically, the linker sequence between the above-mentioned VH and VL can be a sequence in which the GGGS sequence is repeated 2 to 6 times, preferably 2 to 4 times, and more preferably 3 times.

[0031] Furthermore, the above-mentioned low-molecular-weight antibody used as the masking moiety according to the present disclosure is preferably a deletion type in which 1 to 5 amino acid residues are deleted from the C-terminus, more preferably a deletion type in which 1 to 3 amino acid residues are deleted from the C-terminus, and even more preferably a deletion type in which 1 amino acid residue is deleted from the C-terminus. By deleting 1 to 5 amino acid residues from the C-terminus of the low-molecular-weight antibody, the N-terminus, which is the antigen recognition site of the masking moiety, and the second antigen recognition site can be brought into close proximity, resulting in a higher masking effect.

[0032] <Binding Region> In the multispecific antibody of the present disclosure, the binding region comprises a first antigen-binding site that binds to cancer cells and a second antigen-binding site that binds to T cells, and functions as a so-called bispecific antibody. Furthermore, in the multispecific antibody of the present disclosure, it is preferable that the first antigen-binding site specifically binds to cancer cells. Similarly, it is preferable that the second antigen-binding site specifically binds to T cells. The multispecific antibody of the present disclosure can bring cancer cells and T cells closer together via the binding region, provided that the binding of the second antigen-binding site to the antigen is not inhibited by the masking moiety.

[0033] In the multispecific antibody of the present disclosure, the binding region may be a full-length antibody molecule that functions as the bispecific antibody described above, or a low molecular weight antibody containing a portion of a full-length antibody molecule. As with the masking moiety described above, the low molecular weight antibody is not particularly limited, and examples thereof include Fv, Fab, Fab', Fab'-SH, and F(ab'). ２Examples of suitable scFvs include diabodies, linear antibodies, single-chain antibody molecules (e.g., scFvs), and VHHs. In particular, in the present disclosure, the binding region is preferably an scFv that functions as a bispecific antibody as described above. An scFv that functions as a bispecific antibody is a single-chain polypeptide having two sets of variable regions, one for each of two different antigens. The two variable regions may be linked via a linker or the like as needed. Furthermore, each set may also be linked via a linker or the like as needed. The two variable regions contained in an scFv are typically one VH and one VL, but may also be two VHs or two VLs. Generally, the linker connecting the VH and VL is a peptide linker of 10 amino acids or more in length. However, the linker of the scFv in the present disclosure is not limited to such peptide linkers, as long as it does not interfere with the formation of the scFv.

[0034] Furthermore, the class of the antibody for the binding region is not particularly limited, and may be any of the five major antibody classes: IgA, IgD, IgE, IgG, and IgM. Furthermore, in the present disclosure, the subclass (isotype) of the antibody is not particularly limited, and may be, for example, IgG １ , IgG ２ , IgG ３ , IgG ４ , IgA １ and IgA ２ It may be any of the above.

[0035] In particular, in the multispecific antibody of the present disclosure, the binding region preferably has a first antigen-binding site comprising a first heavy chain variable region (first VH) and a first light chain variable region (first VL), and a second antigen-binding site comprising a second heavy chain variable region (second VH) and a second light chain variable region (second VL), with the second VL, first VH, first VL, and second VH arranged in this order from the N-terminus to the C-terminus. That is, in this case, the binding region is a single-chain antibody arranged in this order from the N-terminus to the C-terminus. By forming the binding region as a single-chain antibody, the multispecific antibody of the present disclosure can be easily produced.

[0036] In the binding region of the present disclosure, the first antigen-binding site binds to cancer cells. The antigen targeted by the first antigen-binding site is not particularly limited as long as it is expressed on cancer cells. For example, it is preferable that the first antigen-binding site binds to a cancer cell surface receptor. That is, it is preferable that the first antigen-binding site of the multispecific antibody of the present disclosure binds to a cancer cell surface receptor.

[0037] Here, examples of cancer cell surface receptors targeted by the first antigen-binding site include EGFR, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, FGF-2, FGFR1, FGFR2, FGFR3, FGFR4, HER2 / neu, DLL4, NOTCHR1, IL1B, IL1R, IL2, IL2R, IL4, IL6, IL12, IL13, IL15, IL18, IL23, IGF, IGF1R, ERBB3, VCAM-1, CXCR4, CD3, CD11a, CD19, CD20, CD22, and CD 25, CD28, CD30, CD33, CD40, CD40L, CD41, CD44, CD52, CD80, CD86, CTLA-4, TNFα, TNFR, TRAIL-R1, TRAIL-R2, IgE, IgE receptor, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, GPIIB / IIIA, CLAUDIN-3, CLAUDIN-4, C5 complement, RSV F protein, glycoprotein IIb / IIIa receptor, α4β1 integrin, and α4β7 integrin.

[0038] In particular, it is preferable that the first antigen-binding site targets EGFR. EGFR is an epidermal growth factor receptor and is highly expressed in many cancer cells. The first antigen-binding site can be designed based on known antibodies that use EGFR as an antigen. Known antibodies that use EGFR as an antigen include anti-EGFR humanized monoclonal antibody 528, cetuximab, panitumumab, etc. The amino acid sequences of the first VH and first VL can be designed based on these known antibodies.

[0039] As an example, the first antigen-binding site can be an antibody against EGFR: an anti-EGFR antibody. As an example of an anti-EGFR antibody, the VH and VL of the known anti-EGFR humanized monoclonal antibody 528 can be used. It is also known that the affinity with FGFR is improved in the VH of anti-EGFR humanized monoclonal antibody 528 by substituting Y at position 52, as specified by the Kabat number, with W (Y52W) (Asano, R. et al., Sci. Rep., 2020, 10, 1-8). The amino acid sequence of the VH constituting anti-EGFR humanized monoclonal antibody 528, which has Y52W, is shown in SEQ ID NO: 3, and the amino acid sequence of the VL constituting anti-EGFR humanized monoclonal antibody 528 is shown in SEQ ID NO: 4.

[0040] That is, in the present disclosure, the first antigen-binding site can be a single-chain antibody having a VH comprising the amino acid sequence shown in SEQ ID NO: 3 and a VL comprising the amino acid sequence shown in SEQ ID NO: 4. Note that the VH used as the first antigen-binding site according to the present disclosure can be an antibody having: a VH comprising an amino acid sequence that is 90% or more identical, preferably 93% or more identical, more preferably 95% or more identical, and even more preferably 98% or more identical to the amino acid sequence of SEQ ID NO: 3, which VH has the ability to bind to EGFR expressed on the surface of cancer cells together with the VL; and a VL comprising an amino acid sequence that is 90% or more identical, preferably 93% or more identical, more preferably 95% or more identical, and even more preferably 98% or more identical to the amino acid sequence of SEQ ID NO: 4, which VL has the ability to bind to EGFR expressed on the surface of cancer cells together with the VH.

[0041] Furthermore, in the first antigen-binding site, a desired linker sequence can be inserted between the VH and VL described above. The linker sequence is not particularly limited, and a GGGS sequence can be used. Specifically, the linker sequence inserted between the VH and VL described above can be a sequence in which the GGGS sequence is repeated 2 to 8 times, preferably 3 to 5 times, and more preferably 4 times.

[0042] In the present disclosure, the first antigen-binding site may be designed based on the following antibodies that bind to antigens other than EGFR: For example, the first antigen-binding site may be designed based on an antibody selected from the group consisting of bevacizumab, ranibizumab, trastuzumab, infliximab, adalimumab, efalizumab, gemtuzumab ozogamicin, tositumomab, ibritumomab tiuxetan, eculizumab, alemtuzumab, rituximab, abciximab, daclizumab, basiliximab, gemtuzumab, eculizumab, natalizumab, omalizumab, ipilimumab, tremelimumab, and palivizumab.

[0043] On the other hand, in such a binding region of the present disclosure, the second antigen-binding site binds to a T cell. The antigen targeted by the second antigen-binding site is not particularly limited as long as it is expressed on T cells. For example, it is preferable that the second antigen-binding site binds to a T cell surface molecule. That is, it is preferable that the multispecific antibody of the present disclosure binds to a T cell surface molecule through the second antigen-binding site.

[0044] Here, the T cell surface molecule targeted by the second antigen-binding site is not particularly limited, but can include one molecule selected from the group consisting of CD2, CD3, CD5, CD7, CD43, CD4, and CD8.

[0045] In particular, it is preferable that the second antigen-binding site targets CD3. CD3 is a protein complex that directly binds to the T cell receptor (TCR) and is a molecule that has traditionally been used as a T cell marker. The second antigen-binding site can be designed based on a known antibody that uses CD3 as an antigen. An example of a known antibody that uses CD3 as an antigen is human CD3 antibody (clone number: OKT3). The amino acid sequences of the second VH and second VL can be designed based on the human CD3 antibody (clone number: OKT3).

[0046] As an example, the second antigen-binding site may be an antibody against CD3: an anti-CD3 antibody. As an example of an anti-CD3 antibody, the VH and VL of a known human CD3 antibody (clone number: OKT3) may be used. The amino acid sequence of the VH constituting the human CD3 antibody (clone number: OKT3) is shown in SEQ ID NO: 5, and the amino acid sequence of the VL is shown in SEQ ID NO: 6.

[0047] That is, in the present disclosure, the second antigen-binding site can be a single-chain antibody having a VH comprising the amino acid sequence shown in SEQ ID NO: 5 and a VL comprising the amino acid sequence shown in SEQ ID NO: 6. Note that the VH used as the second antigen-binding site according to the present disclosure can be an antibody having a VH comprising an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 5, preferably 93% or more identical, more preferably 95% or more identical, and even more preferably 98% or more identical, and that has, together with the VL, the ability to bind to CD3 expressed on the surface of T cells, and a VL comprising an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 6, preferably 93% or more identical, more preferably 95% or more identical, and even more preferably 98% or more identical, and that has, together with the VH, the ability to bind to CD3 expressed on the surface of T cells.

[0048] When the multispecific antibody of the present disclosure is a single-chain antibody arranged in the order of second VL, first VH, first VL, and second VH from the N-terminus to the C-terminus as described above, the first VH and first VL described above are arranged between the second VL and the second VH. In this case, a desired linker sequence can be arranged between the second VL and the first VH and between the first VL and the second VH. The linker sequence is not particularly limited, and an SGGG sequence can be used. Specifically, the linker sequence arranged between the VH and VL described above can be an SGGG sequence, or it may be a sequence in which the SGGG sequence is repeated twice.

[0049] <Cleavable sequence> In the multispecific antibody of the present disclosure, a cleavable sequence is disposed between the above-described masking moiety and the binding region. The cleavable sequence is preferably a sequence that is cleaved with high frequency in the vicinity of cancer cells and is not cleaved easily in the vicinity of normal cells. Such a cleavable sequence is preferably a sequence that is cleaved by an enzyme that is highly expressed in cancer cells, or a recognition sequence for that enzyme.

[0050] Examples of enzymes that are highly expressed in cancer cells include proteases, including, but not limited to, MMP1, MMP2, MMP3, MMP8, MMP9, MMP14, plasmin, PSA, PSMA, cathepsin D, cathepsin K, cathepsin Examples of the cleavable enzyme include Caspase-S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. That is, the cleavable sequence can be a recognition sequence for these enzymes.

[0051] More specifically, the protease that cleaves the cleavable sequence is preferably a matrix metalloprotease. Matrix metalloproteases are proteases secreted by cancer cells. Examples of matrix proteases include MMP1, MMP2, MMP3, MMP8, MMP9, and MMP14. That is, in the multispecific antibody of the present disclosure, the cleavable sequence is preferably a recognition sequence for a matrix protease. As an example, GPLGIAGQ (SEQ ID NO: 7) can be used as the cleavable sequence. In the matrix protease recognition sequence shown in SEQ ID NO: 7, the matrix protease cleaves between G and I.

[0052] Furthermore, the cleavable sequence is not limited to the above-described enzyme recognition sequence, and may contain other sequences in addition to the enzyme recognition sequence. For example, other sequences may be present between the cleavable sequence and the masking portion and / or between the cleavable sequence and the binding region. Examples of other sequences include linker sequences consisting of a GGGS sequence or the like. By providing other sequences, the distance between the cleavable sequence and the masking portion and / or between the cleavable sequence and the binding region can be increased. However, since it is preferable to shorten the distance between the binding region and the masking portion, it is preferable that the cleavable sequence does not have other sequences such as a linker sequence.

[0053] <Modifications> The multispecific antibodies of the present disclosure are not limited to the above-described configuration, and may include a so-called hinge region and be dimerized via disulfide bonds between the hinge regions. When the multispecific antibodies of the present disclosure are single-chain antibodies, they may have a hinge region at the C-terminus (Figure 1). When the multispecific antibodies of the present disclosure are antibodies composed of a heavy chain variable region (VH) and a light chain variable region (VL), they may have a hinge region at the C-terminus of the heavy chain variable region (Figure 2). The hinge region may be any of the five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. As an example, an IgG having an H237Y mutation (Kabat numbering) may have a hinge region at the C-terminus of the heavy chain variable region (Figure 2). １ An example of the hinge region of this protein is DKTYTCPPCP (SEQ ID NO: 8). The H237Y mutation (Kabat number) is known to suppress fragmentation during storage (Suzuki, S. et al., Sci. Rep. 2018, 8, 17253-17263).

[0054] Furthermore, the multispecific antibody of the present disclosure is not limited to the above-described configuration, and may also have an Fc region (Fragment of crystalline region) comprising heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3). When the multispecific antibody of the present disclosure is a single-chain antibody, it may have an Fc region at the C-terminus ( FIG. 1 ). When the multispecific antibody of the present disclosure is an antibody composed of a heavy chain variable region (VH) and a light chain variable region (VL), it may have an Fc region at the C-terminus of the heavy chain variable region ( FIG. 2 ). The Fc region may be from any of the five major antibody classes: IgA, IgD, IgE, IgG, and IgM. As an example, an IgG １ An example of an Fc region of the multispecific antibody of the present disclosure is an Fc region (SEQ ID NO: 9) comprising CH2 and CH3. By comprising an Fc region, the multispecific antibody of the present disclosure can acquire effector activity such as antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. Furthermore, by comprising an Fc region, the multispecific antibody of the present disclosure can be purified using Protein A, and by binding to the neonatal Fc receptor, it can avoid transport to lysosomes and degradation, and its increased molecular weight can prolong its in vivo half-life.

[0055] In particular, it is more preferable that the multispecific antibody of the present disclosure be configured to include both the above-described hinge region and Fc region, in which case the multispecific antibody of the present disclosure can combine the effects of the hinge region and the effects of the Fc region.

[0056] <Examples of Multispecific Antibodies> An example of a multispecific antibody of the present disclosure configured as described above is a polypeptide having the amino acid sequence shown in SEQ ID NO: 10. A multispecific antibody comprising the amino acid sequence of SEQ ID NO: 10 comprises GPLGIAGQ (SEQ ID NO: 7) as a cleavable sequence. Furthermore, the multispecific antibody of the present disclosure comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 10, preferably 93% or more identical, more preferably 95% or more identical, and even more preferably 98% or more identical, and can be a polypeptide that costimulates T cells and exerts the effect of inhibiting the proliferation of cancer cells.

[0057] Another example of a multispecific antibody of the present disclosure is a polypeptide having the amino acid sequence shown in SEQ ID NO: 11. A multispecific antibody comprising the amino acid sequence of SEQ ID NO: 11 comprises an amino acid sequence in which the C-terminal lysine residue constituting the masking moiety is deleted from the VL (SEQ ID NO: 2) of the anti-human 4-1BB antibody scFv. The multispecific antibody of the present disclosure comprises the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 11, preferably 93% or more identical, more preferably 95% or more identical, and even more preferably 98% or more identical, and can be a polypeptide that costimulates T cells and exerts the effect of inhibiting the proliferation of cancer cells.

[0058] Another example of a multispecific antibody of the present disclosure is a polypeptide having the amino acid sequence shown in SEQ ID NO: 12. A multispecific antibody comprising the amino acid sequence of SEQ ID NO: 12 comprises an amino acid sequence in which a GGGGS linker sequence is arranged between the cleavable sequence portion and the masking moiety. Furthermore, the multispecific antibody of the present disclosure comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 12, preferably 93% or more identical, more preferably 95% or more identical, and even more preferably 98% or more identical, and can be a polypeptide that costimulates T cells and exerts the effect of inhibiting the proliferation of cancer cells.

[0059] <Cancer Growth Inhibitor> The multispecific antibody of the present disclosure has an excellent effect of inhibiting the growth of cancer cells. Thus, the present disclosure can provide a cancer growth inhibitor comprising the above-described multispecific antibody. The cancer cell growth inhibitory effect of the cancer growth inhibitor can be evaluated, for example, by a reduction in the number of tumor cells, a reduction in tumor size, tumor disappearance, a reduction in tumor burden, etc. in a mammalian, e.g., human, subject.

[0060] The cancer growth inhibitor according to the present disclosure can be used as a composition for cancer treatment. The amount of the cancer growth inhibitor contained in the composition can be adjusted appropriately depending on the type, location, and severity of the cancer, as well as the age, weight, and condition of the subject to be treated. The composition may be administered four times a day, three times a day, twice a day, once a day, every other day, every second day, every third day, every fourth day, every fifth day, once a week, every seventh day, every eighth day, every nine days, twice a week, once a month, or twice a month. The total number of administrations may be, for example, 1 to 30 times, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 to 30 times, but may also be administered more than 30 times.

[0061] The composition can be administered to a subject in need of cancer treatment using methods known to those skilled in the art, for example, by oral administration, local injection (including catheter administration), systemic injection, intravenous injection, or parenteral administration (e.g., transdermal administration, transmucosal administration, more specifically, nasal drops, eye drops, sublingual administration, suppositories, patches, etc.). For ease of handling, the composition may be formulated into a unit-dose injectable form (solution, suspension, emulsion). More specific examples of administration methods include intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intramedullary, intracardiac, intraarticular, intrasynovial, intracranial, intrathecal, and subarachnoid (spinal fluid) injection.

[0062] The composition can be administered as a sterile liquid preparation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which may be buffered to a predetermined pH. The liquid preparation may be a liquid preparation for injection. Furthermore, to extend the contact time with a specific tissue, the liquid preparation may be in the form of a viscous composition having a viscosity within an appropriate viscosity range. The liquid preparation may contain a solvent or dispersion medium such as water, saline, phosphate-buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and mixtures thereof.

[0063] The liquid preparations can be prepared by combining the cancer growth inhibitor of the present disclosure with various amounts of other ingredients in an appropriate amount of a suitable solvent. The liquid preparations may contain a suitable carrier, diluent, or excipient. The liquid preparations may also be lyophilized. Depending on the desired route of administration, the liquid preparations may further contain various auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity-enhancing additives, preservatives, flavoring agents, coloring agents, etc. For information on ingredients that may be contained in liquid preparations, please refer to "REMINGTON'S PHARMACEUTICAL SCIENCE," 17th Edition, 1985.

[0064] The liquid preparation may further contain various additives that enhance the stability and sterility of the liquid preparation, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid, may be used to prevent the action of microorganisms. The vehicle, diluent, or additive used in the liquid preparation must be compatible with the immunoresponsive cell A and / or immunosuppressive inhibitor of the present disclosure. The liquid preparation may be isotonic with blood. Isotonicity can be achieved by including sodium chloride or other pharmaceutically acceptable osmotic agents (e.g., dextrose, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes) in the liquid preparation.

[0065] When a composition containing the cancer growth inhibitor of the present disclosure is used to treat cancer, the subject of treatment may be, for example, any mammal, such as a primate, or more specifically, a human. The subject of treatment may also be a pet or livestock animal, such as a dog, cat, pig, cow, horse, sheep, or goat. The cancer to be treated may be either a solid cancer or a blood cancer, with solid cancer being particularly preferred. Examples of cancers to be treated include adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, lung cancer, tracheal cancer, bronchial cancer, colon cancer, small intestine cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, and ovarian cancer; cancers of bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue; sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma; blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma; germ cell tumors; lymphomas; and leukemia.

[0066] In particular, compositions containing the cancer growth inhibitor of the present disclosure are preferably used against solid cancers, for which so-called immunotherapy has been shown to be ineffective. Solid cancers are formed by the accumulation of cancer cells, and it has been pointed out that there are few immune cells infiltrating into the solid cancer, and furthermore, the immune cells are in a dysfunctional state known as "exhaustion." The multispecific antibody of the present disclosure described above is capable of activating T cells via the masking moiety, and therefore has an excellent effect of inhibiting the growth of cancer cells. Therefore, compositions containing the cancer growth inhibitor of the present disclosure are particularly effective in treating solid cancers.

[0067] Furthermore, a composition containing the cancer growth inhibitor of the present disclosure may be administered prophylactically to a subject in a situation where the presence of cancer cells in the subject is suspected, even before a definitive diagnosis of cancer has been made. In the present disclosure, such a use form is also encompassed in the concept of use for cancer treatment.

[0068] <Polynucleotides, Expression Vectors> To produce the multispecific antibodies of the present disclosure, first, an amino acid sequence containing the masking moiety, binding region, cleavable sequence, optionally a hinge region, and optionally an Fc region is designed, and then a nucleotide sequence encoding the amino acid sequence is designed. A polynucleotide containing the designed nucleotide sequence can be chemically synthesized in its entirety. Alternatively, the polynucleotide can be obtained by a technique such as a nucleic acid amplification reaction using an available vector as a template. The obtained polynucleotide can be stored as an expression vector capable of being expressed in an appropriate host cell.

[0069] Here, a host cell is a cell that can take up an expression vector into its cytoplasm and is used to maintain, replicate and amplify it. Host cells include mammalian, plant, insect, or fungal cells, such as bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; fungal cells such as yeast cells and Pichia pastoris; insect cells such as Drosophila and Spodoptera Sf9 cells; CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bowes melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells), or PERC. Examples of such cells include animal cells such as human retinal cells; or plant cells.

[0070] Furthermore, the expression vector is retained in any of the above-mentioned host cells and expresses a multispecific antibody having an amino acid sequence encoded by the above-mentioned polynucleotide. Specific examples of expression vectors can be selected from the group consisting of commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, and Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμμ, T-even, T2, T3, and T7; and plant viruses. Methods for introducing expression vectors into host cells are not particularly limited, and include calcium phosphate transfection, viral infection, DEAE-dextran-mediated transfection, lipofectamine transfection, and electroporation.

[0071] Multispecific antibodies expressed in host cells can be purified by known methods. Multispecific antibodies can be separated and purified using methods commonly used for polypeptides. For example, multispecific antibodies can be separated and purified by appropriately selecting and combining methods such as affinity chromatography columns, filters, ultrafiltration, salting out, dialysis, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing. The concentration of the obtained multispecific antibodies can be measured by measuring absorbance or enzyme-linked immunosorbent assay (ELISA), for example.

[0072] The present disclosure will be described in more detail below using examples, but the technical scope of the present disclosure is not limited to the following examples.

[0073] Example 1 Preparation of Multispecific Antibody 1 In this example, multispecific antibody 1 was prepared by linking an anti-4-1BB antibody (scFv) via a cleavable sequence. Multispecific antibody 1 is a diabody-type bispecific antibody prepared by combining the variable regions of anti-EGFR humanized monoclonal antibody 528 and anti-CD3 humanized monoclonal antibody OKT3, and the Fv sequence was humanized and Y52W, which improves affinity for EGFR, was introduced. Multispecific antibody 1 also has a hinge region and Fc region into which the H237Y mutation, which suppresses fragmentation during storage, has been introduced.

[0074] Multispecific antibody 1 is designated α-4-1BB scFv-MMP-Ex3-Fc(H237Y)-LH(Y52W), and its amino acid sequence is shown in SEQ ID NO: 10. The 22 amino acids from the N-terminus in SEQ ID NO: 10 are a signal peptide. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 10 is shown in SEQ ID NO: 13. The expression vector pCAGEN-α-4-1BB scFv-MMP-Ex3-Fc(H237Y)-LH(Y52W) containing the polynucleotide encoding multispecific antibody 1 was constructed by overlap PCR using pCAGEN-Ex3-Fc(H237Y)-LH(Y52W) and pRA1-anti-4-1BB scFv(HL) as templates. pCAGEN-Ex3-Fc(H237Y)-LH(Y52W) was used as disclosed in Asano, R. et al., Sci. Rep., 2020, 10, 1-8. pRA1-anti-4-1BB scFv(HL) was prepared by ligating anti-4-1BB scFv(HL), which was synthesized based on the sequence information (clone: ​​94G1) disclosed in U.S. Pat. No. 10,774,151B2, to pRA1.

[0075] As shown in FIG. 4 , pCAGEN-α-4-1BB scFv-MMP-Ex3-Fc(H237Y)-LH(Y52W) prepared in this example encodes a polypeptide in which, from the N-terminus to the C-terminus, the VH of anti-human-4-1BB antibody scFv (SEQ ID NO: 1), the VL of anti-human-4-1BB antibody scFv (SEQ ID NO: 2), a cleavable sequence (hereinafter referred to as the MMP recognition sequence; SEQ ID NO: 7), the VL of OKT3 (SEQ ID NO: 6), the VH constituting 528 and having Y52W (SEQ ID NO: 3), the VL constituting 528 (SEQ ID NO: 4), the VH of OKT3 (SEQ ID NO: 5), the hinge region (SEQ ID NO: 8), and the Fc region (SEQ ID NO: 9) are arranged in this order.

[0076] In this example, the resulting pCAGEN-α-4-1BB scFv-MMP-Ex3-Fc(H237Y)-LH(Y52W) was used to transform Expi293F cells, and multispecific antibody 1 was expressed using a transient expression system. As a preliminary preparation for the transformation, 27 mL of Expi293 Expression Medium was dispensed into a 50 mL conical tube and warmed in a 37°C incubator. Next, 120 μL of PEI MAX (1 mg / mL) [1:4 (w / w) DNA:PEI] was added to 1.5 mL of Opti-MEM medium and allowed to stand for 5 minutes. This was then mixed with 1.5 mL of Opti-MEM medium containing 30 μg of the above-mentioned expression vector and allowed to stand for 20 minutes. 75×10 ６ The mixture was added to 27 mL of Expi293F cells. ２ After culturing for 20 hours at 125 rpm, 75 μL of 0.5 M sodium valproate, 120 μL of 1 M sodium propionate, and 750 μL of 20% (w / v) tryptone were added as enhancers (Day 1), and the culture was continued for another 144 hours. The supernatant obtained by centrifugation at 300 × g for 5 minutes was used as the culture supernatant (Day 7).

[0077] The multispecific antibody 1 of interest was then purified from the culture supernatant of the resulting transformant by protein A affinity chromatography. This affinity chromatography method utilizes the specific binding of protein A to the Fc region of IgG at near-neutral pH, and is capable of eluting proteins having an Fc region under acidic conditions.

[0078] The culture supernatant was centrifuged at 4°C and 10,000 x g for 20 minutes to remove aggregated proteins. The pH of the resulting supernatant was adjusted by adding 1 M Tris-HCl (pH 9.2, final concentration 50 mM). First, 6 CV of MQ and equilibration buffer (pH 8.0) were added to an open column packed with rProtein A Sepharose. Next, the culture supernatant was added, followed by 6 CV of wash buffer (pH 8.0). 2 CV of Pierce ＴＭIgG elution buffer (pH 2.8) was added, and the fraction was collected in a container to which 150 μL of 1 M Tris-HCl (5% volume of the eluted fraction) had been added beforehand. After collection, 6 CV of equilibration buffer (pH 8.0) and 6 CV of MQ were added to the column, in that order, followed by the addition of 20% EtOH and storage at 4°C. The protein concentration of the eluted fraction was calculated by absorbance measurement. The protein concentration was calculated using the extinction coefficient and molecular weight obtained from the ProtParam tool (https: / / web.expasy.org / protparam / ) of Expasy, a website that calculates the physical and chemical parameters of proteins using amino acid sequences, using the following formula: Protein concentration (mg / mL) = {(A280-A320) / extinction coefficient} x molecular weight

[0079] As a result of SDS-PAGE after protein A affinity chromatography, bands were confirmed at around 104,000 and 105,000, which are the theoretical molecular weights of multispecific antibody 1 (not shown).

[0080] Example 2 Production of multispecific antibody 2 Multispecific antibody 2 was designed with the same configuration as in Example 1, except that a GGGGS sequence was inserted between the VL (SEQ ID NO: 2) and the cleavable sequence (MMP recognition sequence, SEQ ID NO: 7) of the anti-human 4-1BB antibody scFv. Transformants were then prepared in the same manner as in Example 1, and the multispecific antibody 2 of interest was purified from the culture supernatant of the transformant by protein A affinity chromatography in the same manner as in Example 1. The amino acid sequence of multispecific antibody 2 is shown in SEQ ID NO: 12. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 12 is shown in SEQ ID NO: 14.

[0081] Example 3 Production of multispecific antibody 3 Multispecific antibody 3 was designed with the same configuration as in Example 1, except that the C-terminal lysine was deleted from the VL (SEQ ID NO: 2) of the anti-human 4-1BB antibody scFv. A transformant was then prepared in the same manner as in Example 1, and the multispecific antibody 3 of interest was purified from the culture supernatant of the transformant by protein A affinity chromatography in the same manner as in Example 1. The amino acid sequence of multispecific antibody 3 is shown in SEQ ID NO: 11. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 11 is shown in SEQ ID NO: 15.

[0082] Comparative Example 1 Preparation of multispecific antibody 4 Multispecific antibody 4 was designed with the same configuration as in Example 1, except that the MMP recognition sequence (SEQ ID NO: 7) was replaced with a recognition sequence for the 3C protease (LEVLFQGP) derived from human rhinovirus type 14, a protease whose expression is not elevated in the vicinity of cancer cells. Transformants were then prepared in the same manner as in Example 1, and the multispecific antibody 4 of interest was purified from the culture supernatant of the transformant by protein A affinity chromatography in the same manner as in Example 1. The amino acid sequence of multispecific antibody 4 is shown in SEQ ID NO: 16. The nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 is shown in SEQ ID NO: 17.

[0083] [Cleavage test with collagenase] Collagenase type IV was used to evaluate the cleavage of multispecific antibodies 1 to 4. The collagenase type IV used in this example was derived from Clostridium histolyticum and was purchased from Abnova. This collagenase type IV is said to have high activity of MMP-2 and MMP-9, which are matrix proteases.

[0084] In this test, collagenase type IV was first added to 250 nM of multispecific antibodies 1 to 4 to a final concentration of 0.1 mg / mL (16 U / mL), and the mixture was incubated at 37°C for 0 to 24 hours. The multispecific antibodies 1 to 4 used were purified monomer fractions. After incubation, Western blotting was performed. Polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to standard methods. The gel, filter paper, and membrane were equilibrated with 1x transfer buffer. The filter paper, membrane, gel, and filter paper were stacked on the anode plate in this order, and the cathode plate was used to cover. Then, the mixture was subjected to transblotting using a Turbo ＴＭ The gel was transferred to a membrane using a transfer system. The membrane was blocked with 25 mL of 5% skimmed milk / PBS-T. After washing with PBS-T, 15 mL of 5% skimmed milk / PBS-T containing 1 μL of HRP-labeled anti-human IgG antibody was added and incubated for 30 minutes. After washing with PBS-T, chemiluminescence was detected using a detection reagent containing luminol and hydrogen peroxide.

[0085] <Results> Western blot results showed that for multispecific antibody 1 and multispecific antibody 3, the band intensity increased around 78,000, the theoretical molecular weight of the C-terminal fragment relative to the MMP recognition sequence, in an incubation time-dependent manner (not shown). Furthermore, after cleavage, a band was also observed around 26,000, the theoretical molecular weight of the N-terminal fragment relative to the MMP recognition sequence (not shown). These results demonstrate that the MMP recognition sequences in multispecific antibody 1 and multispecific antibody 3 were cleaved by collagenase type IV. Note that for multispecific antibody 2, in addition to the C-terminal and N-terminal fragments relative to the MMP recognition sequence, bands were also observed due to cleavage of the linker portion connecting VH and VL at 528 by collagenase type IV (not shown).

[0086] On the other hand, in the case of multispecific antibody 4, no cleavage of the masking moiety was detected at the incubation time when the MMP recognition sequences in multispecific antibodies 1 to 3 were cleaved, but nonspecific removal of the masking moiety was confirmed as the incubation time increased (not shown).

[0087] [Evaluation of binding ability to cancer cells and T cells] The binding activity to cancer cells and T cells was evaluated by flow cytometry using purified fractions of multispecific antibodies 1 to 4 and Ex3-Fc(H237Y)-LH(Y52W) (Asano, R. et al., Sci. Rep., 2020, 10, 1-8) before and after collagenase type IV digestion. In the assessment of binding ability to cancer cells, all of multispecific antibodies 1 to 3 showed binding ability similar to that of Ex3-Fc(H237Y)-LH(Y52W) both before and after collagenase type IV digestion (Figure 5). This suggests that masking by the masking moiety does not inhibit binding to cancer cells.

[0088] On the other hand, it was confirmed that multispecific antibodies 1 to 3 had reduced T cell binding ability compared to Ex3-Fc(H237Y)-LH(Y52W) before collagenase type IV digestion, whereas after collagenase type IV digestion, they had binding ability equivalent to that of Ex3-Fc(H237Y)-LH(Y52W) ( Figure 6 ). These results demonstrate that multispecific antibodies 1 to 3 have low T cell binding ability before cleavage of the MMP recognition sequence, and that their T cell binding ability becomes apparent after cleavage of the MMP recognition sequence. Furthermore, multispecific antibody 4 after collagenase type IV digestion was also observed to have T cell binding ability, although not as strong as multispecific antibodies 1 to 3. As described in the results of the collagenase cleavage test, this is presumed to be due to nonspecific removal of the masking moiety. Furthermore, multispecific antibodies 1 to 3 had similar binding abilities to T cells, suggesting that the length of the portion containing the MMP recognition sequence does not affect the masking effect.

[0089] [Evaluation of binding ability before and after freeze-thawing] To evaluate the storage stability of multispecific antibodies 1 to 3, flow cytometry was performed before and after freeze-thawing of the Ex3-Fc(H237Y)-LH(Y52W), multispecific antibodies 1, 3, and 4. First, the antibody concentration decreased by about 30% before and after freeze-thawing in all cases, suggesting the possibility that some of the antibodies had aggregated.

[0090] Subsequently, in an assessment of binding ability to cancer cells, both multispecific antibody 1 and multispecific antibody 3 showed binding ability equivalent to that of Ex3-Fc(H237Y)-LH(Y52W) both before and after collagenase type IV digestion, regardless of whether or not they were freeze-thawed ( Figure 7 ). These results suggest that multispecific antibodies 1 to 3 are not affected by freeze-thawing and that masking by the masking moiety does not inhibit binding to cancer cells. In contrast, multispecific antibody 4 showed decreased binding ability to cancer cells after freeze-thawing. These results suggest that multispecific antibody 4 may have lower structural stability than multispecific antibodies 1 to 3.

[0091] On the other hand, the binding ability of Ex3-Fc(H237Y)-LH(Y52W) to T cells did not change before and after freeze-thawing ( Figure 8 ). In contrast, the binding ability of multispecific antibodies 1, 3, and 4 decreased after freeze-thawing. However, after collagenase type IV digestion, they retained binding ability equivalent to that before freeze-thawing. Therefore, it is possible that the masking moieties in multispecific antibodies 1, 3, and 4 have a structure that further covers the antigen-binding site consisting of the VH and VL of OKT3 in the freeze-thawed samples. In particular, frozen storage is more useful due to the excellent masking effect of the masking moieties. These findings suggest that the structural stability of multispecific antibodies 1 to 3 is maintained even after freeze-thawing, and that freeze-thawing may improve the masking effect.

[0092] [T cell proliferation test] The effects of multispecific antibodies 1 to 3 on T cell proliferation were evaluated. In this example, a T cell proliferation test was performed using WST-8. In this test, first, 5.0 × 10 cells were placed in each well of a 96-well plate. ４ T-LAK cells were seeded at 100 cells / 50 μL and incubated at 37°C, 5% CO ２ The plates were incubated overnight at 37°C, 5% CO. Then, 50 μL of the sample, the concentration of which had been adjusted with the medium, was added to each well (the amount of PBS carried over from the sample to the medium was 10% or less). Additionally, wells containing 50 μL of T-LAK cells and 50 μL of medium were also prepared to measure the basal activity of T-LAK cells. ２ After incubation at 450 nm for about 48 hours, 10 μL of the detection reagent WST-8 was added, and the mixture was further incubated for 18 hours at 37° C. The effect on T cell proliferation was evaluated by measuring the absorbance at 450 nm and 655 nm using a plate reader.

[0093] As a result, before collagenase type IV digestion, multispecific antibodies 1 to 3 all had lower T cell proliferation potency than Ex3-Fc(H237Y)-LH(Y52W) due to the masking effect of the masking moiety. In contrast, after collagenase type IV digestion, the effect of multispecific antibodies 1 to 3 to proliferate T cells became apparent ( Figure 9 ).

[0094] Furthermore, multispecific antibody 1 and multispecific antibody 3 had higher T cell proliferation potency than Ex3-Fc(H237Y)-LH(Y52W) after collagenase type IV digestion. This was presumably because the released masking moiety was anti-4-1BB scFv(HL), which transmitted a costimulatory signal to T cells. Note that multispecific antibody 2 showed T cell proliferation potency equivalent to that of Ex3-Fc(H237Y)-LH(Y52W) after collagenase type IV digestion.

[0095] These findings strongly suggest the possibility that Multispecific Antibodies 1 to 3 can specifically activate T cells in the vicinity of cancer cells secreting matrix proteases that specifically cleave MMP recognition sequences.

[0096] [Cancer cell cytotoxicity test by MTS assay] Multispecific antibodies 1 to 3 were evaluated for their cytotoxicity against cancer cells by MTS assay. In this test, TFK-1 cells were used as target cells and T-LAK cells as effector cells. First, 5 × 10 TFK-1 cells were cultured per well. ３ The cells were seeded into a 96-well plate at a concentration of 100 μL and incubated at 37°C in 5% CO ２ The cells were incubated overnight at 37°C, 5% CO. Without removing the culture supernatant, T-LAK cells adjusted to the desired E / T ratio (ratio of effector cell number to target cell number) were added at 25 μL / well. Furthermore, samples whose concentrations had been adjusted with medium were added at 25 μL / well (the amount of PBS carried over from the sample to the medium was 10% or less). Also prepared were wells containing 100 μL of MQ as a negative control, 150 μL of medium as a positive control, and 25 μL of T-LAK cells and 25 μL of medium to measure the basal activity of T-LAK cells. The incubation temperature was 37°C, 5% CO. ２ The plates were incubated at 4°C for approximately 20 hours. Next, the culture medium was removed by aspiration, and the plates were washed three times with 100 μL / well of PBS. 100 μL / well of a mixture of 1 mL of MTS reagent and 11 mL of medium was added, and the plates were incubated at 37°C for approximately 30 to 60 minutes, after which the absorbance at 490 nm was measured using a plate reader. The wells containing only the medium were incubated until the absorbance reached approximately 0.8, and the cancer cell cytotoxicity was calculated using the detected data.

[0097] The results of cancer cytotoxicity tests using an MTS assay showed that multispecific antibodies 1 to 3 had lower cancer cytotoxicity than Ex3-Fc(H237Y)-LH(Y52W) before collagenase type IV digestion due to the masking effect of the masking moiety, but their cytotoxicity against cancer cells became apparent after collagenase type IV digestion ( Figure 10 ). Furthermore, multispecific antibodies 1 and 3 exhibited higher cancer cytotoxicity than Ex3-Fc(H237Y)-LH(Y52W) after collagenase type IV digestion. This is presumably because the released masking moiety is anti-4-1BB scFv(HL), which further activated costimulated T cells, thereby enhancing their cytotoxicity against cancer cells. Furthermore, multispecific antibody 2 exhibited cancer cell cytotoxicity equivalent to that of Ex3-Fc(H237Y)-LH(Y52W) after collagenase type IV digestion. These findings strongly suggest that multispecific antibodies 1 to 3 may specifically activate T cells in the vicinity of cancer cells secreting matrix proteases that specifically cleave MMP recognition sequences, and as a result, exhibit superior cytotoxicity against cancer cells.

[0098] [Evaluation of cleavage by matrix proteases secreted by cancer cells] Cleavage of the MMP recognition sequences contained in multispecific antibodies 1 to 3 by matrix proteases secreted by cancer cells was evaluated. In this test, HT1080 cells, A431 cells, and TFK-1 cells were used as cancer cells. 5 × 10 cells were used per well. ３ The cells were seeded into a 96-well plate at a concentration of 100 μL and incubated at 37°C in 5% CO ２ The cells were incubated overnight at 37°C in 5% CO. Then, the culture supernatant was collected and centrifuged at 300 x g for 5 minutes. The supernatant after centrifugation and 100 nM of multispecific antibody 3 or multispecific antibody 4 were added, and the cells were incubated at 37°C in 5% CO. ２The cells were incubated overnight at RT-PCR. Western blotting was then performed using an HRP-labeled anti-human IgG antibody as a detection antibody. As controls, 100 nM of multispecific antibody 3 or multispecific antibody 4 was added before incubation and in the absence of cells, and the cells were incubated in the same manner.

[0099] Western blot results showed that when HT1080 cells and A431 cells were used, a band corresponding to Ex3-Fc(H237Y)-LH(Y52W) was observed for multispecific antibody 3 (not shown). This indicates that matrix proteases secreted from HT1080 cells and A431 cells specifically cleaved the MMP recognition sequence. On the other hand, when TFK-1 cells were used, no band corresponding to Ex3-Fc(H237Y)-LH(Y52W) was observed (not shown). This is thought to be due to the low amount of matrix proteases secreted from TFK-1 cells.

[0100] Furthermore, cancer cytotoxicity tests against HT1080 cells and A431 cells were performed on multispecific antibody 3 and multispecific antibody 4 according to the method described above. The results showed that multispecific antibody 3 exhibited higher cytotoxicity against cancer cells than multispecific antibody 4 ( FIG. 11 ). These results indicate that the MMP recognition sequence contained in multispecific antibody 3 was cleaved by matrix proteases secreted from cancer cells, while the 3C protease recognition sequence contained in multispecific antibody 4 was not cleaved.

[0101] Furthermore, multispecific antibody 3 exhibited higher cytotoxic activity against cancer cells than Ex3-Fc(H237Y)-LH(Y52W). This is thought to be because the masking moiety cleaved by matrix protease is anti-4-1BB scFv, which functions as a costimulatory molecule and further activates T cells. When TFK-1 cells were used, multispecific antibody 3 and multispecific antibody 4 exhibited similar levels of cytotoxic activity, with no significant difference observed.

[0102] From the above, it was demonstrated that in multispecific antibodies 1 to 3, the MMP recognition sequence is cleaved in the vicinity of cancer cells secreting matrix proteases, and the released masking moiety can function as a costimulatory molecule for T cells. As a result, it was revealed that multispecific antibodies 1 to 3 exhibit excellent cytotoxic activity against the cancer cells.

[0103] The disclosure of Japanese Patent Application No. 2024-085882, filed on May 27, 2024, is incorporated herein by reference in its entirety. In addition, all documents, patent applications, and technical standards mentioned in this disclosure are incorporated by reference into this disclosure to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

[0104] 1...first antigen-binding site, 2...second antigen-binding site, 3...binding region, 4...cleavable sequence portion, 5...masking portion, 6...cancer cell, 7...protease, 8...T cell

Claims

1. A multispecific antibody comprising: a binding region comprising a first antigen-binding site that binds to cancer cells and a second antigen-binding site that binds to T cells; a cleavable sequence linked to the binding region; and a masking moiety that, when the binding region is composed of a single-chain antibody, is linked to the N-terminus of the single-chain antibody via the cleavable sequence, or, when the binding region is composed of a heavy chain and a light chain, is linked to the C-terminus of the light chain via the cleavable sequence, wherein the masking moiety functions as an agonist of a costimulatory molecule for the T cells or an antagonist of a co-inhibitory molecule for the T cells when the cleavable sequence is cleaved.

2. The multispecific antibody of claim 1, wherein the masking moiety is a low-molecular-weight antibody having agonistic function corresponding to one ligand selected from the group consisting of 4-1BBL, B7-H2, B7-1, B7-2, CD70, LIGHT, CD40L, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155, and CD112.

3. The multispecific antibody according to claim 2, wherein the low-molecular-weight antibody is a deletion type in which 1 to 5 amino acid residues are deleted from the C-terminus.

4. The multispecific antibody of claim 1, wherein the first antigen-binding site comprises a first heavy chain variable region and a first light chain variable region, the second antigen-binding site comprises a second heavy chain variable region and a second light chain variable region, and the binding region comprises, from N-terminus to C-terminus, the second light chain variable region, the first heavy chain variable region, the first light chain variable region, and the second heavy chain variable region.

5. The multispecific antibody of claim 1, wherein the first antigen-binding site targets a cancer cell surface receptor for binding.

6. The cancer cell surface receptor is EGFR, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, FGF-2, FGFR1, FGFR2, FGFR3, FGFR4, HER2 / neu, DLL4, NOTCHR1, IL1B, IL1R, IL2, IL2R, IL4, IL6, IL12, IL13, IL15, IL18, IL23, IGF, IGF1R, ERBB3, VCAM-1, CXCR4, CD3, CD11a, CD19, CD20, CD22, CD25, CD28, CD30, C 6. The multispecific antibody of claim 5, wherein the receptor is one selected from the group consisting of D33, CD40, CD40L, CD41, CD44, CD52, CD80, CD86, CTLA-4, TNFα, TNFR, TRAIL-R1, TRAIL-R2, IgE, IgE receptor, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ, GPIIB / IIIA, CLAUDIN-3, CLAUDIN-4, C5 complement, RSV F protein, glycoprotein IIb / IIIa receptor, α4β1 integrin, and α4β7 integrin.

7. The multispecific antibody of claim 1, wherein the first antigen-binding site targets EGFR.

8. The multispecific antibody of claim 1, wherein the second antigen-binding site targets a T-cell surface molecule for binding.

9. The multispecific antibody of claim 8, wherein the T cell surface molecule is one molecule selected from the group consisting of CD2, CD3, CD5, CD7, CD43, CD4 and CD8.

10. The multispecific antibody of claim 1, wherein the second antigen-binding site targets CD3.

11. The multispecific antibody according to claim 1, wherein the cleavable sequence has a recognition sequence for a protease secreted by cancer cells.

12. The multispecific antibody of claim 11, wherein the protease is a matrix metalloprotease.

13. The multispecific antibody of claim 1, having an Fc region comprising heavy chain constant region 2 and heavy chain constant region 3.

14. A cancer growth inhibitor comprising the multispecific antibody according to any one of claims 1 to 13.

15. A polynucleotide encoding a multispecific antibody according to any one of claims 1 to 13.

16. An expression vector comprising the polynucleotide of claim 15.

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