Polypeptides comprising immunoglobulin single variable domains targeting glypican-3 and t cell receptor

Abstract

The present invention aims to provide novel drugs for treating subjects suffering from cancer. Specifically, the technology provides polypeptides comprising at least four immunoglobulin single variable domains (ISVDs), characterized in that one ISVD binds to the TCR and at least two ISVDs bind to GPC3. The present invention also provides nucleic acids, vectors, and compositions.

Description

Technical Field

[0001] This invention relates to polypeptides targeting phosphatidylinositol proteoglycan-3 (GPC3) and the T-cell receptor (TCR). The invention also relates to nucleic acid molecules encoding said polypeptides and carriers containing said nucleic acids, as well as compositions containing said polypeptides, nucleic acids, or carriers. The invention further relates to these products in methods for treating subjects suffering from diseases involving abnormal cells such as cancerous cells or infected cells. Furthermore, the invention relates to methods for producing these products. Prior Technology

[0002] Cytotoxic T cells (CTLs) are T lymphocytes that kill cancer cells, infected cells (especially those infected by viruses), or cells damaged in other ways. T lymphocytes (also called T cells) exhibit T cell receptors (TCRs) and CD3 receptors on their cell surface. The αβ TCR-CD3 complex (or "TCR complex") consists of six distinct type I single transmembrane proteins: TCRα and TCRβ chains that form the TCR heterodimer responsible for ligand recognition, and non-covalently associated CD3γ, CD3δ, CD3ε, and ζ chains with cytoplasmic sequence motifs that undergo tyrosine phosphorylation upon receptor activation and recruit a large number of signal transduction components (Call et al. 2004, Molecular Immunology 40: 1295-1305).

[0003] Both the α and β chains of the heterodimeric T cell receptor (TCR) consist of constant and variable domains. T cells are activated upon recognition of a homologous peptide presented by their own MHC molecules by the TCR, accompanied by signaling activation of the CD3 complex via tyrosine phosphorylation, leading to T cell proliferation and differentiation.

[0004] Bispecific antibodies have been engineered to have a tumor-recognizing portion on one arm (the target-binding arm) and an effector-binding arm (the other arm of the molecule) specific for a T-cell antigen (typically CD3). These bispecific antibodies, known as T-cell binders (TCEs), are multi-targeting molecules that enhance a patient's immune response to malignant cells. Co-binding of multispecific antibodies with both T cells and tumor cells leads to the formation of cytolytic synapses between the T cells and tumor cells, which induce T-cell activation and result in tumor cell killing.

[0005] Although most bispecific antibodies targeting activated T cells target the CD3 complex on T cells, some bispecific binders that target the constant domain of the αβ T cell receptor have been described in WO 2016 / 180969 A1.

[0006] Phosphatidylinositol proteoglycan-3 (GPC3) is a GPI-anchored cell surface glycoprotein composed of heparan sulfate GAG ​​chains and a core protein. It is involved in embryogenesis and early development, controlling cell growth and differentiation. Although GPC3 expression is high during development, it is almost absent in normal adult tissues and exhibits moderate / low expression in proximal renal tubules and bronchial cells. Consistent with its role in early development, high levels of GPC3 are also observed in the placenta.

[0007] GPC3 similarly regulates early development through multiple signaling cascades, including the Wnt, Hh, and YAP pathways. Furthermore, GPC3 can interact with essential growth factors such as FGF2 to regulate cell growth. In experimental settings, overexpression of GPC3 can inhibit FGF2-induced cell proliferation. In contrast, GPC3 also negatively regulates the inhibitory growth factor BMP7. In conclusion, GPC3 activity may be highly correlated with background activity, as it not only inhibits cell proliferation but may also promote oncogenesis in liver cancer and other forms of cancer.

[0008] GPC3 is particularly prevalent in tumor tissues from hepatocellular carcinoma (HCC), a disease with significant unmet needs. Soluble GPC3 can also be detected in the serum of HCC patients and has been used to differentiate liver disease from various etiologies.

[0009] Various forms of bispecific antibody constructs have been proposed. For example, bispecific antibody forms may involve the chemical conjugation of two antibodies or fragments thereof (Brennan, M et al., Science, 1985. 229(4708): 81-83; Glennie, MJ et al., J Immunol, 1987. 139(7): 2367-2375).

[0010] However, such bispecific antibody forms have drawbacks including high molecular weight and high viscosity at high concentrations, making subcutaneous administration challenging, for example. Furthermore, each binding unit requires the interaction of two variable domains to achieve specific and high-affinity binding, which impacts peptide stability and production efficiency. Such bispecific antibody forms may also potentially lead to CMC problems associated with low production efficiency, low titers, and / or light chain or heavy chain mismatches.

[0011] Therefore, there is a need for antibody constructs that bind to both target cells and T cells with sufficient affinity to induce cytotoxic responses. Simultaneously, such constructs should not induce cytotoxic responses in non-target cells, i.e., cells that do not express the target antigen or express it only at low levels. This allows for a balance between efficacy and safety. Furthermore, it is desirable that such constructs be efficiently produced, for example, in a microbial host. Ideally, such constructs should also exhibit a sufficiently long half-life in the subjects to be treated to facilitate the spacing of consecutive treatments. Moreover, it is desirable to limit the reactivity of such constructs with pre-existing antibodies in the subjects to be treated (i.e., antibodies present in the subjects prior to the first treatment with the antibody construct). Furthermore, the peptide should not exert undesirable side effects or should exert only minimal undesirable side effects, such as side effects caused by cytotoxic activity against non-target cells. Summary of the Invention

[0012] The inventors of this invention have discovered that peptides that simultaneously and specifically target GPC3 and TCR result in effective T cell-mediated killing of GPC3-expressing cells in vitro. These peptides can be efficiently produced (e.g., in a microbial host). Furthermore, such peptides can exhibit limited reactivity with pre-existing antibodies in the subject to be treated (i.e., antibodies present in the subject prior to initial treatment with the antibody construct). In a preferred embodiment, such peptides exhibit a half-life in the subject to be treated long enough to facilitate the spacing of consecutive treatments. Moreover, such peptides exhibit limited activity only against cells that do not express GPC3 or express low levels of GPC3. This suggests the potential to induce a highly specific T cell-mediated cytotoxic response against GPC3-positive cancer target cells while exhibiting a favorable safety profile.

[0013] In one embodiment, the polypeptide comprises or consists of at least three immunoglobulin single variable domains (ISVDs), wherein at least two ISVDs specifically bind to GPC3 and one ISVD specifically binds to a constant domain of the TCR on T cells. Preferably, the at least two ISVDs specifically binding to GPC3 specifically bind to human GPC3, and the ISVD specifically binding to the TCR specifically binds to human TCR. More preferably, the at least two ISVDs specifically binding to GPC3 are different ISVDs. In another embodiment, the polypeptide comprising or consists of at least three ISVDs preferably further comprises one or more other groups, residues, portions, or binding units, which are optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions, or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, portions, or binding units. For example, the binding unit may be an ISVD that binds to serum proteins, preferably to human serum proteins such as human serum albumin.

[0014] In one embodiment, the present invention provides a polypeptide comprising or composed of at least one immunoglobulin single variable domain (ISVD) specifically bound to GPC3. In another embodiment, the polypeptide of the present invention comprises or composed of at least two ISVDs specifically bound to GPC3, wherein the two ISVDs are optionally linked via peptide linkers. Preferably, the two ISVDs specifically bound to GPC3 are different ISVDs. Furthermore, the polypeptide preferably also comprises one or more other groups, residues, portions, or binding units, which are optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions, or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, portions, or binding units. For example, the binding unit may be an ISVD bound to a serum protein, preferably a human serum protein such as human serum albumin.

[0015] In another embodiment, the polypeptide of the present invention comprises or consists of an ISVD that specifically binds to a constant domain of the TCR on T cells and at least one ISVD that specifically binds to GPC3, wherein the two ISVDs are optionally linked via a peptide linker. Such a polypeptide can be used to redirect T cells to kill cells expressing GPC3. Ideally, the ISVD that binds to the TCR is the only binding portion contained in the polypeptide that specifically binds to, for example, human T cells. Furthermore, examples show that when the ISVD that specifically binds to the TCR is located at the N-terminus of such a polypeptide, better T cell-mediated cytotoxicity is achieved compared to when the same anti-TCR ISVD is not located at the N-terminus. The ISVD that specifically binds to the TCR is therefore preferably located at the N-terminus of at least one ISVD that specifically binds to GPC3. Most preferably, the ISVD that specifically binds to the TCR is located at the N-terminus of the polypeptide containing the ISVD. Furthermore, the polypeptide preferably also comprises one or more other groups, residues, portions, or binding units, which are optionally linked via one or more peptide linkers, wherein the one or more other groups, residues, portions, or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, portions, or binding units. For example, the binding unit may be an ISVD that binds to a serum protein, preferably to a human serum protein such as human serum albumin.

[0016] The invention also provides a nucleic acid molecule capable of representing the polypeptide, a nucleic acid or carrier comprising the nucleic acid, and a composition comprising the polypeptide, the nucleic acid, or the carrier. The composition is preferably a pharmaceutical composition.

[0017] It also provides a host cell or (non-human) host containing a nucleic acid or vector encoding a polypeptide as disclosed herein.

[0018] Furthermore, a method for generating polypeptides as disclosed herein is provided, the method comprising at least the following steps: a. Expressing the nucleic acid sequence encoding the polypeptide in a suitable host cell or host organism or in another suitable expression system; optionally, this is performed subsequently: b. Isolate and / or purify the polypeptide.

[0019] Furthermore, the present invention provides the polypeptide, a composition comprising the polypeptide, or a composition comprising a nucleic acid or carrier containing a nucleotide sequence encoding the polypeptide, for use as a drug. Preferably, the polypeptide or composition is used for the treatment of cancer, such as liver cancer or lung cancer.

[0020] Additionally, a method for treating cancer is provided, wherein the method includes administering to a subject in need a pharmaceutically active amount of a polypeptide or composition according to this disclosure. The cancer is preferably selected from liver cancer or lung cancer. In some embodiments, the method further includes administering one or more additional therapeutic agents.

[0021] Further, the use of the said polypeptide or composition in the preparation of pharmaceutical compositions for the treatment of cancer (preferably liver cancer or lung cancer) is provided.

[0022] Specifically, the present invention provides the following embodiments:

[0023] Example 1. A polypeptide comprising at least three immunoglobulin single variable domains (ISVDs) or thereof, wherein each of the ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the at least three ISVDs are optionally linked via one or more peptide linkers, and wherein: a) The first ISVD contains i. CDR1 as the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 6; ii. CDR2 as the amino acid sequence of SEQ ID NO: 10 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 10; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 14 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 14; b) The second ISVD contains iv. CDR1 as the amino acid sequence of SEQ ID NO: 7 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 7; v. CDR2 as the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 11; and vi. CDR3 as the amino acid sequence of SEQ ID NO: 15 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 15; and c) The third ISVD contains vii. CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 8; viii. CDR2 as the amino acid sequence of SEQ ID NO: 12 or having an amino acid sequence that differs from SEQ ID NO: 12 in two or one amino acid; and ix. CDR3, which is the amino acid sequence of SEQ ID NO: 16 or has an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16. The order of the ISVDs indicates the relative positions of the ISVDs to each other from the N-terminus to the C-terminus of the polypeptide, wherein the first ISVD is optionally located at the N-terminus of the polypeptide.

[0024] Example 2. The polypeptide according to Example 1, wherein: a) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 6, CDR2 as the amino acid sequence of SEQ ID NO: 10, and CDR3 as the amino acid sequence of SEQ ID NO: 14; b) The second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15; and c) The third ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16.

[0025] Example 3. A polypeptide according to any one of Examples 1 or 2, wherein: a) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 2; b) The amino acid sequence of the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 3; and c) The amino acid sequence of the third ISVD exhibits more than 90% sequence identity with SEQ ID NO: 4.

[0026] Example 4. The polypeptide according to any one of Examples 1 to 3, wherein: a) The first ISVD consists of the amino acid sequence of SEQ ID NO: 2; b) The second ISVD consists of the amino acid sequence of SEQ ID NO: 3; and c) The third ISVD consists of the amino acid sequence of SEQ ID NO: 4.

[0027] Example 5. A polypeptide according to any one of Examples 1 to 4, wherein the first ISVD and the second ISVD are linked to each other via a linker consisting of fewer than 10 amino acids, preferably fewer than 6 amino acids, wherein the linker is preferably a 5GS linker.

[0028] Example 6. A polypeptide according to any one of Examples 1 to 5, wherein the polypeptide further comprises one or more other groups, residues, portions or binding units, the one or more other groups, residues, portions or binding units optionally being linked via one or more peptide linkers, wherein the one or more other groups, residues, portions or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, portions or binding units.

[0029] Example 7. The polypeptide according to Example 6, wherein the one or more other groups, residues, portions or binding units that provide the polypeptide with an increased half-life are selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc portions, and small proteins or peptides that can bind to serum proteins.

[0030] Example 8. A polypeptide according to any one of Examples 6 or 7, wherein the one or more other binding units providing the increased half-life of the polypeptide are selected from binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

[0031] Example 9. The polypeptide according to Example 8, wherein the binding unit that provides the increased half-life of the polypeptide is an ISVD bound to human serum albumin.

[0032] Example 10. The polypeptide according to Example 9, wherein the ISVD bound to human serum albumin comprises i. CDR1 as the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 9; ii. CDR2 as the amino acid sequence of SEQ ID NO: 13 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 13; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 17 or having an amino acid sequence that differs from SEQ ID NO: 17 in two or one amino acid.

[0033] Example 11. The polypeptide according to any one of Examples 9 or 10, wherein the ISVD bound to human serum albumin comprises CDR1 as the amino acid sequence of SEQ ID NO: 9, CDR2 as the amino acid sequence of SEQ ID NO: 13, and CDR3 as the amino acid sequence of SEQ ID NO: 17.

[0034] Example 12. The polypeptide according to any one of Examples 9 to 11, wherein the amino acid sequence of the ISVD bound to human serum albumin exhibits more than 90% sequence identity with SEQ ID NO: 5.

[0035] Example 13. The polypeptide according to any one of Examples 9 to 12, wherein the ISVD bound to human serum albumin consists of the amino acid sequence of SEQ ID NO: 5.

[0036] Example 14. A polypeptide according to any one of Examples 1 to 13, wherein the polypeptide comprises or is composed of an amino acid sequence exhibiting more than 90% sequence identity with SEQ ID NO: 1.

[0037] Example 15. A polypeptide according to any one of Examples 1 to 14, wherein the polypeptide comprises or is composed of the amino acid sequence of SEQ ID NO: 1.

[0038] Example 16. A polypeptide comprising at least one immunoglobulin single variable domain (ISVD) or thereof, wherein the ISVD comprises three complementarity-determining regions (CDR1 to CDR3, respectively), and wherein the at least one ISVD comprises: a) CDR1 as the amino acid sequence of SEQ ID NO: 7 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 7; CDR2 is the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 11; and CDR3 is the amino acid sequence of SEQ ID NO: 15 or has an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 15, or b) CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 8; CDR2 is the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 12; and CDR3 is the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16.

[0039] Example 17. The polypeptide according to Example 16, wherein the at least one ISVD comprises: a) CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15, or b) CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16.

[0040] Example 18. A polypeptide according to any one of Examples 16 or 17, wherein the amino acid sequence of the at least one ISVD comprises: a) Sequence identity with SEQ ID NO: 3 exceeding 90%, or b) Sequence identity with SEQ ID NO: 4 exceeding 90%.

[0041] Example 19. A polypeptide according to any one of Examples 16 to 18, wherein the at least one ISVD comprises or is composed of the following: a) The amino acid sequence of SEQ ID NO: 3, or b) The amino acid sequence of SEQ ID NO: 4.

[0042] Example 20. A polypeptide comprising at least two ISVDs or thereof, wherein each of the ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the at least two ISVDs are optionally linked via one or more peptide linkers, and wherein: a) The first ISVD and the second ISVD contain i. CDR1 as the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 7; ii. CDR2 as the amino acid sequence of SEQ ID NO: 11 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 11; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 15 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 15. b) The first ISVD and the second ISVD contain i. CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 8; ii. CDR2 as the amino acid sequence of SEQ ID NO: 12 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 12; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 16 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16. c) The first ISVD contains i. CDR1 as the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 7; ii. CDR2 as the amino acid sequence of SEQ ID NO: 11 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 11; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 15 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 15, and The second ISVD contains iv. CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 8; v. CDR2 as the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 12; and vi. CDR3 as the amino acid sequence of SEQ ID NO: 16 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16. d) The first ISVD contains i. CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 8; ii. CDR2 as the amino acid sequence of SEQ ID NO: 12 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 12; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 16 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16, and The second ISVD contains iv. CDR1 as the amino acid sequence of SEQ ID NO: 7 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 7; v. CDR2 as the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 11; and vi. CDR3 as the amino acid sequence of SEQ ID NO: 15 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 15. e) The first ISVD contains i. CDR1 as the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 6; ii. CDR2 as the amino acid sequence of SEQ ID NO: 10 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 10; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 14 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 14, and The second ISVD contains iv. CDR1 as the amino acid sequence of SEQ ID NO: 7 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 7; v. CDR2 as the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 11; and vi. CDR3 as the amino acid sequence of SEQ ID NO: 15 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 15. f) The first ISVD contains i. CDR1 as the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 7; ii. CDR2 as the amino acid sequence of SEQ ID NO: 11 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 11; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 15 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 15, and The second ISVD contains iv. CDR1 as the amino acid sequence of SEQ ID NO: 6 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 6; v. CDR2 as the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 10; and vi. CDR3 as the amino acid sequence of SEQ ID NO: 14 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 14. g) The first ISVD contains i. CDR1 as the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 6; ii. CDR2 as the amino acid sequence of SEQ ID NO: 10 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 10; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 14 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 14, and The second ISVD contains iv. CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 8; v. CDR2 as the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 12; and vi. CDR3 as the amino acid sequence of SEQ ID NO: 16 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16, or h) The first ISVD contains i. CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 8; ii. CDR2 as the amino acid sequence of SEQ ID NO: 12 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 12; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 16 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16, and The second ISVD contains iv. CDR1 as the amino acid sequence of SEQ ID NO: 6 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 6; v. CDR2 as the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 10; and vi. CDR3 as the amino acid sequence of SEQ ID NO: 14 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 14. The order of the ISVDs indicates their relative positions to each other, from the N-terminus to the C-terminus of the polypeptide.

[0043] Example 21. The polypeptide according to Example 20, wherein: a) The first ISVD and the second ISVD comprise CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15. b) The first ISVD and the second ISVD comprise CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16. c) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15, and the second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16. d) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16, and the second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15. e) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 6, CDR2 as the amino acid sequence of SEQ ID NO: 10, and CDR3 as the amino acid sequence of SEQ ID NO: 14, and the second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15. f) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15, and the second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 6, CDR2 as the amino acid sequence of SEQ ID NO: 10, and CDR3 as the amino acid sequence of SEQ ID NO: 14. g) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 6, CDR2 as the amino acid sequence of SEQ ID NO: 10, and CDR3 as the amino acid sequence of SEQ ID NO: 14, and the second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16, or h) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16, and the second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 6, CDR2 as the amino acid sequence of SEQ ID NO: 10, and CDR3 as the amino acid sequence of SEQ ID NO: 14.

[0044] Example 22. The polypeptide according to any one of Examples 20 or 21, wherein: a) The amino acid sequences of the first ISVD and the second ISVD exhibit more than 90% sequence identity with SEQ ID NO: 3. b) The amino acid sequences of the first ISVD and the second ISVD exhibit more than 90% sequence identity with SEQ ID NO: 4. c) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 3, and the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 4. d) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 4, and the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 3. e) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 2, and the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 3. f) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 3, and the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 2. g) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 2, and the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 4, or h) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 4, and the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 2.

[0045] Example 23. The polypeptide according to any one of Examples 20 to 22, wherein: a) The first ISVD and the second ISVD consist of the amino acid sequence of SEQ ID NO: 3. b) The first ISVD and the second ISVD consist of the amino acid sequence of SEQ ID NO: 4. c) The first ISVD consists of the amino acid sequence of SEQ ID NO: 3, and the second ISVD consists of the amino acid sequence of SEQ ID NO: 4. d) The first ISVD consists of the amino acid sequence of SEQ ID NO: 4, and the second ISVD consists of the amino acid sequence of SEQ ID NO: 3. e) The first ISVD consists of the amino acid sequence of SEQ ID NO: 2, and the second ISVD consists of the amino acid sequence of SEQ ID NO: 3. f) The first ISVD consists of the amino acid sequence of SEQ ID NO: 3, and the second ISVD consists of the amino acid sequence of SEQ ID NO: 2. g) The first ISVD consists of the amino acid sequence of SEQ ID NO: 2, and the second ISVD consists of the amino acid sequence of SEQ ID NO: 4, or h) The first ISVD consists of the amino acid sequence of SEQ ID NO: 4, and the second ISVD consists of the amino acid sequence of SEQ ID NO: 2.

[0046] Example 24. A polypeptide according to any one of Examples 20 to 23, wherein the polypeptide comprises or is composed of an amino acid sequence selected from or consisting of SEQ ID NO: 1, 49-72 and 78-81.

[0047] Example 25. A polypeptide according to any one of Examples 16 to 24, wherein the polypeptide further comprises one or more other groups, residues, portions or binding units, the one or more other groups, residues, portions or binding units optionally being linked via one or more peptide linkers, wherein the one or more other groups, residues, portions or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, portions or binding units.

[0048] Example 26. The polypeptide according to Example 25, wherein the one or more other groups, residues, portions or binding units providing the increased half-life of the polypeptide are selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc portions, and small proteins or peptides that can bind to serum proteins.

[0049] Example 27. The polypeptide according to any one of Examples 25 to 26, wherein the one or more other binding units providing the increased half-life of the polypeptide are selected from binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

[0050] Example 28. The polypeptide according to Example 27, wherein the binding unit that provides the increased half-life of the polypeptide is an ISVD bound to human serum albumin.

[0051] Example 29. The polypeptide according to Example 28, wherein the ISVD bound to human serum albumin comprises i. CDR1 as the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 9; ii. CDR2 as the amino acid sequence of SEQ ID NO: 13 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 13; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 17 or having an amino acid sequence that differs from SEQ ID NO: 17 in two or one amino acid.

[0052] Example 30. The polypeptide according to any one of Examples 28 to 29, wherein the ISVD bound to human serum albumin comprises CDR1 as the amino acid sequence of SEQ ID NO: 9, CDR2 as the amino acid sequence of SEQ ID NO: 13, and CDR3 as the amino acid sequence of SEQ ID NO: 17.

[0053] Example 31. The polypeptide according to any one of Examples 28 to 30, wherein the amino acid sequence of the ISVD bound to human serum albumin exhibits more than 90% sequence identity with SEQ ID NO: 5.

[0054] Example 32. The polypeptide according to any one of Examples 28 to 31, wherein the ISVD bound to human serum albumin consists of the amino acid sequence of SEQ ID NO: 5.

[0055] Example 33. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of Examples 1 to 32, preferably according to any one of Examples 1 to 15.

[0056] Example 34. A host or host cell comprising the nucleic acid according to Example 33.

[0057] Example 35. A method for generating a polypeptide according to any one of Examples 1 to 32, preferably according to any one of Examples 1 to 15, said method comprising at least the following steps: a) Explicitly express the nucleic acid as described in Example 33 in a suitable host cell or host organism or in another suitable expression system; optionally, this is done subsequently: b) Isolate and / or purify the polypeptide according to any one of Examples 1 to 32, or optionally according to any one of Examples 1 to 15.

[0058] Example 36. A composition comprising at least one polypeptide according to any one of Examples 1 to 32, optionally according to any one of Examples 1 to 15, or a nucleic acid according to Example 33.

[0059] Example 37. The composition according to Example 36 is a pharmaceutical composition that further comprises at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprises one or more other pharmaceutically active peptides and / or compounds.

[0060] Example 38. A polypeptide according to any one of Examples 1 to 32, optionally according to any one of Examples 1 to 15, or a composition according to Example 36 or 37, used as a pharmaceutical.

[0061] Example 39. A polypeptide according to any one of Examples 1 to 32, optionally according to any one of Examples 1 to 15, or a composition according to Example 36 or 37, for the treatment of cancer, preferably liver cancer or lung cancer.

[0062] Example 40. A polypeptide or composition for the purpose described in Example 39, wherein the liver cancer is hepatocellular carcinoma (HCC).

[0063] Example 41. A polypeptide or composition for the purpose according to Example 39, wherein the lung cancer is non-small cell lung cancer (NSCLC), preferably squamous cell carcinoma (SCC).

[0064] Example 42. A method for treating cancer, preferably liver cancer or lung cancer, wherein the method comprises administering to a subject in need a pharmaceutically active amount of a polypeptide according to any one of Examples 1 to 32, preferably according to any one of Examples 1 to 15, or a composition according to Examples 36 or 37.

[0065] Example 43. The method according to Example 42, wherein the liver cancer is hepatocellular carcinoma.

[0066] Example 44. The method according to Example 42, wherein the lung cancer is non-small cell lung cancer (NSCLC), preferably squamous cell carcinoma (SCC).

[0067] Example 45. Use of the polypeptide according to any one of Examples 1 to 32, preferably according to any one of Examples 1 to 15, or the composition according to Example 36 or 37 in the preparation of a pharmaceutical product.

[0068] Example 46. Use of the polypeptide according to any one of Examples 1 to 32, preferably according to any one of Examples 1 to 15, or the composition according to Example 36 or 37, for the preparation of a pharmaceutical composition for treating cancer, preferably liver cancer or lung cancer.

[0069] Example 47. Use of the polypeptide or composition according to Example 46, wherein the liver cancer is hepatocellular carcinoma.

[0070] Example 48. Use of the polypeptide or composition according to Example 46, wherein the lung cancer is non-small cell lung cancer (NSCLC), preferably squamous cell carcinoma (SCC). Simple Explanation of the Diagram

[0071] [picture] [1] [:] In the Incucyte-based human TDC (T cell-dependent cytotoxicity) HepG2-Nuclight green assay, dose-dependent killing of the trispecific GPC3 T cell conjugates A022600027 (Fig. 1A), A022600031 (Fig. 1B solid line), and the construct with reference Ab1 (Fig. 1B dashed line) was analyzed at 60 h post-inoculation using an effector-to-target ratio of 15:1. Controls (left) were: (solid square) no compound and (solid triangle) 1 µM brefidobacterium A for 100% killing.

[0072] [picture] [2] [:] In the Incucyte-based human TDC HepG2-Nuclight green assay, dose-dependent killing of trispecific GPC3 ISVD T cell conjugates was analyzed at 72 h post-inoculation using an effector-to-target ratio of 15:1. The graph represents step 1 of the optimized format, where the conjugate length of the trispecific trivalent T cell conjugate form differs between anti-TCR ISVD and anti-GPC3 ISVD. Controls (left) are: no compound (hollow square) and a reference for 100% killing (hollow circle).

[0073] [picture] [3] [:] In the Incucyte-based human TDC HepG2-Nuclight green assay, dose-dependent killing of trispecific GPC3 ISVD T cell conjugates was analyzed at 60 h post-inoculation using an effector-to-target ratio of 15:1. The graph represents step 2 of the optimized format, where the orientation and conjugate length of the trispecific tetravalent T cell conjugate form differ between bicomplementary GPC3-binding ISVDs. Controls (left) are: no compound (hollow squares) and a reference for 100% killing (hollow circles).

[0074] [picture] [4] Step 3 of GPC3 T cell conjugate format optimization: Anti-TCR ISVD T017000624 was replaced by the sequenced optimized variant TCE01 in trivalent and tetravalent forms. Dose-dependent killing of the trispecific GPC3 ISVD T cell conjugate was analyzed at 60 h post-inoculation using an effector to target ratio of 15:1 in Incucyte-based human TDC HepG2-Nuclight green assay (Fig. 4A) and xCELLigence-based human TDC Huh7 assay (Fig. 4B). Controls (left) are: no compound (hollow square) and a reference for 100% killing (hollow circle).

[0075] [picture] [5] [:] Dose-dependent killing of the trispecific GPC3 ISVD T cell conjugate was analyzed at 60 h post-inoculation using an effector to target ratio of 15:1 in Incucyte-based human TDC HepG2-Nuclight green assay (Fig. 5A) and xCELLigence-based human TDC Huh7 assay (Fig. 5B). Step 4, which optimizes the GPC3 T cell conjugate format, involves altering the orientation of anti-TCR ISVD with anti-GPC3 ISVD or with anti-albumin ISVD. Controls (left) are: (hollow square) no compound and (hollow circle) reference for 100% killing.

[0076] [picture] [6] [:] Dose-dependent killing of the trispecific GPC3 ISVD T cell conjugate was analyzed at 60 h post-inoculation using an effector-to-target ratio of 15:1 in Incucyte-based human TDC HepG2-Nuclight green assay (Fig. 6A) and xCELLigence-based human TDC Huh7 assay (Fig. 6B). Step 5 shows the optimized GPC3 T cell conjugate format, where the conjugate length is variable. Controls (left) are: (hollow square) no compound and (hollow circle) reference for 100% killing.

[0077] [picture] [7] [:] The effect of soluble GPC3 (sGPC3) on the cytotoxicity of the trispecific GPC3 ISVD T cell conjugate analyzed at a 15:1 effector-to-target ratio in a xCELLigence-based human TDC Huh7 assay at 60 h post-inoculation (Fig. 7A), and an assessment of its effect on T cell activation of the trispecific GPC3 ISVD T cell conjugate in the presence (Fig. 7B) and absence (Fig. 7C) of target cells (Huh7). Controls (left) are: no compound (hollow circle, Fig. 7A) and isotype control (hollow circle, Fig. 7B and Fig. 7C).

[0078] [picture] [8] [:] Time course of T cell conjugate internalization (Fig. 8A) and GPC3 expression (Fig. 8B) measured at 37ºC at time points of 0.5h, 3h, 24h, and 48h. The control (left) is the measurement at 4ºC for each compound at 0.5h. [。]

[0079] [picture] [9] [:] In xCELLigence-based human TDC assays, dose-dependent killing of different tumor cell lines with reduced GPC3 expression levels was performed using an effector-to-target ratio of 15:1: HepG2 (Fig. 9A) analyzed at 60 h, NCI-H661 (Fig. 9B) analyzed at 75 h, Huh-7 (Fig. 9C) analyzed at 60 h, MKN-45 (Fig. 9D) analyzed at 65 h, BxPC-3 (Fig. 9E) analyzed at 65 h, and NCI-H292 (Fig. 9F) analyzed at 60 h. The control (left) is: (hollow square) no compound (effector and T cells only).

[0080] [picture]

[10] [:] In a xCELLigence-based human TDC assay, using an effector-to-target ratio of 15:1 and analyzed over 60 h, five selected trispecific GPC3 ISVD-based T-cell conjugates demonstrated dose-dependent killing of two tumor cell lines, NCI-H661 (Fig. 10A) and BxPC-3 (Fig. 10B). Controls (left) were: no compound (hollow square, effectors and T cells only) and 30 nM T017000698 (hollow rhombus).

[0081] [picture]

[11] [:] The median and interquartile range of pre-existing antibody reactivity from 96 normal human serum samples, for the ISVD-based GPC3 T cell conjugate forms selected for tetravalent (Fig. 11A) and trivalent (Fig. 11B).

[0082] [picture]

[12] [:] Study design for efficacy model. Huh-7 tumor cells were subcutaneously injected into NOG mice. Tumors grew until an average tumor volume of approximately 150 mm³ was reached. At this point, in vitro expanded T cells were injected intraperitoneally into each mouse (D0). Treatment with intravenously injected A022600424 began on D0, 3 h after T cell injection, and continued on D3, D6, D9, and D12 (q3d). Four dose levels of A022600424 (0.1 mg / kg, 0.2 mg / kg, 0.7 mg / kg, and 2 mg / kg) were tested. Control T017000698 was injected at 2 mg / kg in the control group on D0, D3, D6, D9, and D12 (q3d). Viable blood samples were collected on D6 and D12 prior to use of the test compound. All mice were sacrificed on D15, and blood and tumor samples were collected.

[0083] [picture]

[13] Results of the efficacy model. Four dose levels of A022600424 were tested (0.1 mg / kg, 0.2 mg / kg, 0.7 mg / kg, and 2 mg / kg). Control T017000698 was administered at 2 mg / kg in the control group. Implementation

[0084] The present invention aims to provide novel drugs for the treatment of cancers such as liver cancer or lung cancer.

[0085] The inventors of this invention have discovered that peptides that simultaneously and specifically target GPC3 and TCR lead to effective T cell-mediated killing of GPC3-expressing cells in vitro. These peptides can be efficiently produced (e.g., in a microbial host). Furthermore, such peptides can exhibit limited reactivity with pre-existing antibodies in the subject to be treated (i.e., antibodies present in the subject prior to initial treatment with the antibody construct). In a preferred embodiment, such peptides exhibit a sufficiently long half-life in the subject to be treated to facilitate the spacing of consecutive treatments. Moreover, such peptides exhibit limited activity only against cells that do not express GPC3 or express low levels of GPC3. This suggests the potential to induce a highly specific T cell-mediated cytotoxic response against GPC3-positive target cells.

[0086] In addition to the above, the GPC3 combined with ISVD disclosed herein provides a high affinity for human and cynomolgus monkey GPC3, and can therefore be readily used in monovalent or multivalent form for other applications in which a combination with GPC3 is required. [5.1] [Polypeptide] [, Single specific , ] [, - , ] [, Monovalent peptides , ] [, , ]

[0087] In one state, the polypeptide is single-specific and monovalent.

[0088] The term "monospecific" refers to binding to one or more target molecules of a specific type. Monospecific peptides thus specifically bind to GPC3.

[0089] The term "unitary price" indicates the presence of a binding unit / building block for only one (specific) target molecule, such as ISVD.

[0090] Therefore, in one embodiment, the present invention provides an ISVD comprising a single-specific monovalent polypeptide specifically bound to GPC3, preferably incorporated into GPC3, wherein the ISVD comprises three complementarity-determining regions (CDR1 to CDR3). The ISVD may be selected from the following ISVDs: a) CDR1 as the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 in 2 or 1 amino acids; CDR2 as the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from SEQ ID NO: 11 in 2 or 1 amino acids; and CDR3 as the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence that differs from SEQ ID NO: 15 in 2 or 1 amino acids; or b) CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 in 2 or 1 amino acids; CDR2 as the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that differs from SEQ ID NO: 12 in 2 or 1 amino acids; and CDR3 as the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence that differs from SEQ ID NO: 16 in 2 or 1 amino acids.

[0091] Preferably, the ISVD that specifically binds to GPC3 is selected from ISVDs comprising: a) CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15; or b) CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16.

[0092] In another embodiment of this aspect of the technology, the ISVD specifically bound to GPC3 is selected from ISVDs comprising: a) An amino acid sequence having more than 90% sequence identity with SEQ ID NO: 3, preferably wherein the ISVD comprises or consists of the amino acid sequence of SEQ ID NO: 3; or b) An amino acid sequence having more than 90% sequence identity with SEQ ID NO: 4, preferably wherein the ISVD comprises or is composed of the amino acid sequence of SEQ ID NO: 4.

[0093] In another embodiment, the present invention provides an ISVD or a single-specific monovalent polypeptide comprising a constant domain that specifically binds to a TCR (preferably a human TCR) on T cells, said ISVD comprising three complementarity-determining regions (CDR1 to CDR3, respectively). The ISVD may be an ISVD comprising: CDR1 as the amino acid sequence of SEQ ID NO: 6 or having an amino acid sequence differing from SEQ ID NO: 6; CDR2 as the amino acid sequence of SEQ ID NO: 10 or having an amino acid sequence differing from SEQ ID NO: 10; and CDR3 as the amino acid sequence of SEQ ID NO: 14 or having an amino acid sequence differing from SEQ ID NO: 14.

[0094] Preferably, the ISVD that specifically binds to the TCR comprises the following ISVDs: CDR1 as the amino acid sequence of SEQ ID NO: 6, CDR2 as the amino acid sequence of SEQ ID NO: 10, and CDR3 as the amino acid sequence of SEQ ID NO: 14.

[0095] In another embodiment of this aspect of the technology, the ISVD that specifically binds to the TCR is an ISVD comprising an amino acid sequence having more than 90% sequence identity with SEQ ID NO: 2, preferably wherein the ISVD comprises or is composed of the amino acid sequence of SEQ ID NO: 2.

[0096] These monovalent compounds can also be used as building blocks for multivalent and / or multispecific peptides.

[0097] The ISVD located at the N-terminus of a (monovalent or polyvalent) polypeptide containing an ISVD preferably does not exhibit glutamic acid (E) at its N-terminus. Therefore, in the ISVD located at the N-terminus of the polypeptide, the glutamic acid (E) at position 1 is typically substituted with aspartic acid (D). Thus, for example, if SEQ ID NO: 3, 4, or 5 is located at the N-terminus of the polypeptide, these sequences would typically exhibit an E1D substitution. Conversely, if SEQ ID NO: 2 is not located at the N-terminus, this sequence would typically exhibit a D1E mutation. Therefore, generally, the first position of SEQ ID NO: 2-5 can be E or D, depending on whether these sequences are located at the N-terminus. In a preferred embodiment, the first amino acid of the first ISVD contained in the polypeptide of the present invention is aspartic acid (D). [, , ] [, Single specific , ] [, - , ] [, Multivalent peptides , ] [, , ]

[0098] In another instance, the polypeptide is single-specific and at least bivalent, but may also be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc.

[0099] The terms “divalent,” “trivalent,” “tetravalent,” “pentavalent,” or “hexavalent” all fall under the scope of the term “multivalent” and respectively indicate the presence of two, three, four, five, or six combined units / building blocks such as ISVDs.

[0100] Therefore, in one embodiment, the present invention provides a single-specific-divalent polypeptide comprising two ISVDs specifically bound to GPC3, preferably GPC3, or composed thereof, wherein each of the two ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the two ISVDs are preferably linked via one or more peptide linkers, and wherein: a) The first ISVD and the second ISVD comprise CDR1, which is the amino acid sequence of SEQ ID NO: 7 or has an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids; CDR2, which is the amino acid sequence of SEQ ID NO: 11 or has an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids; and CDR3, which is the amino acid sequence of SEQ ID NO: 15 or has an amino acid sequence that differs from SEQ ID NO: 15 by 2 or 1 amino acids. b) The first ISVD and the second ISVD comprise CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16; c) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having two or one amino acid sequence different from that of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having two or one amino acid sequence different from that of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having two or one amino acid sequence different from that of SEQ ID NO: 15, and The second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having two or one amino acid sequence different from that of SEQ ID NO: 8; CDR2 as the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having two or one amino acid sequence different from that of SEQ ID NO: 12; and CDR3 as the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having two or one amino acid sequence different from that of SEQ ID NO: 16; or d) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 16, and The second ISVD includes CDR1 as the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 15.

[0101] Preferably, the single-specific-bivalent polypeptide comprises or consists of two ISVDs specifically bound to GPC3, wherein: a) The first ISVD and the second ISVD comprise CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15; b) The first ISVD and the second ISVD comprise CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16; c) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15, and the second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16; or d) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16, and the second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15.

[0102] In another embodiment of this aspect of the technology, the single-specific-bivalent polypeptide comprises or consists of two ISVDs specifically bound to GPC3, wherein: a) The amino acid sequences of the first ISVD and the second ISVD exhibit more than 90% sequence identity with SEQ ID NO: 3, wherein the first ISVD and the second ISVD preferably consist of the amino acid sequence of SEQ ID NO: 3; b) The amino acid sequences of the first ISVD and the second ISVD exhibit more than 90% sequence identity with SEQ ID NO: 4, wherein the first ISVD and the second ISVD preferably consist of the amino acid sequence of SEQ ID NO: 4; c) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 3 and the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 4, preferably wherein the first ISVD consists of the amino acid sequence of SEQ ID NO: 3 and the second ISVD consists of the amino acid sequence of SEQ ID NO: 4; or d) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 4 and the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 3, preferably wherein the first ISVD consists of the amino acid sequence of SEQ ID NO: 4 and the second ISVD consists of the amino acid sequence of SEQ ID NO: 3.

[0103] In this regard, the terms "first ISVD" and "second ISVD" only indicate the specific ISVDs that bind to GPC3 and their relative positions to each other, with the numbering starting from the N-terminus of the polypeptide. Therefore, "first ISVD" is closer to the N-terminus than "second ISVD". Consequently, "second ISVD" is closer to the C-terminus than "first ISVD". Because the numbering is not absolute and only indicates the relative positions of the two ISVDs, it does not exclude the possibility that other binding units / building blocks (such as ISVDs binding to GPC3, TCR, or serum albumin, respectively) may be present in the polypeptide. Furthermore, it does not exclude the possibility that other binding units / building blocks, such as ISVDs, may be placed in between. For example, as further described below (see in particular the sections “Multispecific-multivalent peptides” and 5.4 “(in vivo) half-life extension”), the peptide may also contain another ISVD that binds to human serum albumin, which may even be located between the “first ISVD” and the “second ISVD” (such constructs are hereinafter referred to as multispecific, as described in subsequent sections).

[0104] In a preferred embodiment, at least two ISVDs of a monospecific-multivalent polypeptide, particularly the aforementioned monospecific-divalent polypeptide, are linked via peptide linkers. The use of peptide linkers to link two or more (multi)peptides is well known in the art. Exemplary peptide linkers that can be used with monospecific-multivalent polypeptides, particularly with the aforementioned monospecific-divalent polypeptide, are shown in Tables A-5. A common class of peptide linkers is referred to as "Gly-Ser" or "GS" linkers. These are linkers that are essentially composed of glycine (G) and serine (S) residues and typically contain one or more repetitions of a peptide motif such as the GGGGS (SEQ ID NO: 100) motif (e.g., exhibiting the formula (Gly-Gly-Gly-Gly-Ser)n, where n can be 1, 2, 3, 4, 5, 6, 7, or greater). Some common examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 103), the 15GS linker (n=3), and the 35GS linker (n=7). See, for example, Chen et al., Adv. Drug Deliv. Rev. 2013 Oct 15; 65(10): 1357-1369; and Klein et al., Protein Eng. Des. Sel. (2014) 27(10): 325-330. In one embodiment, the ISVDs of a monospecific-multivalent polypeptide, particularly a monospecific-divalent polypeptide, are linked via the linkers shown in Table A-5. In one embodiment, at least two ISVDs are linked via a 35GS or 9GS linker. In a preferred embodiment, at least two ISVDs are linked via one or more 9GS linkers. [, , ] [, Multispecific , ] [, - , ] [, Multivalent peptides , ] [, , ]

[0105] In another embodiment, the polypeptide is at least bispecific, but may also be, for example, trispecific, tetraspecific, pentaspecific, etc. Furthermore, the polypeptide is at least divalent, but may also be, for example, trivalent, tetravalent, pentavalent, hexavalent, etc.

[0106] The terms "bispecificity," "trispecificity," "quadrispecificity," and "pentaspecificity" all fall under the scope of the term "multispecificity," and respectively refer to binding to two, three, four, or five different target molecules.

[0107] The terms “divalent,” “trivalent,” “tetravalent,” “pentavalent,” and “hexavalent” all fall under the scope of the term “multivalent” and respectively indicate the existence of two, three, four, five, six, etc., combined units / building blocks such as ISVDs.

[0108] For example, the polypeptide can be bispecific-bivalent, such as a polypeptide containing two ISVDs or composed thereof, one ISVD specifically binding to GPC3 and the other ISVD specifically binding to a constant domain of the TCR on a T cell, wherein the GPC3 and TCR are preferably human GPC3 and human TCR. The polypeptide can also be bispecific-trivalent, such as a polypeptide containing three ISVDs or composed thereof, wherein two ISVDs specifically bind to GPC3 and the other ISVD specifically binds to a constant domain of the TCR on a T cell, wherein the GPC3 and TCR are preferably human GPC3 and human TCR. In another example, the polypeptide can be trispecific-tetravalent, such as a polypeptide containing four ISVDs or composed thereof, wherein two ISVDs specifically bind to human GPC3, one ISVD specifically binds to a constant domain of the human TCR on a T cell, and one ISVD binds to human serum albumin. Such a polypeptide can also be bicomplementary, for example, in the case where two ISVDs bind to two different epitopes on human GPC3. The term "double complementary site" refers to two different parts (e.g., epitopes) that bind to the same target molecule. A preferred trispecific-tetravalent peptide is, for example, an ISVD construct A022600424 comprising two ISVDs that specifically bind to human GPC3, one ISVD specifically binding to a constant domain of the human TCR on T cells, and one ISVD binding to human serum albumin, and said ISVD construct having double complementary sites for binding to GPC3.

[0109] In one embodiment, the present invention provides a bispecific divalent polypeptide comprising at least two ISVDs or thereof, wherein each of the ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the at least two ISVDs are optionally linked via one or more peptide linkers, and wherein: a) The first ISVD contains i. CDR1 as the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 6; ii. CDR2 as the amino acid sequence of SEQ ID NO: 10 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 10; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 14 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 14, and The second ISVD contains iv. CDR1 as the amino acid sequence of SEQ ID NO: 7 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 7; v. CDR2 as the amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 11; and vi. CDR3 as the amino acid sequence of SEQ ID NO: 15 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 15. b) The first ISVD contains i. CDR1 as the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 7; ii. CDR2 as the amino acid sequence of SEQ ID NO: 11 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 11; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 15 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 15, and The second ISVD contains iv. CDR1 as the amino acid sequence of SEQ ID NO: 6 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 6; v. CDR2 as the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 10; and vi. CDR3 as the amino acid sequence of SEQ ID NO: 14 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 14. c) The first ISVD contains i. CDR1 as the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 6; ii. CDR2 as the amino acid sequence of SEQ ID NO: 10 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 10; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 14 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 14, and The second ISVD contains iv. CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 8; v. CDR2 as the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 12; and vi. CDR3 as the amino acid sequence of SEQ ID NO: 16 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16, or d) The first ISVD contains i. CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 8; ii. CDR2 as the amino acid sequence of SEQ ID NO: 12 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 12; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 16 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16, and The second ISVD contains iv. CDR1 as the amino acid sequence of SEQ ID NO: 6 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 6; v. CDR2 as the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 10; and vi. CDR3 as the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 14.

[0110] In a preferred embodiment, such a polypeptide comprises or consists of at least two ISVDs, each of which contains three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the at least two ISVDs are optionally linked via one or more peptide linkers, and wherein a) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 6, CDR2 as the amino acid sequence of SEQ ID NO: 10, and CDR3 as the amino acid sequence of SEQ ID NO: 14, and the second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15. b) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15, and the second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 6, CDR2 as the amino acid sequence of SEQ ID NO: 10, and CDR3 as the amino acid sequence of SEQ ID NO: 14. c) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 6, CDR2 as the amino acid sequence of SEQ ID NO: 10, and CDR3 as the amino acid sequence of SEQ ID NO: 14, and the second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16, or d) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16, and the second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 6, CDR2 as the amino acid sequence of SEQ ID NO: 10, and CDR3 as the amino acid sequence of SEQ ID NO: 14.

[0111] Therefore, such a polypeptide can be a polypeptide, wherein: a) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 2, and the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 3. b) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 3, and the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 2. c) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 2, and the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 4, or d) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 4, and the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 2.

[0112] Preferably, in such a polypeptide: a) The first ISVD consists of the amino acid sequence of SEQ ID NO: 2, and the second ISVD consists of the amino acid sequence of SEQ ID NO: 3. b) The first ISVD consists of the amino acid sequence of SEQ ID NO: 3, and the second ISVD consists of the amino acid sequence of SEQ ID NO: 2. c) The first ISVD consists of the amino acid sequence of SEQ ID NO: 2, and the second ISVD consists of the amino acid sequence of SEQ ID NO: 4, or d) The first ISVD consists of the amino acid sequence of SEQ ID NO: 4, and the second ISVD consists of the amino acid sequence of SEQ ID NO: 2.

[0113] In another embodiment, the present invention provides a trispecific trivalent polypeptide comprising the aforementioned bispecific divalent polypeptide and a third ISVD (section 5.4; "(in vivo) prolonged half-life") that binds to human serum albumin, as detailed below.

[0114] In one embodiment, the present invention provides a bispecific trivalent polypeptide comprising at least three ISVDs or thereof, wherein each of the ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), wherein the at least three ISVDs are optionally linked via one or more peptide linkers, and wherein: a) The first ISVD specifically binds to the constant domain of the TCR on T cells and includes CDR1 as the amino acid sequence of SEQ ID NO: 6 or having an amino acid sequence that differs from SEQ ID NO: 6 by 2 or 1 amino acids; CDR2 as the amino acid sequence of SEQ ID NO: 10 or having an amino acid sequence that differs from SEQ ID NO: 10 by 2 or 1 amino acids; and CDR3 as the amino acid sequence of SEQ ID NO: 14 or having an amino acid sequence that differs from SEQ ID NO: 14 by 2 or 1 amino acids. b) The second ISVD specifically binds to GPC3 and comprises CDR1 as the amino acid sequence of SEQ ID NO: 7 or having an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acids; CDR2 as the amino acid sequence of SEQ ID NO: 11 or having an amino acid sequence that differs from SEQ ID NO: 11 by 2 or 1 amino acids; and CDR3 as the amino acid sequence of SEQ ID NO: 15 or having an amino acid sequence that differs from SEQ ID NO: 15 by 2 or 1 amino acids; and c) The third ISVD specifically binds to GPC3 and includes CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16. Preferably, the TCR and GPC3 bound by the polypeptide are human TCR and human GPC3, respectively.

[0115] In a preferred embodiment of a multispecific-multivalent polypeptide: a) The first ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 6, CDR2 as the amino acid sequence of SEQ ID NO: 10, and CDR3 as the amino acid sequence of SEQ ID NO: 14; b) The second ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15; and c) The third ISVD comprises CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16.

[0116] Another state of multispecific-multivalent peptides: a) The amino acid sequence of the first ISVD exhibits more than 90% sequence identity with SEQ ID NO: 2, wherein preferably the first ISVD consists of the amino acid sequence of SEQ ID NO: 2; b) The amino acid sequence of the second ISVD exhibits more than 90% sequence identity with SEQ ID NO: 3, wherein preferably the second ISVD consists of the amino acid sequence of SEQ ID NO: 3; and c) The amino acid sequence of the third ISVD exhibits more than 90% sequence identity with SEQ ID NO: 4, wherein preferably the third ISVD consists of the amino acid sequence of SEQ ID NO: 4.

[0117] In this regard, the terms "first ISVD," "second ISVD," "third ISVD," etc., only indicate the relative positions of the ISVDs to each other, where the numbering starts from the N-terminus of the polypeptide. Therefore, the "first ISVD" is closer to the N-terminus than the "second ISVD," and the "second ISVD" is closer to the N-terminus than the "third ISVD." Thus, when viewed from the C-terminus, the ISVD arrangement is reversed. Since the numbering is not absolute and only indicates the relative positions of at least four ISVDs, it does not exclude the possibility that other binding units / building blocks (such as additional ISVDs binding to GPC3 or to another target) may be present in the polypeptide. Furthermore, it does not exclude the possibility that other binding units / building blocks such as ISVDs may be placed in between. For example, as further described below (see in particular, section 5.4 "(in vivo) prolonged half-life"), the polypeptide may also contain another ISVD that binds to human serum albumin, which may even be located between, for example, "third ISVD" and "fourth ISVD".

[0118] In another embodiment, the present invention provides a trispecific-tetravalent polypeptide comprising the aforementioned bispecific-trivalent polypeptide and a fourth ISVD (section 5.4; "(in vivo) prolonged half-life") that binds to human serum albumin, as detailed below.

[0119] In a preferred embodiment, the first ISVD that binds to the TCR is located at the N-terminus of the polypeptide.

[0120] In another embodiment, the present invention provides a bispecific divalent polypeptide comprising an ISVD specifically bound to GPC3 as detailed above for a monospecific monovalent polypeptide (section 5.1; "Monospecific monovalent polypeptide") and an ISVD bound to human serum albumin as detailed below (section 5.4; "(In vivo) prolonged half-life").

[0121] In another embodiment, the present invention provides a bispecific trivalent polypeptide comprising the aforementioned monospecific divalent polypeptide (section 5.1; "Monospecific divalent polypeptide") and an ISVD bound to human serum albumin as detailed below (section 5.4; "(In vivo) prolonged half-life").

[0122] The components of the multispecific-multivalent polypeptides described herein (preferably ISVDs) can be linked together by one or more suitable linkers such as peptide linkers.

[0123] The use of linkers to connect two or more (poly)peptides is well known in the industry. Exemplary peptide linkers are shown in Table A-5. A common class of peptide linkers is referred to as "Gly-Ser" or "GS" linkers. These are linkers that consist essentially of glycine (G) and serine (S) residues and typically contain one or more repetitions of a peptide motif such as the GGGGS (SEQ ID NO: 100) motif (e.g., exhibiting the formula (Gly-Gly-Gly-Gly-Ser)n, where n can be 1, 2, 3, 4, 5, 6, 7, or greater). Some common examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO: 103), the 15GS linker (n=3), and the 35GS linker (n=7). For example, see Chen et al., Adv. Drug Deliv. Rev. 2013 Oct 15; 65(10): 1357-1369; and Klein et al., Protein Eng. Des. Sel. (2014) 27(10): 325-330. In one or more peptides disclosed herein, it is preferable to link the components of the peptide to each other using 5GS and 9GS linkers. Preferably, a first ISVD specifically bound to the TCR is linked to a second ISVD specifically bound to GPC3 using a linker of fewer than 10 amino acids, such as fewer than 6 amino acids (particularly a 5GS linker).

[0124] In one state of the multispecific-multivalent polypeptide, the polypeptide comprising at least three ISVDs or consisting thereof includes at least two ISVDs specifically bound to GPC3 and one ISVD specifically bound to TCR. In this aspect of the technique, the ISVD bound to TCR is linked to one of the at least two ISVDs bound to GPC3 via a 5GS linker, while the at least two ISVDs specifically bound to GPC3 are linked to each other via a 9GS linker. In another state, the multispecific-multivalent polypeptide further comprises an ISVD bound to albumin, which is further linked via a 9GS linker to an ISVD bound to GPC3 that is not linked to the ISVD bound to TCR (as described in Section 5.4, “(In vivo) Half-life Extension”). The inventors have surprisingly found that such a configuration can increase the efficiency of the polypeptide in initiating T cell-mediated cytotoxic responses.

[0125] Therefore, preferably, the polypeptide comprises, or consists of, the following in sequence starting from the N-terminus of the polypeptide: a first ISVD specifically binding to TCR, a second ISVD specifically binding to GPC3, a third ISVD specifically binding to GPC3, and an optional binding unit providing an increased half-life for the polypeptide as defined herein. The binding unit providing the increased half-life for the polypeptide is preferably an ISVD specifically binding to serum albumin.

[0126] Even more preferably, the polypeptide comprises, or consists of, the following in sequence from the N-terminus of the polypeptide: an ISVD specifically bound to the TCR, a linker, a second ISVD specifically bound to GPC3, a linker, a third ISVD specifically bound to GPC3, a linker, and an ISVD bound to human serum albumin, wherein the linker between the first ISVD and the second ISVD is preferably a 5GS linker, and the other linkers are preferably 9GS linkers.

[0127] Such a configuration of the polypeptide can provide potent therapeutic effects for cancer and low binding to pre-existing antibodies.

[0128] Preferably, the multispecific-multivalent peptide exhibits reduced binding to pre-existing antibodies in human serum. For this purpose, in one embodiment, the peptide exhibits lysine (V) at amino acid position 11 and leucine (L) at amino acid position 89 in at least one ISVD (and preferably at the C-terminus of the peptide), but more preferably in each ISVD. In another embodiment, the peptide exhibits an elongation of 1 to 5 (preferably naturally occurring) amino acids at the C-terminus of the C-terminal ISVD, such as a single alanine (A) elongation. The C-terminus of the ISVD is typically VTVSS (SEQ ID NO: 116). In another embodiment, the peptide exhibits lysine (K) or glutamic acid (Q) at position 110 in at least one ISVD (according to Kabat numbering). In another embodiment, in at least one ISVD, the ISVD exhibits lysine (K) or glutamic acid (Q) at position 112 (according to Kabat numbering). In these embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO: 117), VQVSS (SEQ ID NO: 118), VTVKS (SEQ ID NO: 119), VTVQS (SEQ ID NO: 120), VKVKS (SEQ ID NO: 121), VKVQS (SEQ ID NO: 122), VQVKS (SEQ ID NO: 123), or VQVQS (SEQ ID NO: 124), such that after the addition of a single alanine, the C-terminus of the polypeptide exhibits, for example, the sequences VTVSSA (SEQ ID NO: 125), VKVSSA (SEQ ID NO: 126), VQVSSA (SEQ ID NO: 127), VTVKSA (SEQ ID NO: 128), VTVQSA (SEQ ID NO: 129), VKVKSA (SEQ ID NO: 130), VKVQSA (SEQ ID NO: 124), or VQVQS (SEQ ID NO: 125), VKVSSA (SEQ ID NO: 126), VQVSSA (SEQ ID NO: 127), VTVKSA (SEQ ID NO: 128), VTVQSA (SEQ ID NO: 129), VKVKSA (SEQ ID NO: 130), or VKVQSA (SEQ ID NO: 124). 131), VQVKSA (SEQ ID NO: 132) or VQVQSA (SEQ ID NO: 133), preferably VTVSSA (see Table A-7).In another embodiment, the polypeptide exhibits lysine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat designation) in at least one C-terminal ISVD, optionally lysine (K) or glutamic acid (Q) at position 110 (according to Kabat designation) in at least one ISVD, and exhibits an extension of 1 to 5 (preferably naturally occurring) amino acids at the C-terminus of the C-terminal ISVD, such as a single alanine (A) extension (such that the C-terminus of the polypeptide consists, for example, the sequence VTVSSA, VKVSSA, or VQVSSA, preferably VTVSSA). For further information in this regard, see, for example, WO 2012 / 175741 and WO 2015 / 173325.

[0129] In a preferred embodiment, the multispecific-multivalent polypeptide comprises or is composed of an amino acid sequence exhibiting or being identical to SEQ ID NO: 1 by more than 90% (e.g., more than 95% or more than 99%) of the sequence, wherein, more preferably, the CDRs of the four ISVDs are as defined by Abm (or A' to D', in the case of Kabat definition) as described in items A to D in sections “5.2 Immunoglobulin Single Variable Domain” and “5.4 (In Vivo) Half-Life Extension” below, respectively, wherein, in particular: • The first ISVD of the constant domain of the TCR that specifically binds to T cells comprises CDR1 as the amino acid sequence of SEQ ID NO: 6, CDR2 as the amino acid sequence of SEQ ID NO: 10, and CDR3 as the amino acid sequence of SEQ ID NO: 14; • The second ISVD that specifically binds to GPC3 comprises CDR1 as the amino acid sequence of SEQ ID NO: 7, CDR2 as the amino acid sequence of SEQ ID NO: 11, and CDR3 as the amino acid sequence of SEQ ID NO: 15; The third ISVD specifically binding to GPC3 comprises CDR1 as the amino acid sequence of SEQ ID NO: 8, CDR2 as the amino acid sequence of SEQ ID NO: 12, and CDR3 as the amino acid sequence of SEQ ID NO: 16; and • The fourth ISVD bound to human serum albumin comprises CDR1 as the amino acid sequence of SEQ ID NO: 9, CDR2 as the amino acid sequence of SEQ ID NO: 13, and CDR3 as the amino acid sequence of SEQ ID NO: 17. Alternatively, if Kabat is used: • The first ISVD that specifically binds to the TCR comprises CDR1 as the amino acid sequence of SEQ ID NO: 31, CDR2 as the amino acid sequence of SEQ ID NO: 35, and CDR3 as the amino acid sequence of SEQ ID NO: 14; • The second ISVD that specifically binds to GPC3 comprises CDR1 as the amino acid sequence of SEQ ID NO: 32, CDR2 as the amino acid sequence of SEQ ID NO: 36, and CDR3 as the amino acid sequence of SEQ ID NO: 15; The third ISVD that specifically binds to GPC3 comprises CDR1 as the amino acid sequence of SEQ ID NO: 33, CDR2 as the amino acid sequence of SEQ ID NO: 37, and CDR3 as the amino acid sequence of SEQ ID NO: 16; and • The fourth ISVD that binds to human serum albumin comprises CDR1 as the amino acid sequence of SEQ ID NO: 34, CDR2 as the amino acid sequence of SEQ ID NO: 38, and CDR3 as the amino acid sequence of SEQ ID NO: 17.

[0130] In some respects, the polypeptide comprises or is composed of an amino acid sequence selected from or consisting of SEQ ID NO: 1, 49-72 and 78-81.

[0131] Preferably, the polypeptide comprises or is composed of the amino acid sequence of SEQ ID NO: 1 (see Table A-3). In the preferred embodiment, the polypeptide is composed of the amino acid sequence of SEQ ID NO: 1.

[0132] Compared to a polypeptide composed of the amino acids of SEQ ID NO: 1, the polypeptide preferably exhibits at least half the binding affinity for human TCR and at least the same affinity for human GPC3, wherein the binding affinity is measured using the same method such as surface plasma resonance (SPR). [5.2] [Immunoglobulin single variable domain]

[0133] The term "immunoglobulin single variable domain" (ISVD) is used interchangeably with "single variable domain," defining an immunoglobulin molecule in which an antigen-binding site is located on a single immunoglobulin domain and formed therefrom. This distinguishes ISVD from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), where two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and light chain variable domain (VL) interact to form an antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both VH and VL will contribute to the antigen-binding site; a total of six CDRs will participate in the formation of the antigen-binding site.

[0134] Given the above definitions, the antigen-binding domain of a conventional four-chain antibody (such as IgG, IgM, IgA, IgD, or IgE molecules; known in the art) or a Fab fragment, F(ab')2 fragment, Fv fragment (such as a disulfide-linked Fv or scFv fragment) or a biantibody (all known in the art) derived from such a conventional four-chain antibody will generally not be considered as a single immunoglobulin domain because in these cases, it is not a single immunoglobulin domain that binds to the corresponding antigenic epitope, but rather a pair of associated immunoglobulin domains (such as light chain and heavy chain variable domains), i.e., the V HV L pair of immunoglobulin domains, that bind to the corresponding antigenic epitope, which bind together to the corresponding antigenic epitope.

[0135] In contrast, immunoglobulin monovariable domains (MMUs) can specifically bind to antigen epitopes without pairing with other immunoglobulin MMUs. The binding site of an immunoglobulin MMU is formed by a single VH, a single VHH, or a single VL domain.

[0136] Therefore, the single variable domain can be a light chain variable domain sequence (e.g., a VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH sequence or a VHH sequence) or a suitable fragment thereof, as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit, which is essentially composed of a single variable domain such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

[0137] Immunoglobulin single variable domains (ISVDs) can be, for example, heavy chain ISVDs, such as VH, VHH, including camel-like VH or humanized VHH. Preferably, it is a VHH, including camel-like VH or humanized VHH. Heavy chain ISVDs can be derived from conventional four-chain antibodies or heavy chain antibodies.

[0138] For example, an immunoglobulin monovariable domain can be a monovariable antibody (or an amino acid sequence suitable for use as a monovariable antibody), a dAb, or a dAb (or an amino acid sequence suitable for use as a dAb); other monovariable domains, or any suitable fragment of any of them.

[0139] Specifically, an immunoglobulin monovariable domain can be an immunoglobulin monovariable domain (such as VHH, including humanized VHH or camel-like VH) or a suitable fragment thereof. Nanobody®, Nanobodies®, and Nanoclone® are registered trademarks of Ablynx NV.

[0140] The “VHH domain,” also known as VHH, VHH antibody fragment, and VHH antibody, was initially described as a variable domain of an antigen-binding immunoglobulin in “heavy chain antibodies” (i.e., “antibodies without light chains”; Hamers-Casterman et al., Nature 363: 446-448, 1993). The term “VHH domain” has been chosen to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (referred to herein as the “VH domain”) and the light chain variable domains present in conventional 4-chain antibodies (referred herein as the “VL domain”). For a further description of VHH, refer to Muyldermans’ review article (Reviews in Molecular Biotechnology 74: 277-302, 2001).

[0141] Typically, immunoglobulin production involves immunizing laboratory animals, fusing immunoglobulin-producing cells to create hybridomas, and screening for desired specificity. Alternatively, immunoglobulins can be generated by screening immature or synthetic libraries, for example, via phage display.

[0142] The generation of immunoglobulin sequences has been extensively described in various publications, with WO 94 / 04678, Hamers-Casterman et al. (1993), and Muyldermans et al. (2001) serving as examples. In these methods, camels are immunized with a target antigen to induce an immune response against the target antigen. A library of VHHs obtained from the immunization is then further screened for VHHs that bind to the target antigen.

[0143] In these cases, antibody production requires purified antigens for immunization and / or screening. The antigens can be purified from natural sources or during recombinant production.

[0144] Immunization and / or screening of immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0145] The present invention can utilize immunoglobulin sequences from various sources, including mouse, rat, rabbit, donkey, human, and camelid immunoglobulin sequences. The technology also includes fully human, humanized, or chimeric sequences. For example, the technology includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camel-like domain antibodies, such as camel-like dAbs as described by Ward et al. (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996)). Furthermore, the technique also utilizes fused immunoglobulin sequences, such as those forming multivalent and / or multispecific constructs (for reference on multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, see also Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001, and, for example, WO 96 / 34103 and WO 99 / 23221), and immunoglobulin sequences containing tags or other functional parts (e.g., toxins, labels, radiochemical substances, etc.) that can be derived from the immunoglobulin sequences of the present invention.

[0146] "Humanized VHH" comprises an amino acid sequence corresponding to the naturally occurring VHH domain but which has been "humanized," i.e., by replacing one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (and particularly in the structural sequence) with one or more amino acid residues appearing at one or more corresponding positions in the VH domain of a conventional 4-chain antibody from humans (e.g., as noted above). This can be done in ways known per se, as will be clear to those skilled in the art, for example, based on further description herein and prior art (e.g., WO 2008 / 020079). Likewise, it should be noted that such humanized VHH can be obtained in any suitable manner known per se, and is therefore not strictly limited to peptides already obtained using peptides containing the naturally occurring VHH domain as starting material.

[0147] "Camelization of VH" comprises an amino acid sequence corresponding to the naturally occurring VH domain but which has been "camelized," i.e., by replacing one or more amino acid residues in the naturally occurring VH domain of a conventional 4-chain antibody with one or more amino acid residues appearing at one or more corresponding positions in the VHH domain of the heavy chain antibody. This can be done in a manner known per se, as will be clear to those skilled in the art, for example, based on further description herein and prior art (e.g., WO 2008 / 020079). As defined herein, such "camelization" substitutions are preferably inserted at amino acid positions forming and / or present at the VHVL interface and / or at so-called cameloid marker residues, as defined herein (see, for example, WO 94 / 04678 and Davies and Riechmann (1994 and 1996), ibid.). Preferably, the VH sequence used as the starting material or origin for generating or designing camellia-like VHs is a VH sequence derived from mammals, more preferably a human VH sequence, such as the VH3 sequence. However, it should be noted that such camellia-like VHs can be obtained in any suitable manner known per se, and are therefore not strictly limited to peptides obtained using peptides containing naturally occurring VH domains as starting materials.

[0148] A preferred structure of an immunoglobulin single variable domain sequence can be considered to consist of four architecture regions ("FR"), referred to in the art and herein as "architecture region 1" ("FR1"), "architecture region 2" ("FR2"), "architecture region 3" ("FR3"), and "architecture region 4" ("FR4"), which are interrupted by three complementarity-determining regions ("CDR"), referred to in the art and herein as "complementarity-determining region 1" ("CDR1"), "complementarity-determining region 2" ("CDR2"), and "complementarity-determining region 3" ("CDR3").

[0149] As further described in paragraphs q) on pages 58 and 59 of WO 08 / 020079, the amino acid residues of the immunoglobulin single variable domain can be numbered according to the general numbering given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, 91), as applied to the VHH domain from camels in the article Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see, for example, Figure 2 of that publication). It should be noted that, as is well known in the art for the VH domain and for the VHH domain, the total number of amino acid residues in each CDR can vary and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat number). This means that, generally, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. The total number of amino acid residues in the VH domain and the VHH domain will generally be in the range of 110 to 120, and typically between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0150] In this application, unless otherwise stated, the CDR sequence is determined according to the AbM definition as described in Kontermann and Dübel (edited 2010, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 contains amino acid residues at positions 1-25, CDR1 contains amino acid residues at positions 26-35, FR2 contains amino acid residues at positions 36-49, CDR2 contains amino acid residues at positions 50-58, FR3 contains amino acid residues at positions 59-94, CDR3 contains amino acid residues at positions 95-102, and FR4 contains amino acid residues at positions 103-113.

[0151] The determination of the CDR region can also be performed using different methods. According to Kabat's CDR determination, FR1 of the immunoglobulin monovariable domain (IMDV) contains amino acid residues at positions 1-30, CDR1 of the IMDV contains amino acid residues at positions 31-35, FR2 of the IMDV contains amino acid residues at positions 36-49, CDR2 of the IMDV contains amino acid residues at positions 50-65, FR3 of the IMDV contains amino acid residues at positions 66-94, CDR3 of the IMDV contains amino acid residues at positions 95-102, and FR4 of the IMDV contains amino acid residues at positions 103-113.

[0152] In such immunoglobulin sequences, the architecture sequence can be any suitable architecture sequence, and examples of suitable architecture sequences will be clear to those skilled in the art, for example, based on standard manuals and other public disclosures and prior art mentioned herein.

[0153] The architecture sequence is preferably an immunoglobulin architecture sequence (a suitable combination), or (e.g., through humanization or camelification) an architecture sequence derived from an immunoglobulin architecture sequence. For example, the architecture sequence may be an architecture sequence derived from a light chain variable domain (e.g., a VL sequence) and / or a heavy chain variable domain (e.g., a VH sequence or a VHH sequence). In a particularly preferred aspect, the architecture sequence is an architecture sequence derived from a VHH sequence (wherein the architecture sequence may optionally be partially or fully humanized), or a conventional VH sequence that has been camelified (as defined herein).

[0154] In particular, the architectural sequence present in the ISVD sequence used in the technique may contain one or more marker residues (as defined herein) such that the ISVD sequence is V HH, including humanized V HH or camelified V H. Some preferred but non-limiting examples of such architectural sequences (suitable combinations) will become clear from other published texts herein.

[0155] Similarly, as described in this article in general terms of immunoglobulin sequences, suitable fragments (or combinations of fragments) of any of the foregoing may be used, such as fragments containing one or more CDR sequences, which are appropriately side-joined by one or more architecture sequences and / or linked via one or more architecture sequences (e.g., in the same order as these CDR and architecture sequences may appear in the full-size immunoglobulin sequence from which the fragment is derived).

[0156] However, it should be noted that the techniques are not limited regarding the source of the ISVD sequence (or the nucleotide sequence used to represent the ISVD sequence) and the manner in which the ISVD sequence or nucleotide sequence is generated or obtained (or has been generated or obtained). Therefore, the ISVD sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In certain but not limiting aspects, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including, but not limited to, "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and particularly partially or fully humanized VHH sequences), "camelized" (as defined herein) immunoglobulin sequences, and immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting from a synthetic, random, or naturally occurring immunoglobulin sequence), CDR transplantation, faceting, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineered immunoglobulin sequences known to those skilled in the art; or any suitable combination of the foregoing.

[0157] Similarly, the nucleotide sequence can be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and can be, for example, a sequence isolated from a suitable naturally occurring template by PCR (e.g., DNA or RNA isolated from cells), a nucleotide sequence that has been isolated from a library (and in particular, an expression library), a nucleotide sequence that has been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence that has been prepared by PCR using overlapping primers, or a nucleotide sequence that has been prepared using DNA synthesis techniques known per se.

[0158] As stated above, ISVD can be Nanobody® or a suitable fragment thereof. For a general description of Nanobodies, refer to the further description below and the prior art cited herein. However, in this regard, it should be noted that this description and prior art primarily describe so-called "VH3 class" Nanobodies (i.e., Nanobodies with high sequence homology to VH3 class phylogenetic sequences such as DP-47, DP-51, or DP-29). However, it should be noted that the techniques, in their broadest sense, can generally use any type of Nanobody, and, for example, also use so-called "VH4 class" Nanobodies (i.e., Nanobodies with high sequence homology to VH4 class phylogenetic sequences such as DP-78), as described, for example, in WO 2007 / 118670.

[0159] Typically, nanobodies (especially VHH sequences, including (partially) humanized VHH sequences and camel-like VH sequences) are characterized by the presence of one or more "marker residues" (as described herein) in one or more architectural sequences (again, as further described herein). Therefore, a nanobody can generally be defined as an immunoglobulin sequence having the following (general) structure. FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 FR1 to FR4 refer to architecture regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively, and one or more of the marker residues are as further defined herein.

[0160] In particular, a nanobody can be an immunoglobulin sequence having the following (general) structure. FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 FR1 to FR4 refer to architecture regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively, and the architecture sequence is as further defined herein.

[0161] More specifically, a nanobody can be an immunoglobulin sequence having the following (general) structure. FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 FR1 to FR4 refer to architecture regions 1 to 4, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively. According to the Kabat number, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 are selected from the marker residues mentioned in Table A-0 below.

[0162] [surface] [A-0] [:] [Nanobodies] [Marker residues in] [Location] [people] [V, H , 3 ] [Marker residues] 11 L, V; mainly L L, S, V, M, W, F, T, Q, E, A, R, G, K, Y, N, P, I; preferably L 37 V, I, F; usually V F(1), Y, V, L, A, H, S, I, W, C, N, G, D, T, P, preferably F(1) or Y 44 (8) G E (3), Q (3), G (2), D, A, K, R, L, P, S, V, H, T, N, W, M, I; G (2), E (3) or Q (3) are preferred; G (2) or Q (3) is the best. 45 (8) L L (2), R (3), P, H, F, G, Q, S, E, T, Y, C, I, D, V; preferably L (2) or R (3). 47 (8) W, Y F (1), L (1) or W (2)G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferably W (2), L (1) or F (1) 83 R or K; usually R R, K (5), T, E (5), Q, N, S, I, V, G, M, L, A, D, Y, H; K or R is preferred; K is the best. 84 A, T, D; mainly A P (5), S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E; preferably P 103 W W (4), R (6), G, S, K, A, M, Y, L, F, T, N, V, Q, P (6), E, ​​C; preferably W 104 G G, A, S, T, D, P, N, E, C, L; G is preferred. 108 L, M, or T; primarily L Q, L (7), R, P, E, K, S, T, M, A, H; preferably Q or L (7). [Notice:] (1) Specifically, but not exclusively, in combination with KERE or KQRE at positions 43 to 46. (2) GLEW is usually located at positions 44 to 47. (3) Typically, positions 43 to 46 are KERE or KQRE, for example, positions 43 to 47 are KEREL, KEREF, KQREL, KQREF, KEREG, KQREW, or KQREG. Alternatively, sequences such as TERE (e.g., TEREL), TQRE (e.g., TQREL), KECE (e.g., KECEL or KECER), KQCE (e.g., KQCEL), RERE (e.g., REREG), RQRE (e.g., RQREL, RQREF, or RQREW), QERE (e.g., QEREG), QQRE (e.g., QQREW, QQREL, or QQREF), KGRE (e.g., KGREG), and KDRE (e.g., KDREV). Some other possible but less preferred sequences include, for example, DECKL and NVCEL. (4) Having both GLEW at positions 44 to 47 and KERE or KQRE at positions 43 to 46. (5) The position of the naturally occurring VHH domain is usually KP or EP at position 83 to 84. (6) Specifically, but not exclusively, it is combined with GLEW at positions 44 to 47. (7) The premise is that when positions 44 to 47 are GLEW, in the (non-humanized) VHH sequence that also contains W at position 103, position 108 is always Q. (8) The GLEW group also contains GLEW-like sequences at positions 44 to 47, such as GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and ELEW.

[0163] The technique particularly utilizes ISVDs that can bind to a constant structural domain of the TCR or GPC3. In the context of the present invention, "bind to" a target molecule has the common meaning understood in the industry, such as in the case of antibodies and their corresponding antigens.

[0164] A multispecific, multivalent polypeptide may contain one or more ISVDs that specifically bind to GPC3. For example, the polypeptide may contain two ISVDs that specifically bind to GPC3 and an ISVD that specifically binds to TCR.

[0165] The ISVDs used in the technique can form part of a polypeptide containing or consisting of at least two ISVDs, such that the polypeptide can specifically bind to GPC3 and TCR.

[0166] Therefore, the target molecules of ISVD used in this technique are the constant structural domains of GPC3 and TCR, respectively. Binding to the TCR can be achieved, for example, by binding to the TCRα subunit and / or TCRβ subunit. Examples are mammalian GPC3 and TCR. Although human GPC3 (Uniprot accession number P51654, see Table A-8) and human TCR (see Table A-8) are preferred, forms from other species are also suitable for the present invention, such as GPC3 and TCR from the following species: mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates such as cynomolgus monkeys (also referred to herein as "cyno"), or camels such as llamas or alpacas.

[0167] Specific examples of ISVDs that can be used in the aforementioned technology to specifically bind to the constant domain of the TCR on T cells are described in item A below:

[0168] A. An ISVD that specifically binds to human TCR and comprises the following i. CDR1 as the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 6; ii. CDR2 as the amino acid sequence of SEQ ID NO: 10 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 10; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 14 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 14. Preferably, CDR1 is the amino acid sequence of SEQ ID NO: 6, CDR2 is the amino acid sequence of SEQ ID NO: 10, and CDR3 is the amino acid sequence of SEQ ID NO: 14.

[0169] In a preferred embodiment, the ISVD binds to the constant domains of human TCR-α (SEQ ID NO: 135) and / or TCR-β (SEQ ID NO: 136) or their polymorphic variants or subtypes.

[0170] Preferred examples of ISVDs that specifically bind to human TCRs include one or more (and preferably all) architectural regions (and CDRs as defined in item A above) as indicated for ISVD TCE01 in Table A-2, and preferably consist of the complete amino acid sequence of ISVD TCE01 (SEQ ID NO: 2; see Tables A-1 and A-2).

[0171] Similarly, in a preferred embodiment, the amino acid sequence of the ISVD that specifically binds to the human TCR can exhibit more than 90% (e.g., more than 95% or more than 99%) sequence identity with SEQ ID NO: 2, wherein the CDR is as defined in item A above. In particular, the ISVD that specifically binds to the TCR is preferably the amino acid sequence of SEQ ID NO: 2.

[0172] When such an ISVD that binds to the TCR exhibits a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence (item A above) in at least one CDR, the ISVD preferably exhibits at least half the binding affinity to the human TCR compared to the construct TCE01 shown in SEQ ID NO: 2, more preferably at least the same binding affinity, wherein the binding affinity is measured using the same method such as SPR.

[0173] Specific examples of ISVDs that can be specifically bound to GPC3 in the aforementioned technology are described in Items B and C below:

[0174] B. An ISVD that specifically binds to human GPC3 and comprises the following i. CDR1 as the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 7; ii. CDR2 as the amino acid sequence of SEQ ID NO: 11 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 11; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 15 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 15. Preferably, CDR1 is the amino acid sequence of SEQ ID NO: 7, CDR2 is the amino acid sequence of SEQ ID NO: 11, and CDR3 is the amino acid sequence of SEQ ID NO: 15.

[0175] C. An ISVD that specifically binds to human GPC3 and comprises the following i. CDR1 as the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 8; ii. CDR2 as the amino acid sequence of SEQ ID NO: 12 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 12; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 16 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16. Preferably, CDR1 is the amino acid sequence of SEQ ID NO: 8, CDR2 is the amino acid sequence of SEQ ID NO: 12, and CDR3 is the amino acid sequence of SEQ ID NO: 16.

[0176] In a preferred embodiment, the ISVD is bound to human GPC3 of SEQ ID NO: 134.

[0177] Preferred examples of ISVDs that specifically bind to human GPC3 include one or more (and preferably all) architectural regions (and CDRs as defined in items B and C above, respectively) indicated for ISVDs A022600351 and A022600314 in Table A-2, and preferably consist of the complete amino acid sequence of ISVD A022600351 or A022600314 (SEQ ID NO: 3 or 4, see Tables A-1 and A-2).

[0178] Similarly, in a preferred embodiment, the amino acid sequence of one or more ISVDs specifically binding to human GPC3 can exhibit more than 90% (e.g., more than 95% or more than 99%) sequence identity with SEQ ID NO: 3 or 4, wherein the CDRs are as defined in item B or item C above. In particular, the ISVD binding to human GPC3 is preferably the amino acid sequence of SEQ ID NO: 3 or 4.

[0179] When such an ISVD bound to human GPC3 exhibits a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence (item B or item C above) in at least one CDR, the ISVD preferably exhibits at least half the binding affinity to human GPC3, and more preferably at least the same binding affinity, compared to the constructs A022600351 or A022600314 shown in SEQ ID NO: 3 and 4, respectively, wherein the binding affinity is measured using the same method such as SPR.

[0180] Preferably, each of the ISVDs defined under items A through C above is included in the polypeptide.

[0181] Compared to a polypeptide composed of the amino acids of SEQ ID NO: 1, such a polypeptide comprising each of the ISVDs as defined under items A to C above preferably exhibits at least half the binding affinity to human TCR and at least the same binding affinity to human GPC3, wherein the binding affinity is measured using the same method such as SPR.

[0182] The SEQ ID NOs mentioned in items A through C above and item D below (see Section 5.4 "(In vivo) Half-life Extension") are based on the CDR definition as defined by AbM (see Table A-2). It should be noted that the SEQ ID NOs defined according to the same CDR as defined by Kabat (see Table A-2-1) can also be used for items A through C above and item D below (see Section 5.4 "(In vivo) Half-life Extension").

[0183] Therefore, specific examples of constant domains of TCRs or ISVDs of GPC3 that can be used in the technology specifically binding to T cells, as described above using the AbM definition, can also be described using the Kabat definition shown in Projects A' through C' below:

[0184] A'. A specific binding agent to human TCR and comprising the following ISVD i. CDR1 as the amino acid sequence of SEQ ID NO: 31 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 31; ii. CDR2 as the amino acid sequence of SEQ ID NO: 35 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 35; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 14 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 14. Preferably, CDR1 is the amino acid sequence of SEQ ID NO: 31, CDR2 is the amino acid sequence of SEQ ID NO: 35, and CDR3 is the amino acid sequence of SEQ ID NO: 14.

[0185] Preferred examples of ISVDs that specifically bind to human TCRs include one or more (and preferably all) architectural regions (and CDRs as defined in item A' above) indicated for ISVD TCE01 as shown in Table A-2-1, and preferably consist of the complete amino acid sequence of ISVD TCE01 (SEQ ID NO: 2; see Tables A-1 and A-2-1).

[0186] B'. An ISVD that specifically binds to human GPC3 and comprises the following i. CDR1 as the amino acid sequence of SEQ ID NO: 32 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 32; ii. CDR2 as the amino acid sequence of SEQ ID NO: 36 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 36; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 15 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 15. Preferably, CDR1 is the amino acid sequence of SEQ ID NO: 32, CDR2 is the amino acid sequence of SEQ ID NO: 36, and CDR3 is the amino acid sequence of SEQ ID NO: 15.

[0187] C'. An ISVD that specifically binds to human GPC3 and comprises the following i. CDR1 as the amino acid sequence of SEQ ID NO: 33 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 33; ii. CDR2 as the amino acid sequence of SEQ ID NO: 37 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 37; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 16 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 16. Preferably, CDR1 is the amino acid sequence of SEQ ID NO: 33, CDR2 is the amino acid sequence of SEQ ID NO: 37, and CDR3 is the amino acid sequence of SEQ ID NO: 16.

[0188] Preferred examples of such specific binding to one or more ISVDs of human GPC3 include one or more (and preferably all) architectural regions (and CDRs as defined in items B' and C' above, respectively) indicated for ISVDs A022600351 and A022600314 in Table A-2-1, and preferably ISVDs consisting of the complete amino acid sequence of ISVD A022600351 or A022600314 (SEQ ID NO: 3 or 4, see Tables A-1 and A-2-1).

[0189] The percentage of "sequence identity" between the first amino acid sequence and the second amino acid sequence can be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], where each deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence (compared to the first amino acid sequence) is considered a difference of a single amino acid residue (i.e., at a single position).

[0190] Typically, for the purpose of determining the percentage of “sequence identity” between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is designated as the “first” amino acid sequence, and the other amino acid sequence is designated as the “second” amino acid sequence.

[0191] As used herein, "amino acid difference" refers to the deletion, insertion, or substitution of a single amino acid residue relative to a reference sequence, and preferably substitution.

[0192] Amino acid substitution is preferably conservative substitution. Such conservative substitution is substitution in which one amino acid from the following groups (a)-(e) is replaced by another amino acid residue from the same group: (a) small aliphatic, nonpolar, or micropolar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) acetylamines: Asp, Asn, Glu, and Gln; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0193] The particularly preferred conservative substitutions are as follows: Ala becomes Gly or Ser; Arg becomes Lys; Asn becomes Gln or His; Asp becomes Glu; Cys becomes Ser; Gln becomes Asn; Glu becomes Asp; Gly becomes Ala or Pro; His becomes Asn or Gln; Ile becomes Leu or Val; Leu becomes Ile or Val; Lys becomes Arg, becomes Gln or Glu; Met becomes Leu, becomes Tyr or Ile; Phe becomes Met, becomes Leu or Tyr; Ser becomes Thr; Thr becomes Ser; Trp becomes Tyr; Tyr becomes Trp; and / or Phe becomes Val, becomes Ile or Leu. [5.3] [Specificity]

[0194] The terms "specific," "specifically binding," or "specifically binding" refer to the number of different target molecules (such as antigens) from the same organism that a particular binding unit (such as an ISVD) can bind with sufficiently high affinity (see below). "Specific," "specifically binding," or "specifically binding" may be used interchangeably with "selective," "selectively binding," or "selectively binding" herein. Binding units such as ISVD bind more specifically to their designated targets.

[0195] The specificity / selectivity of the binding unit can be determined based on affinity. Affinity represents the strength or stability of molecular interactions. Affinity is usually given by KD or the dissociation constant, expressed in mol / L (or M). Affinity can also be expressed as the association constant KA, which is equal to 1 / KD and expressed in (mol / L)⁻¹ (or M⁻¹).

[0196] Affinity is a measure of the strength of binding between a target molecule and its binding sites: the lower the KD value, the stronger the binding between the target molecule and the target site.

[0197] Typically, the binding unit (such as ISVD) used in the present invention binds to its target with a dissociation constant (KD) of 10⁻⁵ to 10⁻¹² mol / L or lower, and preferably 10⁻⁷ to 10⁻¹² mol / L or lower, and more preferably 10⁻⁸ to 10⁻¹² mol / L (i.e., an association constant (KA) of 10⁵ to 10¹² L / mol or higher, and preferably 10⁷ to 10¹² L / mol or higher, and more preferably 10⁸ to 10¹² L / mol).

[0198] It is generally believed that any KD value greater than 10⁻⁴ mol / L (or any KA value less than 10⁴ L / mol) indicates nonspecific binding.

[0199] KD, which is considered to have specific biological interactions (such as the binding of immunoglobulin sequences to antigens), is typically in the range of 10⁻⁵ mol / L (10,000 nM or 10 µM) to 10⁻¹² mol / L (0.001 nM or 1 pM) or lower.

[0200] Therefore, specific / selective binding may mean that, using the same measurement method such as SPR, the binding unit (or the peptide containing it) binds to TCR and / or GPC3 with a KD value of 10⁻⁵ to 10⁻¹² mol / L or less, and binds to the relevant target with a KD value greater than 10⁻⁴ mol / L. Examples of relevant targets for GPC3 are GPC1, GPC2, GPC4, GPC5, or GPC6. Thus, in embodiments of the technique, the peptide contains an ISVD of 10⁻⁵ to 10⁻¹² mol / L or less to bind to GPC3, and binds to GPC1, GPC2, GPC4, GPC5, and GPC6 of the same species with a KD value greater than 10⁻⁴ mol / L.

[0201] Therefore, compared with the polypeptide composed of the amino acids of SEQ ID NO: 1, the polypeptide preferably exhibits at least half the binding affinity to human TCR and at least the same affinity to human GPC3, wherein the binding affinity is measured using the same method such as SPR.

[0202] Specific binding to a specific target from a particular species does not preclude the binding unit from specifically binding to similar targets from different species. For example, specific binding to human TCRs does not preclude the binding unit (or a polypeptide containing the binding unit) from specifically binding to TCRs from cynomolgus monkeys. Similarly, specific binding to human GPC3, for example, does not preclude the binding unit (or a polypeptide containing the binding unit) from specifically binding to GPC3 from cynomolgus monkeys.

[0203] The ISVD having SEQ ID NO: 2, which binds to human TCR and is contained in a polypeptide of the present technology, exhibits improved binding characteristics compared to ISVD T0170056G05 described in WO 2016 / 180969 A1. More specifically, the ISVD having SEQ ID NO: 2 is derived from T017056G05 and contains specific mutations in CDR1 and CDR3 that result in improved cross-reactivity with human TCR and non-human primate (e.g., cynomolgus monkey) TCR compared to ISVD T0170056G05.

[0204] When it is said that an ISVD exhibits “improved cross-reactivity with human TCRs and nonhuman primate TCRs compared to another ISVD”, it means that for the ISVD, the ratio of binding activity (e.g., expressed as KD or k-off) to human TCRs and nonhuman primate TCRs is lower than that ratio calculated for other ISVDs in the same assay.

[0205] The favorable cross-reactivity with human TCRs and non-human primate TCRs allows for the evaluation of the toxicity of multispecific T-cell conjugating peptides in preclinical studies in non-human primates.

[0206] The specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assays, as well as different variants of these known in the art; and other techniques mentioned herein.

[0207] As will be apparent to those skilled in the art, the dissociation constant can be either actual or apparent. Methods for determining the dissociation constant will be clear to those skilled in the art, and include, for example, the techniques mentioned below. In this regard, it will also be clear that dissociation constants greater than 10⁻⁴ mol / L or 10⁻³ mol / L (e.g., 10⁻² mol / L) may not be measured. Optionally, as will be clear to those skilled in the art, the (actual or apparent) dissociation constant can be calculated based on the (actual or apparent) association constant (KA) using the relationship [KD = 1 / KA].

[0208] The affinity of molecular interactions between two molecules can be measured using various techniques known to them, such as the well-known surface plasma resonance (SPR) biosensor technique (see, for example, Ober et al. 2001, Intern. Immunology 13: 1551-1559). As used herein, the term "surface plasma resonance" refers to an optical phenomenon that allows for the analysis of instantaneous, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and another molecule passes over the immobilized molecule under flow conditions, resulting in k-on, k-off measurements, and thus KD (or KA) values. This can be performed, for example, using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscatave, New Jersey). For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51: 19-26), Jonsson et al. (1991 Biotechniques 11: 620-627), Johnson et al. (1995, J. Mol. Recognit. 8: 125-131) and Johnson et al. (1991, Anal. Biochem. 198: 268-277).

[0209] Another well-known biosensor technique for determining the affinity of biomolecular interactions is bio-layer interferometry (BLI) (see, for example, Abdiche et al. 2008, Anal. Biochem. 377: 209-217). As used herein, the term "bio-layer interferometry" or "BLI" refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an inner reference layer (reference beam) and a layer of immobilized proteins on the biosensor tip (signal beam). Variations in the number of molecules bound to the biosensor tip result in a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the surface of the biosensor tip. Because interactions can be measured instantaneously, association and dissociation rates and affinities can be determined. For example, BLI can be performed using the well-known Octet® system (ForteBio, a division of Pall Life Sciences, Menlo Parker, USA).

[0210] Alternatively, affinity can be measured in a kinetic rejection assay (KinExA) using the KinExA® platform (Sapidyne Instruments Inc., Boise, USA) (see, for example, Drake et al. 2004, Anal. Biochem., 328: 35-43). As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of an antibody / antigen complex is passed through a column containing beads pre-coated with an antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is performed using a fluorescently labeled protein that binds the antibody (or antigen).

[0211] The GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74). [5.4] [Prolonged half-life (in vivo)]

[0212] The polypeptide may also include one or more other groups, residues, portions, or binding units, which are optionally linked via one or more peptide linkers, wherein the other groups, residues, portions, or binding units provide an increased (in vivo) half-life to the polypeptide compared to a corresponding polypeptide without the other groups, residues, portions, or binding units. An extended in vivo half-life means, for example, that the polypeptide exhibits an increased half-life in mammalian subjects, such as human subjects, after administration. Half-life may be expressed, for example, as t1 / 2β.

[0213] The type of group, residue, part or binding unit is generally not limited and can be, for example, selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc parts, and small proteins or peptides that can bind to serum proteins.

[0214] More specifically, the one or more other groups, residues, portions, or binding units that provide an increased half-life for the polypeptide may be selected from binding units that can bind to serum albumin such as human serum albumin or serum immunoglobulin such as IgG, and preferably binding units that can bind to human serum albumin. The binding unit is preferably ISVD.

[0215] For example, WO 04 / 041865 describes Nanobodies® that binds to serum albumin (and particularly to human serum albumin) and can be linked to other proteins (such as one or more other Nanobodies that bind to the desired target) to increase the half-life of said protein.

[0216] International application WO 06 / 122787 describes several Nanobodies® targeting (human) serum albumin. These Nanobodies® include Nanobody® called Alb-1 (SEQ ID NO: 52 in WO 06 / 122787) and its humanized variants, such as Alb-8 (SEQ ID NO: 62 in WO 06 / 122787). Similarly, these can be used to extend the half-life of therapeutic proteins and peptides, as well as other therapeutic entities or portions.

[0217] In addition, WO2012 / 175400 describes another improved form of Alb-1, called Alb-23.

[0218] In a preferred embodiment, the polypeptide comprises a serum albumin-binding moiety selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10, and Alb-23, preferably Alb-8 or Alb-23 or variants thereof, as shown on pages 7-9 of WO 2012 / 175400, and albumin conjugates described in WO 2012 / 175741, WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235, and WO 2018 / 134234. Some preferred serum albumin conjugates are also shown in Tables A-4. Other particularly preferred components of the polypeptide are as described in item D:

[0219] D. An ISVD that binds to human serum albumin and comprises the following: i. CDR1 as the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 9; ii. CDR2 as the amino acid sequence of SEQ ID NO: 13 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 13; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 17 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 17; Preferably, CDR1 is the amino acid sequence of SEQ ID NO: 9, CDR2 is the amino acid sequence of SEQ ID NO: 13, and CDR3 is the amino acid sequence of SEQ ID NO: 17.

[0220] A preferred example of such an ISVD bound to human serum albumin comprises one or more (and preferably all) architectural regions (and CDRs as defined in item D above) as indicated in Table A-2 for ISVD ALB23002, and preferably is an ISVD consisting of the complete amino acid sequence of ISVD ALB23002 (SEQ ID NO: 5, see Tables A-1 and A-2).

[0221] Project D can also be described using Kabat definitions as follows:

[0222] D'. An ISVD that binds to human serum albumin and comprises the following i. CDR1 as the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having two or one amino acid different from that of SEQ ID NO: 34; ii. CDR2 as the amino acid sequence of SEQ ID NO: 38 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 38; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 17 or having an amino acid sequence that differs from one or two amino acids of SEQ ID NO: 17; Preferably, CDR1 is the amino acid sequence of SEQ ID NO: 34, CDR2 is the amino acid sequence of SEQ ID NO: 38, and CDR3 is the amino acid sequence of SEQ ID NO: 17.

[0223] Preferred examples of such ISVDs that bind to human serum albumin include one or more (and preferably all) architectural regions (and CDRs as defined in item D' above) as indicated for ISVD ALB23002 in Table A-2-1, and preferably consist of the complete amino acid sequence of ISVD ALB23002 (SEQ ID NO: 5, see Tables A-1 and A-2-1).

[0224] Similarly, in a preferred embodiment, the amino acid sequence of the ISVD bound to human serum albumin may exhibit more than 90% (e.g., more than 95% or more than 99%) sequence identity with SEQ ID NO: 5, wherein the CDR is as defined in item D or item D' above. In particular, the ISVD bound to human serum albumin is preferably the amino acid sequence of SEQ ID NO: 5.

[0225] When such an ISVD bound to human serum albumin exhibits a difference of 2 or 1 amino acid relative to the corresponding reference CDR sequence (item D or item D' above) in at least one CDR, the ISVD exhibits at least half the binding affinity to human serum albumin, preferably at least the same binding affinity, compared to the construct ALB23002 shown in SEQ ID NO: 5, wherein the binding affinity is measured using the same method such as SPR.

[0226] When such an ISVD bound to human serum albumin is located at the C-terminal position, it can exhibit a C-terminal alanine (A) or glycine (G) extension (preferably A) and is preferably selected from SEQ ID NO: 83, 85, 87, 89, 91, 93, 95, 96 and 98, most preferably SEQ ID NO: 96, which represents SEQ ID NO: 5 with a single alanine extension (see Table A-4 below). In some embodiments, the ISVD bound to human serum albumin is located at a position different from the C-terminal position (i.e., not the C-terminal ISVD of the polypeptide) and is selected from SEQ ID NO: 5, 82, 84, 86, 88, 90, 92, 94 and 97 (see Table A-4 below). [5.5] [Nucleic acid molecules]

[0227] A nucleic acid molecule encoding a polypeptide as disclosed herein is also provided.

[0228] A "nucleic acid molecule" (which may be used interchangeably with "nucleic acid") is a chain of nucleotide monomers linked together by a phosphate backbone to form a nucleotide sequence. Nucleic acids can be used to transform / transfect host cells or host organisms, for example, for the expression and / or production of polypeptides. Suitable hosts or host cells for production purposes will be clear to those skilled in the art and can be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line or any suitable fungus, prokaryotic or eukaryotic organism. Hosts or host cells containing nucleic acids encoding the said polypeptides are also covered by the art.

[0229] Nucleic acids can be, for example, DNA, RNA, or hybrids thereof, and may also contain (e.g., chemically modified) nucleotides, such as PNA. They can be single-stranded or double-stranded, and are preferably in the form of double-stranded DNA. For example, the nucleotide sequence can be genomic DNA or cDNA.

[0230] Nucleic acids can be prepared or obtained in ways known per se, and / or isolated from suitable natural sources. Nucleotide sequences encoding naturally occurring (poly)peptides can, for example, be subjected to site-directed mutagenesis to provide nucleic acid molecules encoding polypeptides with sequence variations. Similarly, as will be apparent to those skilled in the art, for the preparation of nucleic acids, several nucleotide sequences, such as at least one nucleotide sequence encoding a target moiety, can be linked together in a suitable manner with nucleic acids, for example, those encoding one or more linkers.

[0231] The techniques used to generate nucleic acids will be clear to those familiar with the techniques and may include, for example, but not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more portions thereof) to introduce mutations that result in the expression of truncated products; introducing one or more restriction sites (e.g., to produce boxes and / or regions that may be easily digested and / or linked by suitable restriction enzymes); and / or introducing mutations by means of PCR reactions using one or more “mismatched” primers. [5.6] [Carrier]

[0232] Also provided is a vector comprising a nucleic acid molecule encoding a polypeptide as disclosed herein. Vectors, as used herein, are suitable for carrying genetic material into cells. Vectors include naked nucleic acids, such as plasmids or mRNA, or nucleic acids embedded in larger structures such as liposomes or viral vectors.

[0233] Vectors typically contain at least one nucleic acid, optionally linked to one or more regulatory elements, such as, for example, one or more suitable promoters, enhancers, terminators, etc. Vectors are preferably expression vectors, i.e., vectors suitable for expressing encoded polypeptides or constructs under suitable conditions (e.g., when said vector is introduced into cells, e.g., human cells). For DNA-based vectors, this typically includes the presence of elements for transcription (e.g., promoters and polyadenylate signals) and translation (e.g., Kozak sequences).

[0234] Preferably, in the vector, the at least one nucleic acid and the regulatory element are "operably linked" to each other, which generally means that they have a functional relationship with each other. For example, a promoter is considered "operably linked" to a coding sequence if it is capable of initiating or otherwise controlling / regulating transcription and / or expression of the coding sequence (wherein the coding sequence should be understood as "under the control of the promoter"). Typically, when two nucleotide sequences are operably linked, they are in the same orientation and usually also in the same reading frame. They are usually also substantially contiguous, but this may not be necessary.

[0235] Preferably, any regulatory elements of the vector enable it to provide its intended biological function in the intended host cell or host organism.

[0236] For example, a promoter, enhancer, or terminator should be "operable" in the intended host cell or host organism, meaning, for example, that the promoter should be able to initiate or otherwise control / regulate the transcription and / or expression of a nucleotide sequence (e.g., a coding sequence) that is operably linked to it. [5.7] [Composition]

[0237] The technology also provides a composition comprising at least one polypeptide as disclosed herein, at least one nucleic acid molecule encoding a polypeptide as disclosed herein, or at least one carrier comprising such a nucleic acid molecule. The composition may be a pharmaceutical composition. The composition may also comprise at least one pharmaceutically acceptable carrier, diluent, or excipient and / or adjuvant, and optionally comprise one or more other pharmaceutically active polypeptides and / or compounds. [5.8] [Host organism]

[0238] The technology also relates to host cells or host organisms that contain polypeptides as disclosed herein, nucleic acids encoding polypeptides as disclosed herein, and / or carriers containing nucleic acid molecules encoding polypeptides as disclosed herein.

[0239] Suitable host cells or host organisms are clear to those skilled in this art, and are, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, *Escherichia coli*, or *Pichia pastoris*. The optimal host is *Pichia pastoris*. [5.9] [Methods and uses of the polypeptide]

[0240] The technology also provides a method for generating peptides as disclosed herein. The method may include transforming / transfecting a host cell or host organism with a nucleic acid encoding the peptide, expressing the peptide in the host, and optionally subsequently performing one or more isolation and / or purification steps. Specifically, the method may include: a) Expressing the nucleic acid sequence encoding the polypeptide in a suitable host cell or host organism or in another suitable expression system; optionally, this is performed subsequently: b) Isolate and / or purify the polypeptide.

[0241] The suitable host cell or host organism for production purposes will be clear to those skilled in the art, and can be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, *Escherichia coli*, or *Pichia pastoris*. The optimal host is *Pichia pastoris*.

[0242] The polypeptide, nucleic acid molecule, or carrier, or a composition comprising the polypeptide, nucleic acid molecule, or carrier, preferably the polypeptide or a composition comprising the polypeptide, can be used as a drug.

[0243] Therefore, the technology provides peptides, nucleic acid molecules or carriers as described above, or compositions comprising said peptides, nucleic acid molecules or carriers, for use as pharmaceuticals.

[0244] It also provides polypeptides, nucleic acid molecules or carriers as described, or compositions comprising said polypeptides, nucleic acid molecules or carriers, for the treatment of cancer.

[0245] A further method for treating cancer is provided, wherein the method includes administering to a subject in need a pharmaceutically active amount of, as described above, a polypeptide, nucleic acid molecule or carrier, or a composition comprising said polypeptide, nucleic acid molecule or carrier.

[0246] Further, the use of the polypeptide, nucleic acid molecule or carrier as described, or a composition comprising the polypeptide, nucleic acid molecule or carrier, in the preparation of a drug is provided.

[0247] Further, the use of the polypeptide, nucleic acid molecule, or carrier as described, or a composition comprising the polypeptide, nucleic acid molecule, or carrier, in the preparation of a pharmaceutical composition preferably used for treating cancer is provided.

[0248] The cancer can be any type of cancer exhibiting GPC3. The cancer is preferably liver cancer or lung cancer, with liver cancer being more preferred. Liver cancer is more preferably hepatocellular carcinoma. Lung cancer is more preferably non-small cell lung cancer (NSCLC), and most preferably squamous cell carcinoma (SCC).

[0249] Preferably, cancer cells expressing GPC3 express at least half the amount of GPC3 protein (on average) compared to Huh7 cells (on average), and more preferably at least the same amount of GPC3 protein. Huh7 cells are publicly available from, for example, the National Institutes of Biomedical Innovation, Health and Nutrition and the JCRB Cell Bank under accession number JCRB0403.

[0250] The GPC3 represented here preferably refers to GPC3 exposed on the cell surface. The representation (and amount) of (cell surface exposed) GPC3 on the cell can be readily determined by conventional methods generally known in the industry, such as flow cytometry, immunohistochemistry, or as described in the examples.

[0251] As used in the context of the described technology, the “subject” can be any animal, preferably a mammal. Among mammals, a distinction can be made between humans and non-human mammals. Non-human animals can be, for example, companion animals (e.g., dogs, cats), livestock (e.g., cattle, horses, sheep, goats, or pigs) or animals commonly used for research purposes and / or for antibody production (e.g., mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates (e.g., cynomolgus monkeys) or camels (e.g., llamas or alpacas)).

[0252] In cases of prevention and / or treatment, the subject may be any animal, and more specifically any mammal, but preferably a human subject.

[0253] Substances (including polypeptides, nucleic acid molecules, and carriers) or components can be administered to the subject via any suitable route of administration (e.g., enterally (e.g., orally or rectally) or parenterically (e.g., epidermally, sublingually, buccally, nasally, intra-articularly, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, percutaneously, or transmucosally). Parenterial administration, such as intramuscular, subcutaneous, or intradermal administration, is preferred. Subcutaneous administration is the most optimal.

[0254] An effective amount of the peptide, nucleic acid molecule or carrier as described, or a composition containing the peptide, nucleic acid molecule or carrier, may be administered to the subject in order to provide the desired therapeutic outcome.

[0255] One or more doses may be administered. If more than one dose is administered, the doses may be administered at appropriate intervals to maximize the effect of the polypeptide, composition, nucleic acid molecule, or carrier.

[0256] [surface] [A-1] [In pentavalent polypeptides] [A022600424] Different unit prices for internal testing [ISVD] [Amino acid sequence of the building block (」「)] [ID] [ " refers to [as used in this article] [SEQ ID NO] [)] [name] [ID] [Amino acid sequence] TCE01 2 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSS A022600351* 3 EVQLVESGGGVVQPGGSLRLSCAASGFTFSSFAMTWVRRPPGKGLEWVATITNKGVTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYICANARRTGPRAPTDIGSYRGQGTLVTVSS A022600314° 4 EVQLVESGGGVVQPGGSLRLSCAASGSIFRSVFSSSTMEWYRQAPGKKRELVARIAPGEGTYYGALYADSVKGRFTISSRDNAKNTVYLQMNSLRPEDTALYYCASGVAWGQGTLVTVSS ALB23002 5 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS * Sequence-optimized variant of A02260018C08 (SEQ ID NO: 48) ° Sequence-optimized variant of A02260015A08 (SEQ ID NO: 47)

[0257] [surface] [A-2] [:] [CDR] [and the sequence of architectures()] [」「] [ID] [」] [Refers to the given] [SEQ ID NO] [)] [ID] [ISVD] [ID] [FR1] [ID] [CDR1] [ID] [FR2] [ID] [CDR2] [ID] [FR3] [ID] [CDR3] [ID] [FR4] 2 TCE01 18 DVQLVESGGGVVQPGGSLRLSCVAS 6 GYVHKINFYG 20 WYRQAPGKEREKVA 10 HISIGDQTD 24 YADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRA 14 LSRIWPYDY 28 WGQGTLVTVSS 3 A022600351* 19 EVQLVESGGGVVQPGGSLRLSCAAS 7 GFTFSSFAMT 21 WVRRPPGKGLEWVA 11 TITNKGVTS 25 YADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYICAN 15 ARRTGPRAPTDIGSY 29 RGQGTLVTVSS 4 A022600314° 19 EVQLVESGGGVVQPGGSLRLSCAAS 8 GSIFRSVFSSSTME 22 WYRQAPGKKRELVA 12 RIAPGEGTYYGAL 26 YADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCAS 16 GVA 28 WGQGTLVTVSS 5 ALB23002 19 EVQLVESGGGVVQPGGSLRLSCAAS 9 GFTFRSFGMS twenty three WVRQAPGKGPEWVS 13 SISGSGSDTL 27 YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTI 17 GGSLSR 30 SSQGTLVTVSS * Sequence-optimized variant of A02260018C08 (SEQ ID NO: 48) ° Sequence-optimized variant of A02260015A08 (SEQ ID NO: 47)

[0258] [surface] [A-2-1] [:] [CDR] [and the sequence of architectures (""") [ID] [ " refers to the given information. [SEQ ID NO] [)] [ID] [ISVD] [ID] [FR1] [ID] [CDR1] [ID] [FR2] [ID] [CDR2] [ID] [FR3] [ID] [CDR3] [ID] [FR4] 2 TCE01 39 DVQLVESGGGVVQPGGSLRLSCVASGYVHK 31 INFYG 20 WYRQAPGKEREKVA 35 HISIGDQTDYADSAKG 43 RFTISRDESKNTVYLQMNSLRPEDTAAYYCRA 14 LSRIWPYDY 28 WGQGTLVTVSS 3 A022600351* 40 EVQLVESGGGVVQPGGSLRLSCAASGFTFS 32 SFAMT 21 WVRRPPGKGLEWVA 36 TITNKGVTSYADSVKG 44 RFTISRDNAKNTLYLQMNSLRPEDTALYICAN 15 ARRTGPRAPTDIGSY 29 RGQGTLVTVSS 4 A022600314° 41 EVQLVESGGGVVQPGGSLRLSCAASGSIFR 33 SVFSSSTME 22 WYRQAPGKKRELVA 37 RIAPGEGTYYGALYADSVKG 45 RFTISRDNAKNTVYLQMNSLRPEDTALYYCAS 16 GVA 28 WGQGTLVTVSS 5 ALB23002 42 EVQLVESGGGVVQPGGSLRLSCAASGFTFR 34 SFGMS 23 WVRQAPGKGPEWVS 38 SISGSGSDTLYADSVKG 46 RFTISRDNSKNTLYLQMNSLRPEDTALYYCTI 17 GGSLSR 30 SSQGTLVTVSS * Sequence-optimized variant of A02260018C08 (SEQ ID NO: 48) ° Sequence-optimized variant of A02260015A08 (SEQ ID NO: 47)

[0259] [surface] [A-3] [:Selected Multiple Prices] [ISVD] [Amino acid sequence] [name] [ID] [Amino acid sequence] A022600424 1 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEV QLVESGGGVVQPGGSLRLSCAASGFTFSSFAMTWVRRPPGKGLEWVATITNKGVTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGG SGGGSEVQLVESGGGVVQPGGSLRLSCAASGSIFRSVFSSSTMEWYRQAPGKKRELVARIAPGEGTYYGALYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCASGVAWGQGTLVTVSS GGGGSGGGSEVQLVESGGGVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA

[0260] [surface] [A-4] [:serum albumin binding] [ISVD] [sequence(""] [ID] [as used herein] [SEQ ID NO] [)] [Name] [ID] [Amino acid sequence] Alb8 82 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb8-A 83 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA Alb23 84 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23-A 85 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSA Alb83 86 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSS Alb83-A 87 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb132 88 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSS Alb132-A 89 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb73 90 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSS Alb73-A 91 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82 92 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82-A 93 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb199 94 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVKVSS Alb199-A 95 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVKVSSA Alb23002 5 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb223 96 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb216 97 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVKVSS Alb216-A 98 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVKVSSA

[0261] [surface] [A-5] [:connect subsequences()] [」「] [ID] [」] [Refers to what is used in this article] [SEQ ID NO] [)] [name] [ID] [Amino acid sequence] 3A connector 99 AAA 5GS connector 100 GGGGS 7GS connector 101 SGGSGGS 8GS connector 102 GGGGSGGS 9GS connector 103 GGGGSGGGS 10GS connector 104 GGGGSGGGGS 15GS connector 105 GGGGSGGGGSGGGGS 18GS connector 106 GGGGSGGGGSGGGGSGGS 20GS connector 107 GGGGSGGGGSGGGGSGGGGS 25GS connector 108 GGGGSGGGGSGGGGSGGGGSGGGGS 30GS connector 109 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS connector 110 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 40GS connector 111 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS G1 hinge 112 EPKSCDKTHTCPPCP 9GS-G1 hinge 113 GGGGSGGGSEPKSCDKTHTCPPCP Upper hinge area of ​​the llama 114 EPKTPKPQPAAA G3 hinge 115 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

[0262] [surface] [A-6] [Amino acid sequence of the selected multivalent polypeptide (]] [ID] [」refers to a given] [SEQ ID NO] [)] [SEQ] [ID] [sequence] 49 A022600027 DVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGLTFSSYAMGWFRQAPGKERERVVSISRGGGYTYYADSVKGRFTISRDNSENTVYLQMNSLRPEDTALYYCAAARYWATGSEYEFDYWGQGTLVTVSS 50 A022600031 DVQLVESGGGLVQPGGSLRLSCVASGDVHKINFLGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNMVYLQMNSLKPEDTAVYFCRAFSRIYPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDTGTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGGVVQPGGSLRLSCAASGLTFSYAMGWFRQAPGKERERVVSISRGGGYTYYADSVKGRFTISRDNSENTVYLQMNSLRPEDTALYYCAAARYWATGSEYEFDYWGQGTLVTVSS 51 A022600096 DVQLVESGGGLVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTALYCRALSRIWPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRD SVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPDTIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGSEVQLVESGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYCTIGGSLSRSSQGTLVTVSSA 52 A022600102 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSAAAEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDSTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSA 53 A022600103 DVQLVESGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEGGGGSEVQLVESGGGGVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDSTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSA 54 A022600104 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSGGGGSGGGSEGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDSTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSA 55 A022600105 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDSTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSA 56 A022600122 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGGVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 57 A022600131 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGGVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 58 A022600132 DVQLVESGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGRAPDTIGSYRGQGTLVTVSSGGGGSGGGSEGGSEVQLVESGGGLVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSGGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSGGGSEVQLVESGGGGVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 59 A022600133 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGGVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDSTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 60 A022600134 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEGGGGSEVQLVESGGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDSTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 61 A022600135 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTALYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSGGGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDSTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 62 A022600167 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPDETAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDSTLYADSVKGRFTISRDNSKNTLYLQMNSLRPDEDTALYCTIGGGSLSRSSQGTLVTVSSA 63 A022600168 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPDETAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEGGGSEVQLVESGGGLVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDTGTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEGGGGSEVQLVESGGGGVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPDEDTALYCTIGGSLSRSSQGTLVTVSSA 64 A022600169 DVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATINGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDATVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSEVQLVESGGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDTGTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEGGGGSEVQLVESGGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 65 A022600170 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPDETAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDTGTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSGGGGSGGGSEGGSEVQLVESGGVVQGTLVTVSSGGGGSGGGSEVQLPGKGPEWVSSISGSGSDSTLYADSVKGRFTISRDNSKNTLYLQMNSLRPDETATALYCTIGGSLSRSSQGTLVTVSSA 66 A022600172 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPDETAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATINGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGSEGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPDEDTALYCTIGGGSLSRSSQGTLVTVSSGGGGSGGGSEGGSEVQLVESGGGLVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDGTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSA 67 A022600174 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPDETAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPDIGSYRGQGTLVTVSSGGGGSGGGSEGGSEVQLVESGGGLVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSGGGGSGGGSEGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPDEDTALYCTIGGSLSRSSQGTLVTVSSA 68 A022600175 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPDETAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGRAPDTIGSYRGQGTLVTVSSGGGGSGGGSEGGSEVQLVESGGGLVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEGGGGSEVQLVESGGGGVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPDEDTALYCTIGGSLSRSSQGTLVTVSSA 69 A022600178 DVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSEVQLVESGGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEGGGGSEVQLVESGGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDSTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 70 A022600179 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 71 A022600370 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGVVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLRPEDTALYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGC 72 A022600372 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGVVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLRPEDTALYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDGTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLRPEDTGLYFCASGVAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGC 73 A022600373 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGC 74 T017000698 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 78 A022600412 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSSFAMTWVRRPPGKGLEWVATITNAGVTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 1 A022600424 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSSFAMTWVRRPPGKGLEWVATITNKGVTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSIFRSVFSSSTMEWYRQAPGKKRELVARIAPGEGTYYGALYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCASGVAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 79 A022600425 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGSIFRSVFSSSTMEWYRQAPGKKRELVARIAPGEGTYYGALYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCASGVAWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSSFAMTWVRRPPGKGLEWVATITNAGVTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 80 A022600426 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGSIFRSVFSSSTMEWYRQAPGKKRELVARIAPGEGTYYGALYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCASGVAWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSSFAMTWVRRPPGKGLEWVATITNKGVTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA 81 A022600427 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFSSFAMTWVRRPPGKGLEWVATITNAGVTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYICANARRTGPRAPTDIGSYRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSIFRSVFSSSTMEWYRQAPGKKRELVARIAPGEGTYYGALYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCASGVAWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA

[0263] [Table] [A-7] [: with or without] [C] [with a terminal extension] [C] [terminal (」「] [ID] [」 as used herein refers to a given] [SEQ ID NO] [)] [ID] [amino acid sequence] 116 VTVSS 117 VKVSS 118 VQVSS 119 VTVKS 120 VTVQS 121 VKVKS 122 VKVQS 123 VQVKS 124 VQVQS 125 VTVSSA 126 VKVSSA 127 VQVSSA 128 VTVKSA 129 VTVQSA 130 VKVKSA 131 VKVQSA 132 VQVKSA 133 VQVQSA

[0264] [surface] [A-8] [:and] [GPC3] [and] [TCR] [Relevant amino acid sequences] ID [describe] [Amino acid sequence] 134 Human GPC3 (P51654) MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPGSDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIVVRHAKNYTNAMFKNNYPSLTPQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVIYTQLMNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIEVINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDKYWREYILSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQQRQYRSAYYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSPVAENDTLCWNGQELVERYSQKAARNGMKNQFNLHELKMKGPEPVVSQIIDKLKHINQLLRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDLDVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAISVVCFFFLVH 135 Human TCRα constant domain (derived from P01848) PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSC 136 Human TCRβ constant domain (derived from P01850) EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYALSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADC [6] [Example] [6.1] [Example] [1] [:Specifically binds to] [GPC3] [of] [ISVD] [The discovery]

[0265] Two llamas and one alpaca were immunized with a DNA dual-gene vector containing the sequence encoding the precursor of human phosphatidylinositol proteoglycan-3 isoform 2 [NP_004475; 580 AA; Homo sapiens]. The animals were then boosted with recombinant human phosphatidylinositol proteoglycan-3 (R&D Systems, catalog number 2119-GP).

[0266] Following the final immunogen injection, blood samples were collected and peripheral blood mononuclear cells (PBMCs) were prepared using Ficoll-Hypaque according to the manufacturer’s instructions (Amersham Biosciences, Piscatave, NJ, USA), and total RNA was extracted and stored.

[0267] Total RNA was used as the starting material for RT-PCR to amplify the gene fragment encoding ISVD. These fragments were selected and colonized into the phage vector pAX212. Phages were prepared according to the standard protocol (Antibody Phage Display: Methods and Protocols (1st edition, 2002, edited by O'Brian and Aitken, Humana Press, Totoro, NJ)) and stored after filtration sterilization at 4ºC until further use. Phage libraries were constructed from each immunized animal, yielding library sizes of 3 x 10⁸, 6 x 10⁸, and 8 x 10⁸ CFU.

[0268] Recombinant proteins were used to probe the phage display library. In short, phage particles were added at 50 nM (in PBS supplemented with 2% Marvell and 0.05% Tween 20) to biotinylated antigens (human GPC3 R&D Systems, catalog number 2119-GP; cynomolgus monkey GPC3 DGPI DHS-HIS, in-house manufactured (accession number P51654; Q3-R358; S359-H559, S495A, S509A); ​​all biotinylated using standard protocols). The biotinylated antigens were captured on streptavidin- or avidin-coated magnetic beads (Invitrogen). Unbound phages were washed away (with PBS supplemented with 0.05% Tween 20); bound phages were eluted by adding trypsin (1 mg / mL in PBS). Eluted phages were allowed to infect exponentially growing *E. coli* TG-1 cells for subsequent selection rounds (after rescue with helper phages) and / or for screening individual colonies after plating on agar plates. For this purpose, individual colonies were picked into 96-well dishes containing 0.5 mL of medium and grown overnight. 80 µL of the overnight culture from each colony was mixed with 40 µL of 60% glycerol in 2xTY and stored at -80ºC.

[0269] For small-scale production of ISVDs, the overnight culture was seeded into 96-well plates (1 mL volume). ISVD expression was induced by adding IPTG to a final concentration of 1 mM. Periplasmic extract was prepared by freezing the cell pellet and dissolving it in 100 µL PBS. Cell debris was removed by centrifugation.

[0270] Periplasmic extracts were screened for binding to human and cynomolgus monkey GPC3 in an ELISA assay. 384-well high-binding SpectraPlate (PerkinElmer, 6007509) was plated overnight at 4 ºC with 1 μg / mL protein (in PBS). The plates were then blocked at RT for at least one hour (PBS, 1% casein). A 1:10 dilution of the periplasmic extract (in PBS, 0.1% casein, 0.05% Tween 20) was added, and the plate was held at RT for one hour. Unbound periplasmic extracts were washed away (replenished with 0.05% Tween 20 PBS), and the binding ISVD was detected using mouse anti-FLAG-HRP (Sigma-Aldrich, catalog A8592) and subsequent enzymatic reactions in the presence of substrate esTMB (3,3',5,5'-tetramethylbenzidine; SDT).

[0271] DNA sequencing was performed on positive hits from ELISA, and non-redundant strains were further analyzed based on the dissociation rate with human and cynomolgus monkey GPC3 and the binding with cells expressing human and cynomolgus monkey GPC3.

[0272] The dissociation rate of ISVD was determined using a ProteOn XPR36 instrument (Bio-Rad Laboratories, Inc.). ProteOn GLC sensor chips were coated with cynomolgus monkey phosphatidylinositol proteoglycan-3 ΔGPI-HIS (in-house manufactured; accession number P51654; Q3-R358; S359-H559) and human GPC3 (R&D Systems, catalog number 2119-GP). Periplasmic extracts were diluted 1:10 in ProteOn PBS Tween buffer (PBS, pH 7.4, 0.005% Tween 20 (167-2720, BioRad)). Experiments were conducted at 25 ºC. The obtained data were double-referenced by subtraction of reference lanes and blank buffer injection. The processed curves were used for dissociation rate analysis based on a Langmuir dissociation (dissociation rate analysis) model.

[0273] In flow cytometry, CHO Flp-In cells (Invitrogen catalog number K6010) expressing human GPC3 (accession number P51654; Q25-R358, S359-H580) or cynomolgus monkey GPC3 (accession number P51654; Q3-R358; S359-H559) were used to screen for periplasmic extracts against human and cynomolgus monkey GPC3. Briefly, 1 × 10⁵ cells were incubated in a 1:5 dilution of the periplasmic extract at 4ºC for 30 min, followed by three washes. As a control, the parental CHO Flp-In cell line (Invitrogen catalog number K6010) was included. Next, cells were incubated with 1 µg / mL monoclonal anti-FLAG® M2 antibody (Sigma-Aldrich, catalog number F1804) at 4ºC for 30 min, washed again, and incubated with goat anti-mouse PE-labeled antibody (1:100; Jackson Immunoresearch, catalog number 115-115-164) at 4ºC for 30 min. The samples were washed and resuspended in FACS buffer (D-PBS (Gibco) with 10% FBS (Sigma) and 0.05% sodium azide (Merck)) supplemented with 5 nM TOPRO3 (Molecular Probes, catalog number T3605). The cell suspension was then analyzed on a FACS array. Gating was set for live, intact cells using forward / side scattering and TOPRO3 channel fluorescence parameters. Hit values ​​higher than those obtained for control conditions including those without ISVD binding were indicated.

[0274] Based on dissociation rate analysis and binding to CHO cells expressing human and cynomolgus monkey GPC3 (Table 2), two ISVDs were selected (Table 1).

[0275] [surface] [1] [:anti] [GPC3 ISVD] [Amino acid sequence] [ISVD ID] [sequence] [A0226015A08] [(SEQ ID NO: 47)] EVQLVESGGGLVQAGGSLRLSCVASGSIFRSVFSSSTMEWYRQPPGKKRELVARIAPGDGTNYGALYADSVKGRFTISRDDAKKTVDLQMNSLKPEDTGVYFCASGVAWGQGTLVTVSS [A0226018C08] [(SEQ ID NO: 48)] EVQLVESGGGLVQPGGSLRLSCVASGFTFSSFAMTWVRRPPGKGLEWVATITNGGVTSYRDSVKGRFTISRDNAKNTLYLEMTSLNPEDTAVYICANARRTGPRAPTDIGSYRGQGTLVTVSS

[0276] [surface] [2] [:anti] [GPC3 ISVD A0226015A08] [and] [A0226018C08] [Summary of the screening results] [ISVD ID] k offhGPC3(1 / s) k offcyGPC3(1 / s) RatioMFI hGPC3 CHO / CHO RatioMFI cyGPC3 CHO / CHO [A0226015A08] 3.3E-04 4.0E-04 58.2 13.7 [A0226018C08] 9.7E-04 1.1E-03 158.9 19.9 [6.2] [Example] [2] [Based on three specificities] [GPC3 ISVD] [of] [T] [Generation of cell conjugates]

[0277] The selected anti-GPC3 ISVD (sequences in Table 1) was formatted in a trispecific construct having a fixed T-cell conjugate ISVD (T0170056G05, anti-TCR) with a constant domain binding to the TCR at the N-terminus and a fixed albumin-binding ISVD (ALBX00001) at the C-terminus. The building blocks in the construct were genetically linked via a flexible 35GS (GlySer linker) to obtain the form anti-TCR-35GS-anti-GPC3-35GS-ALBX00001 (Table 3). The amino acid sequences are shown in Tables A-6.

[0278] Multivalent ISVDs were expressed in Pichia pastoris. Pichia pastoris NRRL Y-11430 cells containing the target ISVD construct were grown in BGCM medium. Subsequently, the medium was converted to BMCM, and the construct was further grown by stepwise addition of methanol for induction. Cells were rotated and settled, and the supernatant (containing the secreted ISVD) was collected.

[0279] The multivalent ISVDs were purified on protein A resin, followed by a desalting step and (if necessary) preparative SEC in D-PBS.

[0280] [surface] [3] [:Three Specificities] [ISVD] [Sample of the construct] [ID] [and description] [sample] [ID] [SEQ ID NO] [describe] [A022600027] 49 T0170056G05-35GS-A0226018C08-35GS-ALBX00001 [A022600031] 50 T0170056G05-35GS-A0226015A08-35GS-ALBX00001 [6.3] [Example] [3] [:Three Specificities] [GPC3 ISVD T] [Cell conjugate] [T] [Cell-dependent cytotoxicity]

[0281] Trispecific T-cell conjugates containing anti-GPC3 ISVD were characterized in a T-cell-dependent cytotoxicity assay (TDC) (Table 3). HepG2 (ATCC, colony HB8065), a liver cancer cell line with high GPC3 expression, was labeled with Nuclight Green (Essen Bioscience, catalog number 4624) and used as targets for T-cell killing in the presence of trispecific T-cell conjugate constructs containing A0226015A08 or A0226018C08 (i.e., constructs A022600031 or A022600027, respectively) or in the presence of a construct containing a reference GPC3-binding single-domain antibody (Ab1) presenting as anti-TCR-35GS-anti-GPC3-35GS-HLE (HLE = half-life extension factor). Therefore, the plate (96-well F-bottom, Greiner, catalog number 655180) was pre-blocked with 200 µL / well of assay medium (2 h, 37ºC). Each assay component was added simultaneously at a total volume of 200 µL / well: (1) 50 µL of diluted / titrated compound (Nbs; Brefidecin A (Sigma-Aldrich, catalog B7651); (2) 25 µL of diluted HSA (Sigma-Aldrich, catalog A8763-10G) (final concentration: 30 µM); (3) 25 µL of diluted Cytotox Red (Essen Bioscience, catalog 4632) (final concentration: 250 nM); (4) 50 µL of human T cells (T cells were isolated from the skin-colored red cross using RosetteSep T cell enrichment mixture (StemCell, catalog 15061)), and 50 µL of HepG2 Nuclight green (fresh, in DNEM (high glucose, GlutaMAX, pyruvate), Life Technologies-Gibco, catalog 31966), at 15: 1 ratio. Place the board in IncuCyte ZOOM for reading in all three channels (contrast, green, and red), at 4 or 6-hour intervals, for a total of 72 hours.

[0282] The tested trispecific GPC3 T cell binder induced human T cell-mediated killing of HepG2 Nuclight green in a dose-dependent manner, as shown in Figure 1. IC50 values ​​and maximum percentage kill are shown in Table 4.

[0283] [surface] [4] [Based on] [Incucyte] [People] [TDC HepG2-Nuclight green] [Used during measurement] [15:1] [The ratio of effectors to the target, after inoculation] [60h] [Analysis performed, three specificities] [GPC3 ISVD T] [Cell conjugate] [IC50] [(] [M] [) and maximum damage percentage () [%] [)] [sample] [ID] [IC50] [(] [M] [)] [Maximum Damage] [%] [A022600027] 8.6E-09 80 [A022600031] 1.5E-08 75 [Ab1] 1.3E-08 40 [6.4] [Example] [4] [:] [GPC3] [Epitope partitioning of the conjugate() [Epitope binning] [)]

[0284] Epitope partitioning of anti-GPC3 ISVD was performed by flow cytometry that allowed periplasmic extracts of monovalent ISVD A0226015A08 or A0226018C08 to compete with ISVD in its purified trispecific form. For this purpose, 4 × 10⁴ human GPC3 CHO Flp-In cells (reference, see Example 1) were incubated for 5 h at RT and 300 rpm with 1 / 15 and 1 / 150 dilutions of the periplasmic extract, 150 nM of the competitor (trispecific ISVD form; supersaturation concentration), and 0.2 µg / mL of monoclonal anti-FLAG® M2 antibody (Sigma-Aldrich, catalog number F1804) and rat anti-mouse APC (BD-Pharmingen, catalog number 550874). Samples were read in an iQue screener. No competition was observed between A0226015A08 and A0226018C08, indicating that they bind to different non-overlapping epitopes. [6.5] [Example] [5] [:] [GPC3 ISVD T] [Format optimization of cell conjugates]

[0285] In the T-cell-dependent cytotoxicity (TDC) HepG2 Nuclight green assay, the GPC3 ISVD T-cell conjugates in the form described in Table 3 did not achieve full efficacy, as indicated by the maximum kill percentage (Example 3). To increase efficacy and potency, a trispecific construct was generated in which the anti-GPC3 ISVD was combined with the following: a sequence-optimized variant of the anti-TCR ISVD T0170056G05 at the N-terminus, namely T017000624; an albumin-binding ISVD at the C-terminus, namely ALB23002-A (with a single alanine elongation at the C-terminus, SEQ ID NO: 5); and one or more GPC3-binding ISVDs at the central position of the construct (Tables 5 and 6). The optimization comprises two steps: Step 1 – optimization of the linker length between anti-TCR ISVD and anti-GPC3 ISVD (trispecific trivalent form); Step 2 – generation of a dual complementary GPC3 ISVD T-cell conjugate (trispecific tetravalent form) and optimization of the linker length between the two GPC3-binding ISVDs. Amino acid sequences are shown in Table A-6.

[0286] The generated form was tested in the TDC HepG2 Nuclight green assay, as described in Example 3. Analyses for the forms used in Step 1 and Step 2 were performed at 72 h and 60 h post-inoculation, respectively.

[0287] Tables 5 and 6 show the IC50 values ​​and maximum kill in different formats during the TDC HepG2 Nuclight green assay, in steps 1 and 2 of the format optimization.

[0288] In step 1, as the linker length between anti-TCR ISVD and anti-GPC3 ISVD decreased, the trispecific GPC3 ISVD T cell conjugate showed increased efficacy in tumor killing (increased maximum killing), from 72% (35GS linker) to 94% (AAA linker) (Figure 2). In step 2, the GPC3 dual complementary site T cell conjugate was tested for combining A0226018C08 and A0226015A08 in the same construct and placing them in different orientations with different linker lengths between the two constructs. Here, it was observed that the tested variables had no effect on efficacy (Figure 3).

[0289] [surface] [5] [Based on] [Incucyte] [People] [TDC HepG2-Nuclight green] [Used during measurement] [15 : 1] [The ratio of effectors to the target, after inoculation] [72h] [Analysis performed, three specificities] [GPC3 ISVD T] [Cell conjugate] [IC50] [(] [M] [) and maximum damage percentage () [%] [)] [step] [sample] [ID] [SEQ ID NO] [describe] [IC50] [(] [M] [)] [Maximum Damage] [%] [1] A022600096 51 T017000624-35GS-A0226018C08-35GS-ALB23002-A 1.4E-09 72 A022600105 55 T017000624-20GS-A0226018C08-35GS-ALB23002-A 2.14E-09 85 A022600104 54 T017000624-9GS-A0226018C08-35GS-ALB23002-A 2.13E-09 90 A022600103 53 T017000624-5GS-A0226018C08-35GS-ALB23002-A 2.04E-09 92 A022600102 52 T017000624-AAA-A0226018C08-35GS-ALB23002-A 1.72E-09 94

[0290] [surface] [6] [Based on] [Incucyte] [People] [TDC HepG2-Nuclight green] [Used during measurement] [15 : 1] [The ratio of effectors to the target, after inoculation] [72h] [Analysis performed, three specificities] [GPC3 ISVD T] [Cell conjugate] [IC50] [(] [M] [) and maximum damage percentage () [%] [)] [step] [sample] [ID] [SEQ ID NO] [describe] [IC50] [(] [M] [)] [Maximum Damage] [%] [2] A022600103 53 T017000624-5GS-A0226018C08-35GS-ALB23002-A 5.43E-10 90 A022600122 56 T017000624-5GS-A0226018C08-35GS-A0226015A08-35GS-ALB23002-A 4.45E-10 92 A022600131 57 T017000624-5GS-A0226018C08-20GS-A0226015A08-35GS-ALB23002-A 4.44E-10 94 A022600132 58 T017000624-5GS-A0226018C08-9GS-A0226015A08-35GS-ALB23002-A 7.02E-10 91 A022600133 59 T017000624-5GS-A0226015A08-35GS-A0226018C08-35GS-ALB23002-A 4.08E-10 92 A022600134 60 T017000624-5GS-A0226015A08-20GS-A0226018C08-35GS-ALB23002-A 5.28E-10 92 A022600135 61 T017000624-5GS-A0226015A08-9GS-A0226018C08-35GS-ALB23002-A 6.11E-10 91

[0291] To evaluate the ability to kill hepatocellular carcinoma cell lines exhibiting intermediate GPC3 levels compared to the high level of HepG2, a TDC Huh7 assay was performed. For this purpose, the xCELLigence® (Acea) system was used. First, a 96-well E-plate (Acea, catalog number 5232368001) containing 50 µL of assay medium with 4x concentration (120 µM) of Alburex 20 human serum albumin (CSL Behring, catalog number 2160-979) was placed inside the xCELLigence® for background measurement. (Final assay concentration 30 µM) Following background measurements, each assay component was added to a total volume of 200 µL / well simultaneously: (1) 50 µL of diluted / titrated compound; (2) 50 µL of single-cell suspension of Huh7 (HSRRB, strain JCRB0403); and (3) 50 µL of single suspension of effector cells (human T cells, obtained as described in Example 3) to match an effector:target ratio of 15:1. The plate was placed in xCELLigence® and 400 scans were performed at 15-minute intervals. At appropriate time points (approximately 60 h), the cell index (CI) was analyzed, where a CI of 0 indicated 100% killing.

[0292] In step 3 of the GPC3 T cell conjugate format optimization, the anti-TCR ISVD T017000624 was replaced by the sequenced optimized variant T017000680 (TCE01, SEQ ID NO: 2) in trivalent and tetravalent forms. Potency and efficacy were evaluated in HepG2 and Huh7 TDC assays, as described above. The forms, their descriptions, and functionalities are summarized in Table 7. Data are depicted in Figure 4. The third step of format optimization resulted in a smaller increase in efficacy between constructs. Based on this practice, GPC3 T cell conjugate forms A022600167 and A022600168 were used.

[0293] Step 4 in optimizing the GPC3 T-cell binder format involves altering the orientation of anti-TCR ISVD relative to anti-GPC3 ISVD, but this affects potency and efficacy (Table 8, Figure 5). For the trivalent form, cytotoxic efficacy is lost. For the tetravalent form, functionality is still observed, but potency and efficacy are reduced; cytotoxic efficacy against HepG2 Nuclight green is reduced by 20%, and against Huh7 by 40%. Furthermore, altering the orientation of anti-GPC3 ISVD relative to anti-albumin ISVD also affects cytotoxic efficacy. Based on this practice, no changes are made to GPC3 T-cell binder formats A022600167 and A022600168.

[0294] Step 5 in optimizing the GPC3 T-cell conjugate format involves altering the conjugate length (Table 9, Figure 6). Reducing the conjugate length before the trivalent form of anti-albumin ISVD results in decreased efficacy. This was not observed in the tetravalent form, therefore, in this case, a choice can be made between a 9GS and a 35GS conjugate length.

[0295] The groups A022600167 and A022600168, representing the trispecific trivalent and tetravalent forms with the highest potency and efficacy in TDC assays, represent the highest-potency and most potent forms of the GPC3 T cell conjugate. Although the efficacy is comparable, the trivalent form A022600167 differs in potency from the tetravalent form A022600168. In the HepG2 TDC assay, A022600167 was 5-fold less potent than A022600168, while in the Huh7 TDC assay, this difference increased to 50-fold.

[0296] [surface] [7] [:] [GPC3 ISVD T] [Steps for optimizing cell conjugate format] [3] [Based on] [Incucyte] [People] [TDC HepG2-Nuclight green] [During and based on] [xCELLigence] [People] [TDC Huh7] [Used during measurement] [15 : 1] [The ratio of effectors to the target, after inoculation] [60h] [Analysis performed, three specificities] [GPC3 ISVD T] [Cell conjugate] [IC50] [(] [M] [) and maximum damage percentage () [%] [)] [sample] [ID] [SEQ ID NO] [describe] [Incucyte] [Above] [HepG2 IC50] [(] [M] [)] [Incucyte] [Above] [HepG2] [Maximum Damage] [%] [xCELLigence] [Above] [Huh7 IC50] [(] [M] [)] [xCELLigence] [Above] [Huh7] [Maximum Damage] [%] [A022600103] 53 T017000624-5GS-A0226018C08-35GS-ALB23002-A 1.23E-09 96 7.11E-09 71 [A022600167] 62 TCE01-5GS-A0226018C08-35GS-ALB23002-A 1.92E-09 98 8.61E-09 91 [A022600122] 56 T017000624-5GS-A0226018C08-35GS-A0226015A08-35GS-ALB23002-A 5.3E-10 93 2.61E-10 101 [A022600168] 63 TCE01-5GS-A0226018C08-35GS-A0226015A08-35GS-ALB23002-A 3.67E-10 103 1.98E-10 107 [A022600132] 58 T017000624-5GS-A0226018C08-9GS-A0226015A08-35GS-ALB23002-A 6.83E-10 83 2.9E-10 95 [A022六百零万零一百七十五] 68 TCE01-5GS-A0226018C08-9GS-A0226015A08-35GS-ALB23002-A 5.27E-10 96 5.86E-11 99

[0297] [Table] [8] [:] [GPC3 ISVD T] It should be noted that the content in the original text seems to be a combination of some technical or specific data records, and the translation attempts to maintain the integrity and accuracy of the original information as much as possible. If there are specific requirements or corrections regarding the translation of certain terms, it may need to be adjusted according to the actual context.[Steps for optimizing cell conjugate format] [4] [Based on] [Incucyte] [People] [TDC HepG2-Nuclight green] [During and based on] [xCELLigence] [People] [TDC Huh7] [Used during measurement] [15 : 1] [The ratio of effectors to the target, after inoculation] [60h] [Analysis performed, three specificities] [GPC3 ISVD T] [Cell conjugate] [IC50] [(] [M] [) and maximum damage percentage () [%] [)] [sample] [ID] [SEQ ID NO] [describe] [Incucyte] [Above] [HepG2 IC50] [(] [M] [)] [Incucyte] [Above] [HepG2] [Maximum Damage] [%] [xCELLigence] [Above] [Huh7 IC50] [(] [M] [)] [xCELLigence] [Above] [Huh7] [Maximum Damage] [%] [A022600167] 62 TCE01-5GS-A0226018C08-35GS-ALB23002-A 1.92E-09 98 8.61E-09 91 [A022600178] 69 A0226018C08-5GS-TCE01-35GS-ALB23002-A No effect No effect [A022600168] 63 TCE01-5GS-A0226018C08-35GS-A0226015A08-35GS-ALB23002-A 3.67E-10 103 1.98E-10 107 [A022600169] 64 A0226018C08- 5GS-TCE01-5GS- A0226015A08-35GS-ALB23002-A 6.41E-10 81 1.02E-09 61 [A022600174] 67 TCE01-5GS-A0226018C08-9GS-A0226015A08-9GS-ALB23002-A 6.73E-10 96 6.26E-11 99 [A022600172] 66 TCE01-5GS-A0226018C08-9GS-ALB23002-9GS-A0226015A08-A 6.01E-10 81 6.70E-11 90

[0298] [surface] [9] [:] [GPC3 T] [Steps for optimizing cell conjugate format] [5] [Based on] [Incucyte] [People] [TDC HepG2-Nuclight green] [During and based on] [xCELLigence] [People] [TDC Huh7] [Used during measurement] [15 : 1] [The ratio of effectors to the target, after inoculation] [60h] [Analysis performed, three specificities] [GPC3 ISVD T] [Cell conjugate] [IC50] [(] [M] [) and maximum damage percentage () [%] [)] [sample] [ID] [SEQ ID NO] [describe] [Incucyte] [Above] [HepG2 IC50] [(] [M] [)] [Incucyte] [Above] [HepG2] [Maximum Damage] [%] [xCELLigence] [Above] [Huh7 IC50] [(] [M] [)] [xCELLigence] [Above] [Huh7] [Maximum Damage] [%] [A022600167] 62 TCE01-5GS-A0226018C08-35GS-ALB23002-A 1.92E-09 98 8.61E-09 91 [A022600179] 70 TCE01-5GS-A0226018C08-9GS-ALB23002-A 2.92E-09 76 6.13E-09 66 [A022600168] 63 TCE01-5GS-A0226018C08-35GS-A0226015A08-35GS-ALB23002-A 3.67E-10 103 1.98E-10 107 [A022600170] 65 TCE01-5GS-A0226018C08-35GS-A0226015A08-9GS-ALB23002-A 3.19E-10 97 1.86E-10 103 [A022600175] 68 TCE01-5GS-A0226018C08-9GS-A0226015A08-35GS-ALB23002-A 5.27E-10 96 5.86E-11 99 [A022600174] 67 TCE01-5GS-A0226018C08-9GS-A0226015A08-9GS-ALB23002-A 6.73E-10 96 6.26E-11 99 [6.6] [Example] [6] [In soluble] [GPC3] [Existence under] [T] [Evaluation of Cell Activation Induction]

[0299] GPC3 can be released into circulation in a soluble form, and its levels are increased in up to 50% of HCC patients. Circulating soluble GPC3 antibody aggregates can lead to immune complex deposition and related toxicity. Therefore, it is important to assess the effect of soluble GPC3 on T cell activation in the presence of GPC3 T cell binders.

[0300] The reported serum GPC3 levels varied from 10 to 300 ng / mL, corresponding to 0.1 to 10 nM. The assessment was performed in three ways using 1, 10, and 100 nM soluble GPC3 in the presence of the trispecific GPC3 ISVD T cell conjugates A022600167 and A022600168: (1) cytotoxicity in the presence of target cells (Huh-7) and soluble GPC3 (Fig. 7A): no additional killing was observed compared to the absence of soluble GPC3; (2) CD69 upregulation in the presence of target cells and soluble GPC3 (Fig. 7B): no additional T cell activation was observed compared to the absence of soluble GPC3; (3) CD69 upregulation in the absence of target cells and the presence of soluble GPC3 (Fig. 7C): no T cell activation was observed. Therefore, the risk of toxic effects due to increased serum levels of soluble GPC3 was considered low.

[0301] Cytotoxicity assays against Huh-7 were performed as described in Example 5. CD69 expression on T cells was determined by flow cytometry using anti-CD69 antibody (BD Pharmigen, catalog number 557050) and anti-mouse IgG1 antibody (BD Pharmigen, catalog number 556650). [6.7] [Example] [7] [:] [GPC3] [Mediated] [T] [Internalization of cell conjugates]

[0302] GPC3 is known to be internalized, and its internalization rate may affect the efficacy of compounds. In Huh-7 cells, the internalization rate and GPC3 receptor density were assessed over a 48-hour timeframe in the presence of a trispecific GPC3 ISVD T-cell conjugate equivalent to A022600167 and A022600168, as well as a reference CD3-GPC3 bispecific T-cell conjugate antibody (Ab2) (Table 10).

[0303] Internalization was determined by labeling A022600167 and A022600168, as well as Ab2, with pHAb (Promega, catalog number G9841), a pH-sensitive dye exhibiting low fluorescence at pH > 7 and a significant increase in fluorescence as the solution pH becomes acidic. For this labeling, forms with an additional -GGC at the C-terminus were generated to achieve single-site incorporation of the label (Table 10): A022600167-GGC corresponds to A022600370, A022600168-GGC corresponds to A022600372, and as a control, a GPC3 ISVD-free form, A022600373, was generated. Measurements were performed using a yellow laser on a BD FACSArray (pHAb: excitation maximum at 532 nm, emission maximum at 560 nm). The internalization rate was determined by quantifying internalization at 37°C at different time points (0.5 h, 3 h, 24 h and 48 h) compared to 0.5 h at 4°C (Table 10, Figure 8A).

[0304] Receptor expression was determined by red laser on a BD FACSArray using a fixed concentration of 3xFLAG-His6-labeled ISVD (20 nM) combined with APC-labeled anti-FLAG for detection. The ISVD binds to GPC3 epitopes different from A02260018C08 and A02260015A08 (Table 10, Figure 8B).

[0305] Internalization rate was calculated as the slope of a kinetic curve with arbitrary units. The trispecific GPC3 ISVD T cell conjugate showed a slower internalization rate than the reference bispecific T cell conjugate Ab2. Internalization of the GPC3 ISVD T cell conjugate was GPC3-mediated, as the control form (A022600373) without GPC3 binding to ISVD did not show internalization. No decrease in GPC3 cell surface expression was observed within 48 h (Table 10, Figure 8).

[0306] [surface]

[10] [:] [pHAb] [Trinity of the label] [GPC3 T] [Internalization rate of cell conjugates] [sample] [ID] [SEQ ID NO] [describe] [Tagged] [pHAb] [degree] [Internalization rate (normalized slope)] [n=2] [)] [A022600370] 71 TCE01-5GS-A0226018C08-35GS-ALB23002-GGC 1 194 [A022600372] 72 TCE01-5GS-A0226018C08-35GS-A0226015A08-35GS-ALB23002-GGC 1 297 [A022600373] 73 TCE01-20GS-ALB23002-GGC 1 2 [Ab2] - - 1.4 597 [6.8] [Example] [8] [Cancer cell lines] [GPC3] [Performance Analysis and] [GPC3 T] [Functional associations of cell conjugates]

[0307] The expression levels of GPC3 protein in a group of cancer cell lines were determined by immunocytochemistry (ICC) using QIFIKIT® (Dako, catalog number K0078) according to the manufacturer's instructions (Table 11). Additionally, immunohistochemistry (IHC) was performed on hepatocellular carcinoma and normal kidney samples.

[0308] ICC and IHC were performed using the Ventana discovery XT robot (Ventana medical system, Roche). Cell lines and tissue samples were first fixed in 4% formalin and then embedded in paraffin. After dewaxing, cells were incubated with buffer CC1 standard (Ventana, catalog number 950-124) at 95ºC for 48 minutes, followed by a 4-minute blocking step with each of blocking agents A and B (Ventana, catalog number 760-104). Mouse monoclonal IgG2a anti-GPC3 antibody (Ventana, catalog number 790-4564) was applied at room temperature for 60 minutes, followed by 4 minutes of fixation with 0.05% glutaraldehyde in 5M NaCl (Prolabo, catalog number 20879-238). Biotinylated goat anti-mouse IgG2a antibody (Southern Biotech, catalog 1080-080) diluted 1 / 200 in antibody diluent (Ventana; catalog 760-108) was applied at room temperature for 32 minutes. Detection was performed using the DABMap kit (Ventana; catalog 760-124). Sections were counterstained with hematoxylin II (Ventana, catalog 790-2208) for 4 minutes and then counterstained with blue dye (Ventana, catalog 760-2037) for another 4 minutes, followed by dehydration and mounting with Cytoseal XYL (Richard-Allan Scientific, catalog 8312-4). Immunohistochemical staining was evaluated semi-quantitatively by assessing both cell staining intensity (graded as 0: no staining, 1 (or +): weak, 2 (or ++): moderate; 3 (or +++): strong) and the percentage of positive cells in each intensity category. The tissue score (H score) was calculated according to the following formula:

[0309] H score = 3 x (grade 3 cells%) + 2 x (grade 2 cells%) + 1 x (grade 1 cells%).

[0310] Possible score ranges are 0 to 300, as described in the literature (Detre et al., J Clin Pathol 1995; 48:876-878 and Lui et al., Journal of Latex Class filed, August 2015, Vol. 14, No. 8). The determination of the H score in HCC is based on the evaluation of the membrane performance of GPC3.

[0311] Within the tested cell lines, as determined by QIFIKIT®, Hep-G2 (ATCC, colony HB-8065; 5.2E5 receptors / cell) showed the highest level of GPC3 expression, followed by NCI-H661 (ATCC, colony HTB-183; 3.4E5 receptors / cell) and Huh-7 (HSRRB, colony JCRB0403; 6.8E4 receptors / cell), the latter being considered an intermediate-expression cell line. These cell lines were derived from hepatocellular carcinoma and lung cancer, which are solid tumors with relevant GPC3 expression. Cell lines showing low or very low GPC3 expression that did not show any staining in ICC were MKN-45 (DSMZ, colony ACC409; 1.5E 4 receptors / cell), NCI-H23 (ATCC, colony CRL-5800; 2.6E 3 receptors / cell), BxPC-3 (ATCC, colony CRL-1687; 1.5E 3 receptors / cell), and NCI-H292 (ATCC, colony CRL-1848; 6E 2 receptors / cell).

[0312] For comparison between cancer cell lines and patient tumor samples, H scores were determined. GPC3-positive cancer cell lines in ICC, namely Huh-7, NCI-H661, and HepG2, showed H scores better than 80 (Table 11), corresponding to a mean H score of 80-75 determined for GPC3-positive hepatocellular carcinoma (HCC) samples in IHC (Table 12). Normal kidney GPC3-positive samples showed a mean H score of 0-75 (Table 12), while cancer cell lines MKN-45, NCI-H23, BxPC-3, and NCI-H292 were negative for GPC3 staining (Table 11). These cell lines were used as representatives of cells exhibiting normal GPC3 expression levels.

[0313] To assess the functionality of the trispecific GPC3 T cell conjugates using concordant cancer cell lines, TDC assays were performed using the xCELLigence system, as described in Example 5; the results are shown in Table 13 and Figure 9. For the high-performance GPC3 cell lines Hep-G2 and NCI-H661, the trispecific GPC3 ISVD T cell conjugates A022600167 and A022600168 showed similar potency (NCI-H661) and 10-fold lower potency (Hep-G2) compared to the bispecific T cell conjugate Ab2. For the intermediate-performance cell line Huh-7, the tetravalent form A022600168, which binds to two complementary sites of GPC3, showed the same potency as Ab2, while the trivalent form A022600167 was 10-fold less potent. For GPC3-low expression cell lines MKN-45 and BxPC-3, Ab2 showed efficacy in the nM range, while the trispecific GPC3 ISVD T cell conjugate showed no cytotoxic effect. For the very low GPC3 expression cell line NCI-H292, no compound showed any effect. The T cell conjugate T017000698, lacking GPC3 binding to ISVD, showed no cytotoxic effect on any cell line, thus confirming the GPC3-specific effect of the trispecific GPC3 T cell conjugate.

[0314] In summary, Ab2 is an effective T-cell conjugate capable of killing cancer cell lines with GPC3 expression levels as low as 1,000 receptors / cells and an H score of 0. In contrast, the trispecific T-cell conjugate form effectively kills high- and intermediate-GPC3-expressing cancer cell lines with H scores similar to those in HCC and large cell lung cancer samples, without killing cell lines with GPC3 expression levels below 10,000 receptors / cells and H scores below the average of normal kidney samples.

[0315] [surface]

[11] [Different cancer cell lines] [GPC3] [Performance level.] [Cell line] [Cancer Organization] [Performance] [RNA] [FPKM] [Expression protein] [#GPC3 / ] [cell] [Expression protein] [ICC] [(] [H] [Score)] [Hep-G2] liver 2253 619006 70%+++, 25%++ (260) [NCI-H661] lung 237 346756 40%++ (80) [Huh-7] liver 549 78027 20%++, 40%+ (80) [MKN-45] Stomach 20.9 7453 0 [NCI-H23] lung 3.17 2255 0 [BxPC3] pancreas 5.3 1332 0 [NCI-H292] lung 0.04 452 0

[0316] [surface]

[12] [Identified by immunohistochemistry of hepatocellular carcinoma and normal kidney samples] [GPC3 H] [Score (based on...)] [GPC3] [Evaluation of membrane performance]. [Total Cases] [H] [Score] [total] [GPC3+] [Case Study] [H] [Score] [HCC] 288 52,22 187 80,75 [Normal Kidney] 35 0,17 8 0.75

[0317] [surface]

[13] [Based on] [xCELLigence] [People] [TDC] [During the measurement, those with decreased performance] [GPC3] [Different tumor cell lines with different performance levels, used] [15:1] [The ratio of effectors to the target,] [GPC3 T] [Cell conjugate] [IC50] [(] [M] [):exist] [60h] [Analysis] [HepG2] [,exist] [75h] [Analysis] [NCI-H661] [,exist] [60h] [Analysis] [Huh-7] [,exist] [65h] [Analysis] [MKN-45] [,exist] [65h] [Analysis] [BxPC-3] [,exist] [60h] [Analysis] [NCI-H292] [。]na = Not available [Sample] [ID] [SEQ ID NO] [describe] [Hep-G2] [NCI-H661] [Huh-7] [MKN-45] [BxPC-3] [NCI-H292] [A022600167] 62 TCE01-5GS-A0226018C08-35GS-ALB23002-A 9.2E-11 1.8E-10 4.7E-10 na na na [A022600168] 63 TCE01-5GS-A0226018C08-35GS-A0226015A08-35GS-ALB23002-A 7.3E-11 1.4E-10 2.4E-11 na na na [T017000698] 74 TCE01-9GS-ALB23002-A na na na na na na [Ab2] - Bispecific CD3-GPC3 1.1E-11 2.8E-11 3.0E-11 2.0E-09 3.6E-09 na [6.9] [Example] [9] [:] [A0226015A08] [and] [A0226018C08] [Sequence optimization]

[0318] Further sequence optimization was performed on ISVD A0226015A08 and A0226018C08.

[0319] Sequence optimization involves replacing one or more specific amino acid residues in the sequence to improve one or more (desired) properties of the ISVD.

[0320] Examples of such sequence optimizations are mentioned in other descriptions in this paper, and include, for example, but are not limited to:

[0321] 1) Substitution in the parental wild-type ISVD sequence to obtain an ISVD sequence more identical to the human VH3-JH germline common sequence is a process known as humanization. To achieve this, specific amino acids in the FR that differ between the ISVD and the human VH3-JH germline common sequence are changed in a manner that preserves the integrity of the protein's structure, activity, and stability, except for so-called marker residues.

[0322] 2) Substitution towards llamas to increase the stability of ISVD, which is defined as llamaization. To this end, the amino acid sequences of the parent wild-type ISVD were compared with the amino acid sequences of the llama IGHV lineage of ISVD (highest hits were identified based on BlastP analysis of ISVD and llama IGHV lineages).

[0323] 3) Substitutions that improve long-term stability or properties under storage, substitutions that increase performance levels in desired host cells or host organisms, and / or substitutions that remove or reduce (unwanted) one or more post-translational modifications (such as glycosylation or phosphorylation), depending on the desired host cell or host organism.

[0324] 4) Mutations at position 11 toward Val and at position 89 toward Leu (according to Kabat) to minimize the binding of any naturally occurring, pre-existing antibodies.

[0325] The sequence optimization of A0226015A08 yielded the final sequence-optimized variant A022600314, which contains 13 amino acid substitutions (i.e., L11V, A14P, V23A, P40A, D52cE, N54Y, D73N, K76N, D79Y, K83R, G88A, V89L, F91Y) compared to the parent ISVD strain A0226015A08 (Table 14).

[0326] Sequence optimization of A0226018C08 yielded two sequence-optimized variants: A022600345, which contains eight amino acid substitutions (L11V, V23A, G54A, R60A, E81Q, T82aN, N83R, V89L) compared to the parental ISVD strain A0226018C08, and A022600351, which contains eight amino acid substitutions (L11V, V23A, G54K, R60A, E81Q, T82aN, N83R, V89L) (Table 14).

[0327] [surface]

[14] [:] [A0226015A08] [and] [A0226018C08] [The optimized form of the amino acid sequence.] [sample] [ID] [SEQ ID NO] [describe] [sequence] [A022600314] 75 (same as 4) A0226015A08(L11V, A14P, V23A, P40A, D52cE, N54Y, D73N, K76N, D79Y, K83R, G88A, V89L, F91Y) EVQLVESGGGVVQPGGSLRLSCAASGSIFRSVFSSSTMEWYRQAPGKKRELVARIAPGEGTYYGALYADSVKGRFTISSRDNAKNTVYLQMNSLRPEDTALYYCASGVAWGQGTLVTVSS [A022600345] 76 A0226018C08(L11V, V23A, G54A, R60A, E81Q, T82aN, N83R, V89L) EVQLVESGGGVVQPGGSLRLSCAASGFTFSSFAMTWVRRPPGKGLEWVATITNAGVTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYICANARRTGPRAPTDIGSYRGQGTLVTVSS [A022600351] 77 (same as 3) A0226018C08(L11V, V23A, G54K, R60A, E81Q, T82aN, N83R, V89L) EVQLVESGGGVVQPGGSLRLSCAASGFTFSSFAMTWVRRPPGKGLEWVATITNKGVTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTALYICANARRTGPRAPTDIGSYRGQGTLVTVSS

[0328] The characteristics of the sequence-optimized variant compared to the parental ISVD are evaluated as follows:

[0329] Variants were evaluated by flow cytometry targeting their binding to HepG2, Huh-7, and CHO Flip-In cynomolgus monkey GPC3 cells, as described in Example 1.

[0330] The thermal stability of variants was tested using a Lightcycler (Roche) in a thermal displacement assay (TSA). In this assay, parental ISVD and its variants were cultured at different pH levels in the presence of SYPRO™ Orange, and temperature gradients were applied. When ISVD began to denature, SYPRO™ Orange binding caused an increase in fluorescence, allowing determination of the melting temperature (Tm) for a given pH.

[0331] The results are summarized in Tables 15 and 16.

[0332] [surface]

[15] [Parental] [ISVD A0226015A08] [Sequence optimization variants] [A0226000314] [The analysis results.] [sample] [ID] [mutation] [EC50] [(] [M] [)] [HepG2] [EC50] [(] [M] [)] [Huh-7] [EC50] [(] [M] [)] [CHO-cGPC3] [Tm] [(] [ºC] [)exist] [pH 7] [Down] [TSA] [A0226015A08] WT 7.5E-09 6.6E-09 7.8E-09 70 [A022600314] L11V, A14P, V23A, P40A, D52cE, N54Y, D73N, K76N, D79Y, K83R, G88A, V89L, F91Y 1.6E-08 9.3E-09 1.2E-08 65

[0333] [surface]

[16] [Parental] [ISVD A0226018C08] [Sequence optimization variants] [A022600345] [and] [A0226000351] [The analysis results.] [sample] [ID] [mutation] [EC50] [(] [M] [)] [HepG2] [EC50] [(] [M] [)] [Huh-7] [EC50] [(] [M] [)] [CHO-cGPC3] [Tm] [(] [ºC] [)exist] [pH 7] [Down] [TSA] [A0226018C08] WT 9.15E-10 6.96E-10 4.9E-10 67 [A022600345] L11V, V23A, G54A, R60A, E81Q, T82aN, N83R, V89L 3.91E-10 2.76E-10 1.5E-10 75 [A022600351] L11V, V23A, G54K, R60A, E81Q, T82aN, N83R, V89L 4.31E-10 4.77E-10 2.1E-10 74

[0334] Compared to the parental ISVD A0226015A08, A022600314 showed a less than 2-fold decrease in binding potency for different cell lines expressing human GPC3 or cynomolgus monkey GPC3. Tm decreased slightly by 5ºC and was within acceptable limits. For A022600314, the architectural identity % in the architectural region was 88.8% based on the AbM definition (see Antibody Engineering, Vol. 2, edited by Kontermann and Dübel, Springer Verlag Heidelberg Berlin, 2010) and 85.1% based on the Kabat definition, compared to the reference hIGHV3-23SO / IGHJ4.

[0335] Compared to the parent ISVD A0226018C08, variants A022600345 and A022600351 showed a 2-fold increase in binding potency to different cell lines expressing human GPC3 or cynomolgus monkey GPC3. The Tm values ​​of variants A022600345 and A022600351 increased to 75ºC and 74ºC, respectively. For both A022600345 and A022600351, the architectural identity percentage in the architectural region was 89.9% based on the AbM definition and 89.7% based on the Kabat definition, compared to the reference hIGHV3-23SO / IGHJ4. [6.10] [Example]

[10] [Three-specificity sequence optimization] [GPC3 ISVD T] [Generation of cell conjugates]

[0336] Sequence optimizations of GPC3 ISVDs A022600314 (an optimized variant of A0226015A08), A022600345, and A022600351 (an optimized variant of A0226018C08) were used to generate five trispecific GPC3 T cell conjugate forms to evaluate the optimal combination of building block and connector lengths, as described in Table 17.

[0337] [surface]

[17] [The different three specificities evaluated] [GPC3 ISVD T] [Choice of Cell Connector Form] [sample] [ID] [SEQ ID NO] [describe] [A022600427] 81 TCE01-5GS-A022600345-9GS-A022600314-9GS-ALB23002-A [A022600424] 1 TCE01-5GS-A022600351-9GS-A022600314-9GS-ALB23002-A [A022600425] 79 TCE01-5GS-A022600314-20GS-A022600345-9GS-ALB23002-A [A022600426] 80 TCE01-5GS-A022600314-20GS-A022600351-9GS-ALB23002-A [A022600412] 78 TCE01-5GS-A022600345-20GS-ALB23002-A

[0338] The functionality of five selected forms was tested using different cancer cell lines in an xCELLingence-based TDC assay, as described in Example 8. The results are depicted in Table 18. For high and intermediate GPC3-expressing cell lines HepG2, NCI-H661, and Huh-7, the tetravalent form was more potent than the trivalent form. For the high GPC3-expressing cell line NCI-H661, the tetravalent forms A022600424 and A022600427 were more effective than A022600425 and A022600426. For the low GPC3-expressing cell lines NCI-H23 and BxPC-3, a lack of cytotoxicity was confirmed for all trispecific GPC3 ISVD T cell conjugate forms.

[0339] Figure 10 shows dose-dependent killing curves based on xCELLingence TDC assays for five selected ISVD forms using three different T cell donors, illustrated by cell lines NCI-H661 and BxPC-3.

[0340] [surface]

[18] [Based on] [xCELLigence] [People] [TDC] [During the assay, different tumor cell lines were used...] [15:1] [The ratio of the effector to the target and in] [60h] [analyze,] [GPC3 T] [Cell conjugate] [IC50] [(] [M] []. na = Unavailable; No fit = No curve fit obtained, IC50 estimated as >1E-7M [sample] [ID] [IC50] [(] [M] [)] [HepG2] [IC50] [(] [M] [)] [NCI-H661] [IC50] [(] [M] [)] [Huh-7] [IC50] [(] [M] [)] [NCI-H23] [IC50] [(] [M] [)] [BxPC-3] [A022600427] 1.03E-10 1.00E-10 1.37E-10 No fitting No fitting [A022600424] 1.15E-10 1.14E-10 1.16E-10 No fitting No fitting [A022600425] 1.11E-10 2.59E-10 1.58E-10 No fitting No fitting [A022600426] 1.25E-10 2.64E-10 1.09E-10 No fitting No fitting [A022600412] 3.82E-10 6.18E-10 1.53E-09 na No fitting [Ab2] 1.49E-11 4.60E-11 8.41E-11 1.28E-09 1.09E-09

[0341] The binding of pre-existing antibodies to five selected forms was evaluated using an SPR-based setup (Example 14). Figure 11 shows that only low levels of binding to the pre-existing antibodies were observed for all forms, consistent with the developmentability requirements.

[0342] The performance of the five forms in *Pichia pastoris* was evaluated, focusing on product potency and purity during upstream processing (USP) at a 5 L fermenter scale, and yield after downstream processing (DSP) (Table 19). A022600424 and A022600426 were identified as superior ISVD development candidates, combining a low percentage of high molecular weight (HMW) species, a low percentage of variants on RPC, with high potency and optimal overall DSP yield.

[0343] [surface]

[19] [:] [5] [Based on the choice] [ISVD] [of] [GPC3 T] [The manifestation of cell conjugate forms in Pichia pastoris.] [sample] [ID] [Valence()] [g / L] [)] [Analytical] [SEC] [(] [HMW%] [)] [RPC] (After the peak) [%] [)] [DSP] [Yield] [(total] [%] [)] [A022600412] 3.9 5.4 3.3 26 [A022600424] 4.9 1.8 1.7 45 [A022600425] 5.6 6.3 2.7 twenty four [A022600426] 5.4 2.4 3.3 32 [A022600427] 4.7 5.1 1.4 32

[0344] Based on the optimal combination of GPC3-driven killing efficacy, reduction of pre-existing antibodies, and performance in Pichia pastoris, A022600424 was selected as a development candidate. [6.11] [Example]

[11] [:] [A022600424] [right] [GPC3] [、] [TCRab] [Binding and affinity with serum albumin]

[0345] Quantification of the affinity (expressed as equilibrium dissociation constant (KD)) of A022600424 for the following parameters using ProteOn XPR36 via surface plasma resonance (SPR): human and cynomolgus monkey GPC3 (R&D... Systems, catalog number 2119-GP and internally produced (accession numbers P51654, Q3-R358, S359-H559)); human and cynomolgus monkey TCRab (both internally produced, with the extracellular domains of the α and β chains fused with zipper peptides for dimerization; accession numbers: human α chain P01848, human β chain P01850; the predicted sequence of the cynomolgus monkey α chain is the same as that of the human, while the predicted sequence of the cynomolgus monkey β chain differs by 4 aa: A125V, E136V, V167M, S177F); and human, cynomolgus monkey and mouse serum albumin (Sigma catalog number A8763, internally produced from animal tissue, DivBioScience catalog number IMSA, respectively).

[0346] Recombinant GPC3 protein was captured on a GLC sensor chip (Biorad) immobilized with THE anti-His antibody (Genscript, catalog number ABIN387699) via amine coupling using EDC and NHS chemicals (run buffer: HBS-EP+, pH 7.4). Purified ISVD was injected at different concentrations (between 0.3 nM and 1000 nM) for 2 minutes (flow rate 45 µL / min), and dissociation was tracked for 900 s. Regeneration was performed by injecting 10 mM glycine-HCl (pH 1.5) for 1 minute (flow rate 45 µL / min). Data were double-referenced by subtracting a reference ligand lane and injecting blank buffer. Processed sensor maps were analyzed using ProteOn Manager 3.1.0 (version 3.1.0.6) software based on a 1:1 interaction model (Langmuir binding model).

[0347] Recombinant TCR protein was immobilized on a GLC sensor chip (Biorad) via amine coupling using EDC and NHS chemicals (run buffer: HBS-EP+, pH 7.4). Purified ISVD was injected at different concentrations (between 0.2 nM and 200 nM) for 2 minutes (flow rate 45 µL / min) and dissociation was tracked for 900 s. Regeneration was performed by injecting 3 M MgCl2 for 3 minutes (flow rate 90 µL / min). Data were double-referenced by subtracting a reference ligand lane and injecting blank buffer. Processed sensor maps were analyzed using ProteOn Manager 3.1.0 (version 3.1.0.6) software based on a 1:1 interaction model (Langmuir binding model).

[0348] Serum albumin was immobilized on a GLC sensor chip (Biorad) via amine coupling using EDC and NHS chemicals (run buffer: HBS-EP+, pH 7.4). Purified ISVD was injected at different concentrations (between 0.24 nM and 500 nM) for 2 minutes (flow rate 45 µL / min), with dissociation tracked for 900 s. Regeneration was performed by injecting 10 mM glycine-HCl (pH 1.5) for 47 seconds (flow rate 100 µL / min). Data were double-referenced by subtracting a reference ligand lane and injecting blank buffer. Processed sensor maps were analyzed using ProteOn Manager 3.1.0 (version 3.1.0.6) software based on a 1:1 interaction model (Langmuir binding model).

[0349] The results (Table 20) show that A022600424 binds to human and cynomolgus monkey GPC3 with high affinity.

[0350] [surface]

[20] [With humans and crab-eating macaques] [GPC3] [Humans and crab-eating macaques] [TCRab] [And the binding affinity of serum albumin from humans, cynomolgus monkeys, and mice]. na = unavailable [antigen] [sample] [ID] [KD] [(] [M] [)] [people] [KD] [(] [M] [)] Crab-eating macaques [KD] [Mouse] [GPC3] [A022600424] < 5.6E-12 < 5.9E-12 na [Ab2] 1.8E-09 1.7E-09 na [TCRab] [A022600424] 6.3E-09 5.4E-09 na [T017000698] 8.5E-09 1.1E-08 na [Serum albumin] [A022600424] 8.3E-10 3.3E-10 6.9E-09 [ALB223] 8.8E-10 5.7E-10 5.3E-09

[0351] For CHO-Flp-In cells and Huh-7 cells that overexpressed human and cynomolgus monkey GPC3, the binding of A022600424 to cell-expressed human and cynomolgus monkey GPC3 was evaluated by flow cytometry, and EC50 values ​​between 1 nM and 2 nM were obtained (Table 21).

[0352] At EC30 concentrations, A022600424 was evaluated for competitive binding to human and cynomolgus T cells against TCRab-binding monovalent ISVDs T017000624 and T017000623 (T0170056G05 variant), respectively. On the day of assay, T cells (human T cells obtained as described in Example 3 and cynomolgus T cells purchased from LPT Laboratories, Germany) were thawed, counted, and diluted to a concentration of 1E+06 cells / mL. 75 µL was then added to the wells of a V-bottom 96-well plate (Greiner, catalog number 651180). Cells were washed once with cold FACS buffer, followed by the addition of 25 µL of Nb and 25 µL of the competing agent to the wells. Dilute T017000624 to a 2x concentration of 4E-08 M (2E-08 M in wells), dilute T017000623 to a 2x concentration of 1E-07 M (5E-08 M in wells), and dilute A022600424 in the air to a final concentration between 8 µM and 7.8 nM. Resuspend the cells and incubate the plate at 4ºC for 90 min, then wash the plate twice with cold FACS buffer. Resuspend the cells in 50 µL of 1 / 1000 dilution of single-line anti-FLAG® M2 (Sigma Aldrich, catalog number F1804) in FACS buffer and incubate at 4ºC for 30 min. Wash the plate twice with cold FACS buffer. Cells were resuspended in 50 µL of 1 / 100 diluted allophycocyanin-conjugated Fc fragment-specific AffiniPure goat anti-mouse IgG (subclass 1+2a+2b+3) (Jackson Immunoresearch, catalog number 115-135-164) in FACS buffer and incubated at 4ºC for 30 min. The plates were washed twice in cold FACS buffer. Cells were resuspended in 55 µL of 1 / 1000 diluted propidium iodide (Sigma-Aldrich, catalog number P4170) in FACS buffer, and data were then acquired on a MACSQuant X (Miltenyi biotec).

[0353] The results are shown in Table 21. A022600424 bound to both human and cynomolgus monkey T cells with an affinity of approximately 200 nM.

[0354] [surface] [twenty one] [:] [A022600424] [Comparison of cellular performance in humans and cynomolgus monkeys] [GPC3] [As well as humans and crab-eating macaques] [TCRab] [A combined assessment.] [antigen] [sample] [ID] [CHO huGPC3] [EC50] [(] [M] [)] [CHO cyGPC3] [EC50] [(] [M] [)] [Huh-7] [EC50] [(] [M] [)] [GPC3] (Combined) [FACS] [)] [A022600424] 1.82E-09 1.11E-09 1.25E-09 [Ab2] 1.69E-08 5.4E-09 5.2E-09 [Original] [hu T] [cell] [IC50] [(] [M] [)] [Original] [cy T] [cell] [IC50] [(] [M] [)] [TCRab] [(compete] [FACS] [)] [A022600424] 1.7E-07 2.5E-07 [T017000698] 1.9E-07 2.5E-07

[0355] To assess the functionality of A022600424 using cynomolgus monkey T cells, a xCELLigence-based TDC assay of Huh-7 was performed using cynomolgus monkey T cells (LPT Laboratory, Germany), as described in Example 8. The IC50 values ​​for cynomolgus monkey and human T cells were found to be comparable (Table 22).

[0356] [surface] [twenty two] [Based on] [xCELLigence] [of] [TDC] [During the measurement, for] [Huh-7] [,by] [15:1] [The ratio of the effector to the target and in] [60h] [Analysis using cynomolgus monkeys] [T] [cell,] [A022600424] [Functionality.] [sample] [ID] [Original] [hu T] [cell] [IC50] [(] [M] [)] [Original] [cy T] [cell] [IC50] [(] [M] [)] [A022600424] 7.59E-11 2.29E-10 [Ab2] 7.88E-11 3.57E-11 [6.12] [Example]

[12] [:] [A022600424] [For combining to] [GPC3] [Selectivity]

[0357] The absence of A022600424 binding to members of the GPC3 family (i.e., GPC1, GPC2, GPC5, and GPC6) was assessed by ELISA, and the absence of A022600424 binding to GPC4 was assessed by SPR (Proteon XPR36).

[0358] Human GPC1 (R&D systems, catalog 4519-GP), human GPC2 (R&D systems, catalog 2304-GP), human GPC3 (R&D systems, catalog 2119-GP), human phosphatidylinositol proteoglycan-5 (R&D systems, catalog 2607-G5), and human phosphatidylinositol proteoglycan-6 (R&D systems, catalog 2845-GP) were directly plated overnight at 4ºC (2 µg / mL, 1xPBS buffer). The following day, the plates were washed 6 times (using an AquaMax microplate washer, Molecular devices) and blocked with 1xPBS + 1% casein for 2 hours at room temperature. After another 6 washes, A022600424 (1xPBS, 0.1% casein, 0.05% TWEEN 20) was added to the plates and incubated at room temperature for 1 hour. Next, the sample was removed, followed by six washes and the addition of anti-ISVD mAb ABH0077 (1xPBS, 0.1% casein, 0.05% TWEEN 20) at a final concentration of 17 nM at room temperature over a 1-hour period. The plate was washed six more times, and HRP-conjugated goat anti-mouse IgG multiclonal antibody (Abcam, catalog number ab97040) (1 / 1250 dilution; 1xPBS, 0.1% casein, 0.05% TWEEN 20) was applied to the plate at room temperature for 1 hour. After six final washes, es(HS)TMB substrate (SDT) was added, and the reaction was terminated by adding 1 M HCl after 10 minutes. The absorbance of the plate was measured at 450 nm and 620 nm on a Clariostar instrument (BMG LABTECH), and OD 450-OD 620 was calculated and plotted for data analysis.

[0359] Recombinant human GPC4 / hFc (R&D Systems, catalog 9195-GP) was captured on a GLC sensor chip (Biorad) immobilized with mouse anti-human IgG1 (GE Healthcare, catalog BR-1008-39) via amine coupling using EDC and NHS chemicals (run buffer: HBS-EP+, pH 7.4). Purified ISVD was injected at different concentrations (between 4 nM and 1000 nM) for 2 minutes (flow rate 45 µL / min), and dissociation was tracked for 900 s. Regeneration was performed by injecting 10 mM glycine-HCl (pH 1.5) for 1 minute (flow rate 45 µL / min). Data were double-referenced by subtracting a reference ligand lane and injecting blank buffer. The processed sensor map was analyzed using ProteOn Manager 3.1.0 (version 3.1.0.6) software based on a 1:1 interaction model (Langmuir binding model).

[0360] No combination of A022600424 with any of the tested GPC3 family members was detected. [6.13] [Example]

[13] [Pre-existing antibody pairs in humans] [A022600424] [Reactivity]

[0361] For normal human serum (n=96), the binding of pre-existing antibodies to A022600424 was assessed using ProteOn XPR36 (Bio-Rad Laboratories, Inc.). PBS / Tween (phosphate-buffered saline, pH 7.4, 0.005% Tween20) was used as the run buffer, and experiments were performed at 25ºC.

[0362] A022600424 was captured on the sensor chip via binding of HSA immobilized on the chip using an ALB23002 building block. To immobilize the HSA, the ligand lanes of the ProteOn GLC sensor chip were activated with EDC / NHS (30 μL / min), and HSA was injected at 100 µL / mL in ProteOn acetate buffer (pH 4.5) to achieve an immobilization level of approximately 2900 RU. Following immobilization, the surface was inactivated with ethanolamine HCl (30 μL / min).

[0363] Subsequently, A022600424 was injected onto the HSA surface at 45 μL / min for 2 min to achieve an ISVD capture level of approximately 800 RU. The sample containing the pre-existing antibody was centrifuged at 14,000 rpm for 2 min, and the supernatant was diluted 1:10 in PBS-Tween 20 (0.005%), then injected at 45 μL / min for 2 min, followed by a subsequent 400-second dissociation step. After each cycle (i.e., before a new ISVD capture and blood injection step), the HSA surface was regenerated by injecting HCl (100 mM) at 45 μL / min for 2 min. A sensor map showing the binding of the pre-existing antibody was obtained after double referencing by subtracting 1) ISVD-HSA dissociation and 2) non-specific binding with the reference ligand lane. The binding level of the pre-existing antibody was determined by setting the reporter point at 125 seconds (5 seconds after the end of association). The percentage reduction in pre-existing antibody binding was calculated relative to the reference ISVD binding level at 125 seconds.

[0364] Compared to the unoptimized tetravalent ISVD form F027301099, the tetravalent ISVD form A022600424, optimized for reduced binding to pre-existing antibodies by introducing mutants L11V and V89A in the anti-TCR building blocks, mutants L11V and V89L in each GPC3 and serum albumin binding building block, and C-terminal alanine, showed significantly less binding to pre-existing antibodies (Figure 11A). [6.14] [Example]

[14] [In soluble] [GPC3] [In existence] [A022600424] [of] [T] [Assessment of cell activation induction]

[0365] As described in Example 6, the induction of T cell activation in A022600424 in the presence of soluble GPC3 was evaluated. A022600424 exhibited the same behavior as its wild-type variant A022600168 (Figure 7). [6.15] [Example]

[15] [In vitro amplification] [T] [The cells have] [Huh-7] [Tumor] [NOG] [In mice] [A022600424] [In vivo proof of concept]

[0366] In an in vivo efficacy study in NOG mice with tumors, hepatocellular carcinoma Huh-7 tumor cells were subcutaneously injected and allowed to grow until an average tumor volume of approximately 150 mm³ was reached. At this point, in vitro expanded T cells were intraperitoneally injected into the mice. Tumor cell killing via ISVD-mediated T cell recruitment was evaluated by measuring tumor volume and analyzing tumor growth kinetics. The in vivo efficacy of A022600424 for tumor cell killing was evaluated and compared with the control T cell binder T017000698 (SEQ ID NO: 74, Table A-6) lacking GPC3 specificity.

[0367] In detail, 2 x 10⁶ Huh-7 tumor cells resuspended in 100 µL HBSS were subcutaneously injected into NOG mice. Tumors grew until they reached an average tumor volume of approximately 150 mm³. At this point, 10⁷ in vitro expanded T cells resuspended in 200 µL PBS were intraperitoneally injected into each mouse (D0). This T-cell injection was performed 24 hours after randomization of mice to different groups. Treatment with intravenously injected A022600424 began on D0, 3 h after T-cell injection, and continued on D3, D6, D9, and D12 (q3d; Fig. 12). Four dose levels of the TCR / GPC3 binding peptide (0.1 mg / kg, 0.2 mg / kg, 0.7 mg / kg, and 2 mg / kg) were tested. T017000698 was injected at 2 mg / kg in the control group on D0, D3, D6, D9, and D12 (q3d). Blood samples were collected from viable mice on days 6 and 12 in heparin-containing tubes to measure antibody exposure. Mice were sacrificed on day 15, and blood and tumor tissue were collected. Blood was used for antibody exposure measurements, and tumor tissue was used for analysis of target performance (GPC3) and T cell infiltration.

[0368] The results of tumor growth kinetics are shown in Figure 13. Mice treated with T017000698 were used as a control group for the analysis at D24, at which point all control mice were alive because they did not reach the endpoint criterion (2000 mm³ tumor volume). A dose-response pattern was observed in the tumor growth profiles of A022600424 and control T017000698 for inducing tumor arrest. The dose levels of 0.7 mg / kg (**, p = 0.0016) and 2 mg / kg (*, p = 0.0415) in A022600424 were significantly different from those in control T017000698. Doses of 0.1 mg / kg and 0.2 mg / kg had a lower effect on controlling tumor growth and were not significantly different from the control group. Statistical analysis was performed using Dunnett's multiple comparison test for analysis via one-way ANOVA.

[0369] In summary, the results indicate that A022600424 can induce statistically significant tumor arrest in a dose-dependent manner in this model. This confirms the concept of peptide-induced T cell-mediated killing via the GPC3 ISVD T cell conjugate, achieved by cross-linking T cells with GPC3 on Huh-7 tumor cells. [7] [Industrial Applicability]

[0370] The polypeptides described herein, the nucleic acid molecules encoding the polypeptides, the carriers containing the nucleic acids, and the compositions thereof can be used, for example, in the treatment of subjects suffering from cancer.

[0371] none

[0372] none.

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390]

[0391]

[0392]

[0393]

[0394]

[0395]

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

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[0408]

[0409]

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[0411]

[0412]

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[0420]

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[0422]

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[0424]

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[0436]

[0437]

Claims

1. A polypeptide comprising at least three immunoglobulin single variable domains (ISVDs) or thereof, wherein each of the ISVDs comprises three complementarity-determining regions (CDR1 to CDR3, respectively), and wherein: a) A first ISVD specifically binds to the constant domain of the TCR on T cells and includes i. CDR1 as the amino acid sequence of SEQ ID NO: 6; ii. CDR2 as the amino acid sequence of SEQ ID NO: 10; and iii. CDR3 as the amino acid sequence of SEQ ID NO: 14; b) A second ISVD specifically binds to GPC3 and includes iv. CDR1 as the amino acid sequence of SEQ ID NO: 7; v. CDR2 as the amino acid sequence of SEQ ID NO: 11; and vi. CDR3 as the amino acid sequence of SEQ ID NO: 15; and c) A third ISVD specifically binds to GPC3 and includes vii. CDR1 as the amino acid sequence of SEQ ID NO: 8; viii. CDR2 as the amino acid sequence of SEQ ID NO: 12; and ix. CDR3 as the amino acid sequence of SEQ ID NO:

16. The order of the ISVDs indicates the relative positions of the ISVDs to each other from the N-terminus to the C-terminus of the polypeptide, wherein the first ISVD is located at the N-terminus of the polypeptide.

2. The polypeptide as claimed in claim 1, wherein: a) The first ISVD consists of the amino acid sequence of SEQ ID NO: 2; b) The second ISVD consists of the amino acid sequence of SEQ ID NO: 3; and c) The third ISVD consists of the amino acid sequence of SEQ ID NO:

4.

3. The polypeptide as claimed in claim 1 or 2, wherein the first ISVD and the second ISVD are linked to each other via linkers consisting of fewer than 10 amino acids.

4. The polypeptide as claimed in claim 1 or 2, wherein the polypeptide further comprises one or more other groups, residues, portions or binding units, wherein the one or more other groups, residues, portions or binding units provide an increased half-life to the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, portions or binding units.

5. The polypeptide of claim 4, wherein the one or more other groups, residues, portions or binding units providing the increased half-life of the polypeptide are selected from the group consisting of: polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc portions, and small proteins or peptides that can bind to serum proteins.

6. The polypeptide as claimed in claim 4, wherein the one or more other binding units providing the increased half-life of the polypeptide are selected from the group consisting of binding units that can bind to serum albumin or serum immunoglobulins.

7. The polypeptide as claimed in claim 6, wherein the binding unit providing the increased half-life of the polypeptide is an ISVD bound to human serum albumin.

8. The polypeptide of claim 7, wherein the ISVD bound to human serum albumin comprises i. CDR1 as the amino acid sequence of SEQ ID NO: 9; ii. CDR2 as the amino acid sequence of SEQ ID NO: 13; and iii. CDR3 as the amino acid sequence of SEQ ID NO:

17.

9. The polypeptide of claim 7, wherein the amino acid sequence of the ISVD bound to human serum albumin consists of the amino acid sequence of SEQ ID NO:

5.

10. The polypeptide as claimed in claim 1 or 2, wherein the polypeptide comprises or is composed of an amino acid sequence exhibiting or consisting of SEQ ID NO:

1.

11. A nucleic acid comprising a nucleotide sequence encoding a polypeptide as described in any one of claims 1 to 10.

12. A method for generating a polypeptide as described in any one of claims 1 to 10, the method comprising at least the following steps: a) expressing the nucleic acid as described in claim 11 in a suitable host cell or host organism or in another suitable expression system; followed by: b) isolating and / or purifying the polypeptide as described in any one of claims 1 to 9.

13. A composition comprising at least one polypeptide as described in any one of claims 1 to 10 or a nucleic acid as described in claim 11.

14. The composition as claimed in claim 13 is a pharmaceutical composition, the pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant.

15. Use of a polypeptide as described in any one of claims 1 to 10 or a composition as described in claim 13 or 14 for the preparation of a medicament for treating cancer.

16. Use of a polypeptide as described in any one of claims 1 to 10 or a composition as described in claim 13 or 14 for the preparation of a pharmaceutical composition for the treatment of liver cancer or lung cancer.

17. The use as described in claim 16, wherein the liver cancer is hepatocellular carcinoma.

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