Conditionally active anti-HER2 antibodies, antibody fragments, their immunoconjugates, and their uses

Abstract

Polypeptides having heavy and / or light chain variable regions that specifically bind to the HER2 protein, antibodies and antibody fragments comprising the heavy and / or light chain variable regions that bind to the HER2 protein, and multispecific antibodies that bind to the HER2 protein and CD3. Pharmaceutical compositions and kits containing the polypeptides, antibodies and antibody fragments, and multispecific antibodies containing the polypeptides are also provided.

Description

[Technical Field]

[0001] This disclosure relates to anti-HER2 antibodies, anti-HER2 antibody fragments, anti-HER2 polyspecific antibodies, and immunoconjugates of such antibodies and antibody fragments, as well as the use of antibodies, antibody fragments, polyspecific antibodies, and immunoconjugates in diagnostic and therapeutic methods. [Background technology]

[0002] Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor family that possesses tyrosine kinase activity. Receptor dimerization leads to autophosphorylation of tyrosine residues within the receptor's cytoplasmic domain, initiating various signaling pathways that contribute to cell proliferation and tumorigenesis. Further details on the role of HER2 in cancer can be found in numerous articles, such as "Human Epidermal Growth Factor Receptor 2 (HER2) in Cancers: Overexpression and Therapeutic Implications," Iqbal, Nida and Iqbal, Naveed, Molecular Biology International, Vol. 2014, Article ID 852748.

[0003] Overexpression of the ERB-B2 gene (also known as the "HER2 gene") occurs in a significant proportion of breast cancers. Overexpression of the HER2 protein is strongly associated with increased disease recurrence and poor prognosis. Drugs targeting the HER2 protein in breast cancer have shown significant positive effects in the treatment of HER2-positive breast cancer. Overexpression of the HER2 protein also occurs in ovarian cancer, gastric cancer, lung adenocarcinoma, invasive forms of uterine cancer, gastric cancer, and salivary duct cancer.

[0004] The HER2 protein is a target of the monoclonal antibody trastuzumab, Herceptin, which has been shown to be effective in cancers where the HER2 protein is overexpressed. Trastuzumab binding to the HER2 protein has been shown to increase p27, a protein that stops cell proliferation. Another monoclonal antibody, pertuzumab, is approved by the FDA for use in combination with trastuzumab. Pertuzumab inhibits the dimerization of HER2 and HER3 receptors. Other therapies targeting the HER2 protein are available or under development.

[0005] There are at least four tests for HER2 overexpression: the ImmunoHistoChemistry test, which determines whether there is too much HER2 protein in cancer cells; the fluorescence in situ hybridization test, which determines whether there is too many copies of the HER2 gene in cancer cells; the subtraction probe technology chromogenic in situ hybridization test, which also determines whether there is too many copies of the HER2 gene in cancer cells; and the Inform Dual In Situ hybridization test, which also determines whether there is too many copies of the HER2 gene in cancer cells.

[0006] The present invention aims to provide anti-HER2 antibodies or antibody fragments with reduced or minimal side effects, particularly suitable for therapeutic and diagnostic use for the diagnosis and treatment of cancer. Some of these anti-HER2 antibodies or antibody fragments may have a higher binding affinity to HER2 proteins in the tumor microenvironment compared to HER2 proteins present in normal tissues. These anti-HER2 antibodies or antibody fragments typically have at least equivalent efficacy to known anti-HER2 antibodies or antibody fragments. In addition, these anti-HER2 antibodies or antibody fragments may exhibit reduced side effects compared to monoclonal anti-HER2 antibodies, including those known in the art for having relatively low binding affinity to HER2 proteins in normal tissues. These advantages can provide more selective targeting of HER2 proteins, and as a result of the selectivity of the antibodies against HER2 proteins present in the tumor microenvironment, it may be possible to use higher doses of these anti-HER2 antibodies or antibody fragments, thereby enabling more effective therapeutic treatment without a corresponding increase in undesirable side effects. [Overview of the Initiative]

[0007] In one embodiment, the present invention provides an isolated polypeptide that specifically binds to the HER2 protein, wherein the polypeptide comprises a heavy chain variable region comprising three anti-HER2 complementarity determining regions, the regions having sequences H1, H2, and H3. The H1 sequence is GFX1IKDTYIH (sequence number 1), The H2 sequence is X2IX3PTX4X5YX6X7YADSVKG (sequence number 2), The H3 sequence is WGGDGFYX8MDY (sequence number 3), In the formula, X1 is N or W, X2 is R or K, X3 is Y, K or D, X4 is N or A, X5 is G or K, X6 is T or D, X7 is R or E, and X8 is A or E, representing a heavy-chain variable region. A light chain variable region comprising three anti-HER2 complementarity-determining regions having sequences L1, L2, and L3, The L1 sequence is RASQDVNTX9VA (sequence number 4), The L2 sequence is SASFLYS (sequence number 5), The L3 array is QQX 10 YTTPPT (sequence number 6) In the formula, X9 is A or D, and X 10 However, it includes a light chain variable region which is H, D, or E, As a premise, if X1 to X8 are N, R, Y, N, G, T, R, and A respectively, then X9 is not A, but X 10 But it's not H.

[0008] The isolated polypeptide of the present invention has an H1 sequence of GFWIKDTYIH (SEQ ID NO: 7) or GFNIKDTYIH (SEQ ID NO: 50), and an H2 sequence of KIYPTNGYTRYADSVKG (SEQ ID NO: 8), RIKPTNGYTRYADSVKG (SEQ ID NO: 9), RIDPTNGYTRYADSVKG (SEQ ID NO: 10), RIYPTAGYTRYADSVKG (SEQ ID NO: 11), RIYPTNKYTRYADSVKG (SEQ ID NO: 12), RIYPTNGYDRYADSVKG (SEQ ID NO: 13), RIYPTNGYTEYADSVKG ( The sequence is one of sequence numbers 14) and RIYPTNGYTRYADSVKG (sequence number 49), the H3 sequence is WGGDGFYEMDY (sequence number 15) or WGGDGFYAMDY (sequence number 51), the L1 sequence is RASQDVNTDVA (sequence number 16) or RASQDVNTAVA (sequence number 52), the L2 sequence is SASFLYS (sequence number 5), and the L3 sequence is QQDYTTPPT (sequence number 17), QQEYTTPPT (sequence number 18), or QQHYTTPPT (sequence number 53).

[0009] In another embodiment, the present invention provides an isolated polypeptide that specifically binds to the HER2 protein, the polypeptide comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs: 19-28, and the light chain variable region has an amino acid sequence selected from SEQ ID NOs: 29-32.

[0010] In yet another aspect, the present invention provides an isolated polypeptide that specifically binds to the HER2 protein, the polypeptide comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs. 33 and 19-28, and the light chain variable region has an amino acid sequence selected from SEQ ID NOs. 30-32.

[0011] In yet another aspect, the present invention provides an isolated polypeptide that specifically binds to the HER2 protein, the polypeptide comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs. 35-39, and the light chain variable region has an amino acid sequence selected from SEQ ID NOs. 41-48.

[0012] In certain embodiments, the present invention provides an isolated polypeptide that specifically binds to the HER2 protein, the polypeptide comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 35 and a light chain variable region having an amino acid sequence selected from any one of SEQ ID NOs: 41 to 48.

[0013] In another particular embodiment, the present invention provides an isolated polypeptide that specifically binds to the HER2 protein, the polypeptide comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 36 and a light chain variable region having an amino acid sequence selected from any one of SEQ ID NOs: 41-48.

[0014] In another embodiment, the present invention provides an isolated polypeptide that specifically binds to the HER2 protein, the polypeptide comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 37 and a light chain variable region having an amino acid sequence selected from any one of SEQ ID NOs: 41-48.

[0015] In yet another embodiment, the present invention provides an isolated polypeptide that specifically binds to the HER2 protein, the polypeptide comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 38 and a light chain variable region having an amino acid sequence selected from any one of SEQ ID NOs: 41 to 48.

[0016] In yet another embodiment, the present invention provides an isolated polypeptide that specifically binds to the HER2 protein, the polypeptide comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 39 and a light chain variable region having an amino acid sequence selected from any one of SEQ ID NOs: 41 to 48.

[0017] In yet another embodiment, the isolated polypeptide comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising three anti-HER2 complementarity determining regions, H1, H2, and H3. The H1 sequence is sequence number 50. The H2 sequence is sequence number 49, sequence number 9, sequence number 12, or sequence number 13. The H3 sequence is sequence number 51. The light chain variable region includes three anti-HER2 complementarity-determining regions, L1, L2, and L3, and six anti-CD3 complementarity-determining regions, L4, L5, L6, L7, L8, and L9. The L4 sequence is GFTFNTYAMN (sequence number 54), The L5 sequence is RIRSKYNNYATYYADSVKD (sequence number 55), L6 array, HX 11 NTE 12 NSKVSWFX 13 Y (sequence number 70), L7 array, RSSX14 It is GAVTTSNYDN (SEQ ID NO: 71), the L8 sequence is GTNKRAP (SEQ ID NO: 58), the L9 sequence is ALWYSNLWV (SEQ ID NO: 59), wherein X 11 is G, S, A, or T, and X 12 is G or P, and X 13 is A or Q, and X 14 is T or A.

[0018] In another embodiment, an isolated polypeptide having nine CDRs, the L6 sequence is any one of SEQ ID NOs: 56 and 60 - 67, and the L7 sequence is SEQ ID NO: 57, 68, or 69.

[0019] In another specific embodiment of the isolated polypeptide, the L4 sequence is selected from SEQ ID NOs: 57, 68, and 69.

[0020] In another aspect, the present invention relates to an anti - HER2 antibody or antibody fragment comprising each of the isolated polypeptides of the foregoing embodiments.

[0021] In the foregoing embodiments, the antibody or antibody fragment may have a higher binding affinity for HER2 protein at the pH in the tumor microenvironment compared to the pH in the non - tumor microenvironment. The pH in the tumor microenvironment can be in the range of 5.0 - 7.0, and the pH in the non - tumor microenvironment can be in the range of 7.2 - 7.8.

[0022] In another aspect, the present invention relates to an antibody or antibody fragment comprising a heavy - chain variable region and a light - chain variable region, the heavy - chain variable region comprising three anti - HER2 complementarity - determining regions, which have the sequences H1, H2, and H3, the H1 sequence is GFX1IKDTYIH (SEQ ID NO: 1), the H2 sequence is X2IX3PTX4X5YX6X7YADSVKG (SEQ ID NO: 2), the H3 sequence is WGGDGFYX8MDY (SEQ ID NO: 3), In the formula, X1 is N or W, X2 is R or K, X3 is Y, K or D, X4 is N or A, X5 is G or K, X6 is T or D, X7 is R or E, and X8 is A or E, representing a heavy-chain variable region. A light chain variable region comprising three anti-HER2 complementarity-determining regions having sequences L1, L2, and L3, The L1 sequence is RASQDVNTX9VA (sequence number 4), The L2 sequence is SASFLYS (sequence number 5), The L3 array is QQX 10 YTTPPT (sequence number 6) In the formula, X9 is A or D, and X 10 However, it includes a light chain variable region which is H, D, or E, As a premise, if X1 to X8 are N, R, Y, N, G, T, R, and A respectively, then X9 is not A, but X 10 But it's not H.

[0023] In certain embodiments of the antibody or antibody fragment, the H1 sequence may be GFWIKDTYIH (SEQ ID NO: 7) or GFNIKDTYIH (SEQ ID NO: 50), the H2 sequence may be KIYPTNGYTRYADSVKG (SEQ ID NO: 8), RIKPTNGYTRYADSVKG (SEQ ID NO: 9), RIDPTNGYTRYADSVKG (SEQ ID NO: 10), RIYPTAGYTRYADSVKG (SEQ ID NO: 11), RIYPTNKYTRYADSVKG (SEQ ID NO: 12), RIYPTNGYDRYADSVKG (SEQ ID NO: 13), RIYPTNGYTEYADSVKG (SEQ ID NO: 14), or RIYPTNGYTRYADSVKG (SEQ ID NO: 49), and the H3 sequence may be WGGDGFYEMDY (SEQ ID NO: 15) or WGGDGFYAMDY (SEQ ID NO: 51).

[0024] In each of the aforementioned embodiments of this aspect of the antibody or antibody fragment, the L1 sequence may be RASQDVNTDVA (SEQ ID NO: 16) or RASQDVNTAVA (SEQ ID NO: 52), the L2 sequence may be SASFLYS (SEQ ID NO: 5), and the L3 sequence may be QQDYTTPPT (SEQ ID NO: 17), QQEYTTPPT (SEQ ID NO: 18), or QQHYTTPPT (SEQ ID NO: 53).

[0025] In some of the aforementioned embodiments of this aspect of the antibody or antibody fragment, the H1 sequence is SEQ ID NO: 50, the H2 sequence is SEQ ID NO: 49, and the H3 sequence is SEQ ID NO: 51.

[0026] In some of the aforementioned embodiments of this aspect of the antibody or antibody fragment, the L1 sequence is RASQDVNTAVA (SEQ ID NO: 52) and the L3 sequence is QQHYTTPPT (SEQ ID NO: 53).

[0027] In certain embodiments of the antibody or antibody fragment, the H1 sequence is SEQ ID NO: 50, the H2 sequence is RIKPTNGYTRYADSVKG (SEQ ID NO: 9), RIYPTNKYTRYADSVKG (SEQ ID NO: 12), RIYPTNGYDRYADSVKG (SEQ ID NO: 13), or RIYPTNGYTRYADSVKG (SEQ ID NO: 49), and the H3 sequence is WGGDGFYEMDY (SEQ ID NO: 15) or WGGDGFYAMDY (SEQ ID NO: 51).

[0028] In each of the aforementioned embodiments of this aspect of the antibody or antibody fragment, the heavy chain variable region may be any one of SEQ ID NOs: 19 to 28, and the light chain variable region may be any one of SEQ ID NOs: 29 to 32.

[0029] In each of the above embodiments of this aspect of the antibody or antibody fragment, the heavy chain variable region may be any one of SEQ ID NOs: 33 and 19-28, and the light chain variable region may be any one of SEQ ID NOs: 30-32.

[0030] In each of the aforementioned embodiments of this aspect of the antibody or antibody fragment, the heavy chain variable region may be any one of SEQ ID NOs: 35 to 39, and the light chain variable region may be any one of SEQ ID NOs: 41 to 48.

[0031] In a particular embodiment, the antibody or antibody fragment has a heavy chain variable region of SEQ ID NO: 19 or 20 and a light chain variable region of SEQ ID NO: 29.

[0032] In other embodiments, the antibody or antibody fragment has a heavy chain variable region which is SEQ ID NO: 33, and the light chain variable region which is one of SEQ ID NOs: 30-32.

[0033] In other embodiments, the antibody or antibody fragment has the light chain variable region of SEQ ID NO: 30, and one of the heavy chain variable regions of SEQ ID NOs: 33 and 19-28.

[0034] In a particular embodiment, the antibody or antibody fragment has a heavy chain variable region of SEQ ID NO: 35 and a light chain variable region selected from any one of SEQ ID NOs: 41 to 48.

[0035] In a particular embodiment, the antibody or antibody fragment has a heavy chain variable region of SEQ ID NO: 36 and a light chain variable region selected from any one of SEQ ID NOs: 41 to 48.

[0036] In other embodiments, the antibody or antibody fragment has a heavy chain variable region of SEQ ID NO: 37 and a light chain variable region selected from any one of SEQ ID NOs: 41 to 48.

[0037] In other embodiments, the antibody or antibody fragment has a heavy chain variable region of SEQ ID NO: 38 and a light chain variable region selected from any one of SEQ ID NOs: 41 to 48.

[0038] In yet another embodiment, the antibody or antibody fragment has a heavy chain variable region of SEQ ID NO: 39 and a light chain variable region selected from any one of SEQ ID NOs: 41 to 48.

[0039] In some embodiments, the antibody or antibody fragment is a heavy chain variable region comprising three anti-HER2 complementarity determining regions, H1, H2, and H3. The H1 sequence is GFX1IKDTYIH (sequence number 1), The H2 sequence is X2IX3PTX4X5YX6X7YADSVKG (sequence number 2), The H3 sequence is WGGDGFYX8MDY (sequence number 3), In the formula, X1 is N or W, X2 is R or K, X3 is Y, K or D, X4 is N or A, X5 is G or K, X6 is T or D, X7 is R or E, and X8 is A or E, representing a heavy-chain variable region. It comprises a light chain variable region containing three anti-HER2 complementarity-determining regions having sequences L1, L2, and L3, and six anti-CD3 complementarity-determining regions, L4, L5, L6, L7, L8, and L9, The L1 sequence is RASQDVNTX9VA (sequence number 4), The L2 sequence is SASFLYS (sequence number 5), The L3 array is QQX 10 YTTPPT (sequence number 6) In the formula, X9 is A or D, and X 10 However, it is either H, D, or E. As a premise, if X1 to X8 are N, R, Y, N, G, T, R, and A respectively, then X9 is not A, but X 10 However, not H The L4 sequence is GFTFNTYAMN (sequence number 54), The L5 sequence is RIRSKYNNYATYYADSVKD (sequence number 55), L6 array, HX 11 NTE 12 NSKVSWFX 13 Y (sequence number 70), L7 array, RSSX 14 GAVTTSNYDN (Sequence ID 71) The L8 sequence is GTNKRAP (sequence number 58), The L9 sequence is ALWYSNLWV (sequence number 59), In the formula, X 11 However, it is G, S, A, or T, and X 12 However, it is G or P, and X 13 However, it is A or Q, and X 14 However, it is a polyspecific antibody or antibody fragment that is either T or A.

[0040] In another embodiment of the antibody or antibody fragment, the antibody or antibody fragment is polyspecific and comprises a heavy chain variable region including three anti-HER2 complementarity determining regions, H1, H2, and H3. The H1 sequence is sequence number 50. The H2 sequence is selected from sequence number 49, sequence number 9, sequence number 12, or sequence number 13. The H3 sequence includes a heavy chain variable region, which is sequence number 51, a light chain variable region containing three anti-HER2 complementarity-determining regions L1, L2, and L3, and six anti-CD3 complementarity-determining regions L4, L5, L6, L7, L8, and L9. The L1 sequence is sequence number 52 or sequence number 16. The L2 sequence is sequence number 5, The L3 sequence is sequence number 53. The L4 sequence is GFTFNTYAMN (sequence number 54), The L5 sequence is RIRSKYNNYATYYADSVKD (sequence number 55), L6 array, HX 11 NTE 12 NSKVSWFX 13 Y (sequence number 70), L7 array, RSSX 14 GAVTTSNYDN (Sequence ID 71) The L8 sequence is GTNKRAP (sequence number 58), The L9 sequence is ALWYSNLWV (sequence number 59), In the formula, X 11 However, it is G, S, A, or T, and X 12 However, it is G or P, and X13 However, it is A or Q, and X 14 However, it is either T or A.

[0041] In another embodiment of the polyspecific antibody or antibody fragment, the L6 sequence is one of sequence numbers 56 and 60-67, and the L7 sequence is sequence number 57, 68, or 69.

[0042] Each of the aforementioned embodiments of the antibody or antibody fragment in this embodiment may have a higher binding affinity to the HER2 protein at the pH in the tumor microenvironment compared to the different pH levels that occur in the non-tumor microenvironment. The pH in the tumor microenvironment may be in the range of 5.0 to 7.0, while the pH in the non-tumor microenvironment may be in the range of 7.2 to 7.8.

[0043] Each of the aforementioned embodiments of the antibody or antibody fragment in this embodiment may have a ratio of binding affinity to the HER2 protein at pH in the tumor microenvironment to binding affinity to the HER2 protein at different pH in the non-tumor microenvironment of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

[0044] In further embodiments, the present invention relates to an immunoconjugate comprising an antibody or antibody fragment in any of the above embodiments. This immunoconjugate may comprise at least one agent selected from chemotherapeutic agents, radioatoms, cell proliferation inhibitors, and cytotoxic agents, or at least two such agents.

[0045] In each of the aforementioned embodiments of the immunoconjugate, at least one agent may be a radioagent, which may be selected from α-ejectors, β-ejectors, and γ-ejectors.

[0046] In each of the aforementioned embodiments of the immunoconjugate, at least one agent may be covalently bound to the linker molecule. In each of the aforementioned embodiments of the immunoconjugate, at least one agent may be selected from mytansinoids, auristatin, drastatin, calicheamicin, pyrrolobenzodiazepines, and anthracyclines.

[0047] In yet another embodiment, the present invention relates to a pharmaceutical composition comprising each of the polypeptides of the above embodiments, each of the antibodies or antibody fragments of the above embodiments, or each of the immunoconjugates of the above embodiments, and a pharmaceutically acceptable carrier.

[0048] The aforementioned embodiments of the pharmaceutical product may include an isotonic agent.

[0049] Each of the aforementioned embodiments of the pharmaceutical composition may further comprise an immune checkpoint inhibitor molecule. The immune checkpoint inhibitor molecule may be an antibody or antibody fragment against an immune checkpoint. The immune checkpoint may be selected from CTLA4, LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, GITR, B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS. Alternatively, the immune checkpoint may be one of CTLA4, PD-1, or PD-L1.

[0050] Each of the aforementioned embodiments of the pharmaceutical composition may further comprise an antibody or antibody fragment against an antigen selected from CTLA4, PD1, PD-L1, AXL, ROR2, CD3, EpCAM, B7-H3, ROR1, SFRP4, and WNT proteins.

[0051] In another embodiment, the present invention relates to a method for treating cancer, comprising the step of administering to a patient having cancer each of the polypeptides of the aforementioned embodiments, each antibody or antibody fragment of the aforementioned embodiments, each immune conjugate of the aforementioned embodiments, or each pharmaceutical composition of the aforementioned embodiments.

[0052] In yet another embodiment, the present invention provides a kit for diagnosis or treatment comprising any of the polypeptides, antibodies or antibody fragments, or immunoconjugates described above. [Brief explanation of the drawing]

[0053] [Figure 1] This shows an exemplary sequence arrangement of the light chain variable region of the anti-HER2 antibody of the present invention. [Figure 2] This shows an exemplary sequence arrangement of the heavy chain variable region of the anti-HER2 antibody of the present invention. [Figure 3A-3E] This figure shows the binding activity of the HER2 benchmark antibody to the human HER2 protein at pH 6.0 and pH 7.4, as measured by enzyme-linked immunosorbent assay (ELISA), compared to an exemplary conditionally active anti-HER2 antibody of the present invention. The benchmark antibody is indicated by BM. For each conditionally active antibody, one of the heavy chain (HC) and light chain (LC) is designated in each figure. An unspecified heavy chain or light chain is the heavy chain or light chain of the benchmark antibody. The Y-axis is the optical density (OD) at 450 nm. The X-axis shows the antibody concentration (log ng / mL) with a starting concentration of 300 ng / mL. [Figure 4] The binding activity of the HER2 benchmark antibody (BM) to the human HER2 protein over a range of pH values, as measured by enzyme-linked immunosorbent assay (ELISA), is shown. For each conditionally active antibody, the heavy chain (HC) and light chain (LC) are designated in the figure. The Y-axis represents the optical density (OD) at 450 nm. The X-axis represents the pH of the incubation buffer and wash buffer (pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4). [Figure 5]The binding activity of the conditionally active anti-HER2 antibody of the present invention to human cancer cell lines (SKBR3) expressing human HER2 protein on the cell surface, measured by fluorescence-activated cell classification (FACS), is shown for four different concentrations of the antibody at pH 6.0 (blue) or pH 7.4 (orange). [Figure 6A-6B] The binding activity of the HER2 benchmark antibody (BM) of the present invention against the human HER2 protein at pH 6.0 (Figure 6A) and pH 7.4 (Figure 6B), as determined by pH affinity ELISA assay, and the conditionally active anti-HER2 binding activity are shown. The substitution numbering referenced in this figure is based on the BAP-130 benchmark antibody in Figure 2. [Figures 7A-7B] The binding activity of the HER2 benchmark antibody (BM) of the present invention against the cynoHER2 protein at pH 6.0 (Figure 7A) and pH 7.4 (Figure 7B), as determined by pH affinity ELISA assay, and the conditionally active anti-HER2 binding activity are shown. The substitution numbering referenced in this figure is based on the BAP-130 benchmark antibody in Figure 2. [Figure 8] This figure shows the binding activity of HER2 benchmark antibodies (BMs) and conditionally active antibodies to the human HER2 protein at various pH values, as determined by a pH range ELISA assay. The substitution numbering referenced in this figure is based on the BAP-130 benchmark antibody in Figure 2. [Figure 9A] The average body weight in grams of the mice in the different treatment groups in Example 7 is shown. Data are expressed as mean ± standard error. [Figure 9B] The relative body weight changes of the different treatment groups of mice in Example 7 are shown as percentages. Percentage changes were calculated based on the animal's body weight on the first day of administration. Data are expressed as mean ± standard error. [Figure 9C] The tumor growth curves for different treatment groups of mice in Example 7 are shown. Data are expressed as mean ± standard error. [Figure 10A]This shows the sequence arrangement of exemplary heavy chain variable regions of the anti-HER2 antibody of the present invention. Exemplary heavy chain variable regions BAP150.24-WT-HC (SEQ ID NO: 34), BAP150.24-02-HC (SEQ ID NO: 35), BAP150.24-05-HC (SEQ ID NO: 36), BAP150.24-06-HC (SEQ ID NO: 37), BAP150.24-07-HC (SEQ ID NO: 38), and BAP150.24-08-HC (SEQ ID NO: 39) are shown. H1, H2, and H3 CDRs are underlined, respectively. [Figure 11A] The sequence arrangement of exemplary light chain variable regions of the anti-HER2 antibody of the present invention is shown. Exemplary light chain variable regions BAP150.24-WT-LC (SEQ ID NO: 40), BAP150.24-BF11-LC (SEQ ID NO: 41), BAP150.24-BF15-LC (SEQ ID NO: 42), BAP150.24-BF19-LC (SEQ ID NO: 43), BAP150.24-BF39-LC (SEQ ID NO: 44), BAP150.24-BF40-LC (SEQ ID NO: 45), BAP150.24-BF42-LC (SEQ ID NO: 46), BAP150.24-BF45-LC (SEQ ID NO: 47), and BAP150.24-BF46-LC (SEQ ID NO: 48) are shown. The L1, L2, L3, L4, L5, L6, L7, L8, and L9 CDRs are underlined, respectively. [Figure 12] This study demonstrates that a bispecific antibody may be a tetravalent homodimer "butterfly" containing CAB CD3, and that such an antibody can be detected by binding to CD3 on a plate. [Figures 13A-13D] The binding activity of WT HER2 × WT CD3, WT HER2 × CAB CD3-BF45, and CAB HER2-24-06 × CAB CD3-BF19 bispecific antibodies is shown compared to isotype × WT CD3 at pH 6.0 (Figures 13A and 13C) and pH 7.4 (Figures 13B and 13D) as determined by pH sandwich ELISA assay. [Figure 14]This shows the binding activity of WT HER2 × WT CD3, WT HER2 × CAB CD3-BF45, and CAB HER2-24-06 × CAB CD3-BF19 bispecific antibodies at various pH values ​​determined by pH range ELISA assays. [Figure 15A-15I] Surface plasmon resonance (SPR) coupling analyses of WT HER2 × WT CD3 with ligands huHER2-His, cyno-HER2-His, and huCD3-His are shown at pH 6.0 (Figures 15A-15C), pH 6.5 (Figures 15D-15F), and pH 7.4 (Figures 15G-15I), respectively. [Figure 16A-16I] Surface plasmon resonance (SPR) coupling analyses of WT HER2×CAB CD3-BF-45 with ligands huHER2-His, cyno-HER2-His, and huCD3-His are shown at pH 6.0 (Figures 16A-16C), pH 6.5 (Figures 16D-16F), and pH 7.4 (Figures 16G-16I), respectively. [Figure 17A-17I] Surface plasmon resonance (SPR) coupling analyses of CAB HER2-24-06×CAB CD3-BF-19 with ligands huHER2-His, cyno-HER2-His, and huCD3-His are shown at pH 6.0 (Figures 17A-17C), pH 6.5 (Figures 17D-17F), and pH 7.4 (Figures 17G-17I), respectively. [Figure 18] This is a schematic structure of a tetravalent polyspecific antibody that is a homodimer, with each arm having a binding site for the antigen (Ag) and a binding site for CD3.

[0054] definition To facilitate understanding of the examples provided herein, certain frequently occurring terms are defined herein.

[0055] In relation to the measured quantity, the term “approximately” as used herein refers to the normal variation of the measured quantity that would be expected by a person skilled in the art to perform the measurement, to perform the measurement in accordance with the purpose of the measurement and the accuracy of the measuring instrument used, and to handle the measurement. Unless otherwise indicated, “approximately” refers to a variation of + / - 10% of the given value.

[0056] As used herein, the term “affinity” refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, “binding affinity” refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0057] As used herein, the term "affinity matured" antibody refers to an antibody having one or more modifications in one or more heavy chain variable regions or light chain variable regions, the modifications resulting in improved antibody affinity to an antigen compared to a parent antibody without such modifications.

[0058] As used herein, the term “amino acid” refers to any organic compound containing an amino group (--NH2) and a carboxyl group (--COOH), preferably as free groups or, alternatively, after condensation as part of a peptide bond. "The alpha-amino acids that form 20 naturally encoded polypeptides" is understood in the art to refer to alanine (ala or A), arginine (arg or R), asparagine (asn or N), aspartic acid (asp or D), cysteine ​​(cys or C), glutamic acid (glu or E), glutamine (gin or Q), glycine (gly or G), histidine (his or H), isoleucine (ile or I), leucine (leu or L), lysine (lys or K), methionine (met or M), phenylalanine (phe or F), proline (pro or P), serine (ser or S), threonine (thr or T), tryptophan (tip or W), tyrosine (tyr or Y), and valine (val or V).

[0059] As used herein, the term “antibody” refers to intact immunoglobulin molecules, as well as fragments of immunoglobulin molecules capable of binding to the epitopes of antigens, e.g., Fab, Fab', (Fab')2, Fv, and SCA fragments. These antibody fragments, which retain some ability to selectively bind to antigens (e.g., polypeptide antigens) of the antibodies from which they are derived, can be prepared using methods well known in the art (see, for example, “Antibodies: A Laboratory Manual, Second Edition,” Greenfield, Edward A., Ed., ISBN 978-1-936113-81-1 (2014)), as further described below. Antibodies can be used to isolate fractional amounts of antigens by immunoaffinity chromatography. Various other uses of such antibodies are for the diagnosis and / or staging of diseases (e.g., tumorigenesis), as well as for therapeutic applications to treat diseases (e.g., tumorigenesis, autoimmune diseases, AIDS, cardiovascular diseases, infections, etc.). Chimeric antibodies, human-like antibodies, humanized antibodies, or fully human antibodies are particularly useful for administration to human patients.

[0060] Fab fragments consist of monovalent antigen-binding fragments of antibody molecules and are produced by digesting the entire antibody molecule with the enzyme papain, yielding fragments consisting of intact light and heavy chain portions.

[0061] The Fab' fragment of an antibody molecule can be obtained by treating the entire antibody molecule with pepsin and then reducing it, yielding a molecule consisting of intact light and heavy chain portions. In this manner, two Fab' fragments are obtained per treated antibody molecule.

[0062] The (Fab')2 fragment of an antibody can be obtained by treating the entire antibody molecule with the enzyme pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0063] An Fv fragment is defined as a genetically modified fragment containing a variable region of the light chain and a variable region of the heavy chain expressed as two separate chains.

[0064] As used herein, the term “antibody fragment” refers to molecules other than intact antibodies, including a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and polyspecific antibodies formed from antibody fragments.

[0065] As used herein, the terms “anti-HER2 antibody,” “HER2 antibody,” and “HER2-binding antibody” refer to an antibody that can bind to the HER2 protein with sufficient affinity to be useful as a diagnostic and / or therapeutic agent when the antibody targets the HER2 protein. In one embodiment, the degree of binding of the anti-HER2 antibody to unrelated non-HER2 proteins is less than about 10% of the binding of the antibody to the HER2 protein, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody that binds to the HER2 protein is less than 1 μM, less than 100 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 0.01 nM, or less than 0.001 nM (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant (Kd) of M). In certain embodiments, the anti-HER2 antibody binds to HER2 proteins from different species, for example, to HER2 protein epitopes conserved between the extracellular domains of HER2 proteins.

[0066] As used herein, the term “binding” refers to the interaction between an antibody’s variable region or Fv and an antigen, which has an interaction depending on the presence of a specific structure on the antigen (e.g., an antigenic determinant or epitope). For example, an antibody’s variable region or Fv generally recognizes and binds to the structure of a specific protein rather than to any protein. As used herein, the terms “specifically binding” or “binding specifically” mean that an antibody’s variable region or Fv binds or associates with a particular antigen more frequently, rapidly, for a longer duration, and / or with higher affinity than other proteins. For example, an antibody’s variable region or Fv binds specifically to that antigen with higher affinity, with higher avidity, and for a longer duration than it would to bind to other antigens. In another example, an antibody’s variable region or Fv binds to a cell surface protein (antigen) with substantially higher affinity than the affinity to the relevant protein or other cell surface protein or antigen generally recognized by a polyreactive native antibody (i.e., a native antibody known to bind to a variety of naturally occurring antigens in humans). However, "specific binding" does not necessarily require exclusive or undetectable binding to another antigen, which is meant by the term "selective binding." For example, "specific binding" of an antibody's variable region or Fv (or other binding region) means that it binds to an antigen, and that the antibody's variable region or Fv binds to the antigen with an equilibrium constant (KD) of 100 nM or less, e.g., 50 nM or less, e.g., 20 nM or less, e.g., 15 nM or less, or 10 nM or less, or 5 nM or less, 2 nM or less, or 1 nM or less.

[0067] As used herein, the terms “cancer” and “malignant” typically refer to or describe a physiological condition in mammals characterized by unregulated cell proliferation / growth. Examples of cancer include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma), blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, as well as various types of head and neck cancers.

[0068] As used herein, the terms “proliferative disorder” and “proliferative disorder” refer to disorders related to a certain degree of abnormal cell proliferation. In one embodiment, the proliferative disorder is cancer.

[0069] As used herein, the term “chemotherapeutic agent” refers to a chemical substance useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocone, metsuredopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamamine; and acetogenins (special (including buratacin and buratacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapacon; lapachol; colchicine; betulinic acid; camptothecin (including synthetic analogues topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; calistatin; CC-1065 (so (including synthetic analogs of adzeresin, karzeresin and bizeresin); podophyllotoxin; podophyllic acid; teniposide; cryptophycin (especially cryptophycin 1 and cryptophycin 8); drastatin; duocalmycin (including synthetic analogs KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictin; spongstatin; chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, me Nitrogen mustards such as chloretamine, mechloretamine oxide hydrochloride, melphalan, novembitine, fenestrine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotosine, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as engine antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega I1 (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33:183-186 (1994)); oral α-4 integrin inhibitor CDP323;Dynemycin containing dynemycin A; esperamicin; and neocardinostatin chromophore and related pigment proteins (endiin antibiotic chromophore), acrasinomycin, actinomycin, autoramycin, azaserin, bleomycin, kactinomycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), peglilated liposomal doxorubicin (CAELYX® and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, keramycin, rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin; methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epotilon, antimetabolites such as 5-fluorouracil (5-FU); denopterin, Folic acid analogs such as methotrexate, pteropterin, and trimethrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as floric acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisantren; edatraxate; defofamine; demecolsin;Diadicone; Elformitin; Erliptinium acetate; Epotilon; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Ronidynin; Mytansinoids such as Mytansin and Anthamitosin; Mitoguazone; Mitoxantrone; Mopidammol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Rosoxantrone; 2-Ethylhydrazide; Procarbazine; PSK (Registered Trademark) Polysaccharide Complex (JHS Natural) Products, Eugene, Oreg.); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadicone; 2,2',2'-Trichlorotriethylamine; Trichothecene (especially T-2 Toxin, Beraclin A, Loridine A, Angidin); Urethane; Vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacitosine; Arabinoside ("Ara-C"); Thiotepa; Taxoids, e.g., Paclitaxel (TAXOL®), Albumin-Modified Nanoparticle Formulation of Paclitaxel (ABRAXANE®), and Docetaxel (TAXOTERE®); Chlorambucil; 6-Thiogunine; Mercaptopurine; Meth Platinum agents such as trexate; cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vinca, which prevents microtubule formation by tubulin polymerization, such as vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMF®); and retinoids such as retinoic acid containing bexarotene (TARGRETIN®);Bisphosphonates such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tildronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog); a Inthisense oligonucleotides, in particular those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, such as ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (Sorafenib, Bayer); SU-11248 (Sunitinib, SUTENT®, Pfizer); Perifosin, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); Bortezomib (VELCADE®); CCI-779; Tipifarnib (R11577); Olafenib, ABT510; Bcl-2 inhibitors such as Oblimersen sodium (GENASENSE®); Pixanthrox EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine / threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such as ronafarnib (SCH6636, SARASAR®); and any pharmaceutically acceptable salts, acids, or derivatives of any of the above; two or more combinations of the above, such as CHOP (an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone);Also mentioned is FOLFOX (an abbreviation for a treatment regimen using oxaliplatin (ELOXATIN®) in combination with 5-FU and leucovorin).

[0070] Chemotherapy agents as defined herein include “anti-hormone agents” or “endocrine therapeutic agents” that act to modulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth. These may be hormones themselves, but are not limited to: anti-estrogens with a mixed agonist / antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), doxifen, doroxifen, raloxifene (EVISTA®), trioxyfen, keoxyfen, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®) and EM800 (agents that can block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestan and exemestane (AROMASIN®), as well as anastrazole (ARIMIDEX®) and letrozole (FEMARA®). This includes nonsteroidal aromatase inhibitors such as aminoglutethimide, as well as other aromatase inhibitors including borozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozol, and 4(5)-imidazole; luteinizing hormone-releasing hormone agonists including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and triptorelin; progestins such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, sex steroids including fluoxymesterone, all-trans lethionic acid, and androgens / retinoids such as fenretinide; onapristone; antiprogesterone; estrogen receptor downregulators (ERD); antiandrogens such as flutamide, nilutamide, and bicalutamide; and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[0071] As used herein, the term “chimeric” antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a particular source or species, and the remainder of the heavy chain and / or light chain originates from a different source or species.

[0072] As used herein, the term “conditionally active antibody” refers to an anti-HER2 antibody that is more active under tumor microenvironmental conditions than under non-tumor microenvironmental conditions. Tumor microenvironmental conditions include lower pH, higher concentrations of lactate and pyruvate, hypoxia, lower concentrations of glucose, and slightly higher temperature compared to the non-tumor microenvironment. For example, a conditionally active antibody is virtually inactive at normal body temperature but is active at higher temperatures in the tumor microenvironment. In yet another embodiment, a conditionally active antibody is less active in normal oxygenated blood but more active in the hypoxic environment present in tumors. In yet another embodiment, a conditionally active antibody is less active at normal physiological pH 7.2–7.8 but more active under the acidic pH 5.0–7.0 present in the tumor microenvironment. Other conditions known to those skilled in the art exist in the tumor microenvironment and can be used as conditions in this invention, under which anti-HER2 antibodies have different binding affinities to the HER2 protein.

[0073] As used herein, the term “cell division inhibitor” refers to a compound or composition that stops cell proliferation either in vitro or in vivo. Therefore, cell division inhibitors may significantly reduce the proportion of cells in the S phase. Further examples of cell division inhibitors include agents that block cell cycle progression by inducing G0 / G1 arrest or M phase arrest. Trastuzumab (HERCEPTIN®), a humanized anti-HER2 antibody, is an example of a cell division inhibitor that induces G0 / G1 arrest. Classical M phase blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors (such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin). Certain drugs that stop G1, such as tamoxifen, prednisone, dacarbazine, mechloretamine, cisplatin, methotrexate, 5-fluorouracil, and DNA alkylating agents like ara-C, also overflow into S-phase arrest. Further information can be found in Mendelsohn and Israel, eds., *The Molecular Basis of Cancer*, Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WBSaunders, Philadelphia, 1995), e.g., p. 13. Taxanes (paclitaxel and docetaxel) are both anticancer drugs derived from the yew tree. Docetaxel derived from European yew (TAXOTERE®, Rhone-Poulenc Rorer) is a semi-synthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel stabilize microtubules by promoting microtubule construction from tubulin dimers and inhibiting depolymerization, leading to inhibition of intracellular mitosis.

[0074] As used herein, the term “cytotoxic agent” refers to a substance that inhibits or blocks cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At). 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Pb 212 Examples of antitumor agents or anticancer agents include radioactive isotopes of Lu, chemotrexate, and Lu, chemotherapeutic agents or chemotherapeutic drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents), growth inhibitors, enzymes such as nucleases and their fragments, antibiotics, toxins such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, as well as various antitumor agents or anticancer agents disclosed below.

[0075] As used herein, the term “diabody” refers to a small antibody fragment having two antigen-binding sites, which are the same polypeptide chain (V H -V L ) Light chain variable domain (V L ) bound to the heavy chain variable domain (V H This includes the use of linkers that are too short to allow pairing between two domains on the same chain, forcing the domains to pair with complementary domains on another chain to generate two antigen-binding sites.

[0076] As used herein, the term “detectably labeled” refers to any substance whose detection or measurement, either directly or indirectly, by physical or chemical means, indicates the presence of an antigen in a sample. Typical examples of useful detectable labels include, but are not limited to,: molecules or ions that are directly or indirectly detectable based on their light absorption, fluorescence, reflectance, light scattering, phosphorescence, or luminescence properties; molecules or ions that are detectable by their radioactive properties; and molecules or ions that are detectable by their nuclear magnetic resonance or paramagnetic properties. For example, among molecules that are indirectly detectable based on light absorption or fluorescence are various enzymes that convert suitable substrates, for example, from non-light-absorbing molecules to light-absorbing molecules, or from non-fluorescent molecules to fluorescent molecules.

[0077] As used herein, the term “diagnosis” refers to determining susceptibility to a disease or disorder in a subject, determining whether a subject is currently suffering from a disease or disorder, determining the prognosis of a subject suffering from a disease or disorder (e.g., identifying a pre-metastatic or metastatic cancer state, the stage of cancer, or the response of cancer to treatment), and determining treatment (e.g., monitoring the subject’s condition to provide information regarding the effectiveness or efficacy of treatment). In some embodiments, the diagnostic methods of the present invention are particularly useful in detecting early-stage cancer.

[0078] As used herein, the term “diagnostic agent” refers to a molecule that can be detected directly or indirectly and used for diagnostic purposes. Diagnostic agents may be administered to a subject or sample. Diagnostic agents may be provided on their own or conjugated to a vehicle such as a conditionally active antibody.

[0079] As used herein, the term “effector function” refers to the biological activity resulting from the Fc region of an antibody, which differs depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0080] As used herein, the term “effective dose” for a drug, for example, a pharmaceutical formulation, refers to an effective amount in terms of the dosage and duration required to achieve the desired therapeutic or prophylactic outcome.

[0081] As used herein, the term “Fc region” is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes the Fc region of the natural sequence and the Fc region of variants. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the lysine (Lys447) at the C-terminus of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system (also known as the EU index), as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0082] As used herein, the term "framework" or "FR" refers to residues in the variable domain other than those in the complementarity-determining region (CDR or H1-3 in the heavy chain, and L1-3 in the light chain). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR sequence and FR sequence are V H (or V LIn general, they appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0083] The terms "full-length antibody," "intact antibody," or "whole antibody" refer to the antigen-binding variable region (V). H or V L This refers to antibodies that include a light chain constant domain (CL) and heavy chain constant domains (CH1, CH2, and CH3). The constant domains may be the constant domains of the natural sequence (e.g., the human natural sequence constant domain) or amino acid sequence variants thereof. Depending on the amino acid sequence of their heavy chain constant domains, full-length antibodies can be assigned to different "classes". There are five main classes of full-length antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0084] As used herein, the term “function-conserving variant” refers to a given amino acid residue in a protein or enzyme that is altered without altering the overall conformation and function of the polypeptide, and includes, but is not limited to, amino acid substitutions with amino acids having similar properties (e.g., polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity, aromaticity, etc.). Amino acids other than those indicated as conserved may differ in the protein, and as a result, the percentage sequence similarity of the protein or amino acids between any two functionally similar proteins may vary, for example, from 70% to 99% when the similarity is determined according to an alignment scheme such as the clustering method based on the MEGALIGN algorithm. A “function-conserving variant” also includes a polypeptide having at least 60% amino acid identity, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95%, when determined by the BLAST or FASTA algorithm, and having the same or substantially similar properties or functions as the native protein or parent protein being compared.

[0085] As used herein, the terms “host cell,” “host cell line,” and “host cell culture” are interchangeable and refer to cells into which exogenous nucleic acids have been introduced (including the offspring of such cells). Host cells include “transformed organisms” and “transformed cells,” and include primary transformed cells and their offspring, regardless of the number of passages. Offspring may not have nucleic acid content that is exactly identical to that of the parent cells and may contain mutations. Mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included herein.

[0086] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an amino acid sequence derived from a non-human source that utilizes the repertoire of human antibodies or the coding sequence of another human antibody. This definition of human antibody explicitly excludes humanized antibodies that contain non-human antigen-binding residues.

[0087] As used herein, the term “humanized” antibody refers to a chimeric antibody containing amino acid residues derived from a non-human CDR and amino acid residues derived from a human FR. In certain embodiments, the humanized antibody contains substantially all of at least one, typically two, variable domains, with all or substantially all of the CDRs corresponding to those of a non-human antibody and all or substantially all of the FRs corresponding to those of a human antibody. The humanized antibody may optionally contain at least a portion of the constant region of an antibody derived from a human antibody. The “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0088] As used herein, the term “immunoconjugate” refers to an antibody conjugated to one or more heterologous molecules, including, but not limited to, cytotoxic agents.

[0089] As used herein, the terms “individual” or “subject” refer to mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans, and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0090] As used herein, the term “inhibition of cell growth or proliferation” means reducing cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death.

[0091] As used herein, the term “isolated” antibody refers to an antibody isolated from its natural environment. In some embodiments, antibodies are purified to a purity of over 95% or over 99%, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion-exchange or reverse-phase high-performance liquid chromatography (HPLC)). For an overview of antibody purity assessment methods, see, for example, Flatman et al., J. Chromatogr. B, vol. 848, pp. 79-87, 2007.

[0092] As used herein, the term “isolated nucleic acid encoding an anti-HER2 antibody” refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of an antibody, including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations within a host cell.

[0093] As used herein, the term “metastasis” refers to all HER2-involved processes that support cancer cells dispersing from a primary tumor, infiltrating lymphatic vessels and / or blood vessels, circulating through the bloodstream, and proliferating in distal lesions (metastases) in normal tissues elsewhere in the body. In particular, it refers to cellular events of tumor cells that underlie metastasis and are stimulated or mediated by the HER2 protein, such as proliferation, migration, anchorage independence, evasion of apoptosis, or secretion of angiogenic factors.

[0094] As used herein, the term “microenvironment” means any part or region of tissue or body that has immutable or transient, physical or chemical differences from other areas of tissue or body. In the case of tumors, as used herein, the term “tumor microenvironment” refers to the environment in which the tumor resides, including non-cellular areas within the tumor and areas immediately outside the tumor tissue but not related to the intracellular compartments of the cancer cells themselves. Tumors and the tumor microenvironment are closely related and constantly interacting. Tumors can alter their microenvironment, and the microenvironment can influence tumor growth and spread. Typically, the tumor microenvironment has a low pH, ranging from 5.0 to 7.0, or from 5.0 to 6.8, or from 5.8 to 6.8, or from 6.2 to 6.8. On the other hand, normal physiological pH is in the range of 7.2 to 7.8 for most tissues. The tumor microenvironment is also known to have lower concentrations of glucose and other nutrients, but higher concentrations of lactate, compared to plasma. Furthermore, the tumor microenvironment may have a temperature 0.3 to 1°C higher than normal physiological temperature. The tumor microenvironment is discussed in Gillies et al., “MRI of the Tumor Microenvironment,” Journal of Magnetic Resonance Imaging, vol.16, pp.430-450, 2002, which is incorporated herein by reference in its entirety. The term “non-tumor microenvironment” refers to the microenvironment in areas other than tumors.

[0095] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies in that population are identical and / or bind to the same epitope, except for variant antibodies that contain, for example, naturally occurring mutations or that may arise during the production of a monoclonal antibody preparation, and such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies specific to different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is specific to a single determinant on an antigen. Therefore, the modifier “monoclonal” should not be interpreted as indicating an antibody characteristic such as that obtained from a substantially homogeneous population of antibodies, and that it requires the production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be produced by a variety of techniques, including, but are not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for producing monoclonal antibodies are described herein.

[0096] As used herein, the term "naked antibody" refers to an antibody that is not conjugated with a heterogeneous moiety (e.g., a cytotoxic moiety) or radiolabeling. Naked antibodies may be present in pharmaceutical formulations.

[0097] As used herein, the term “packaging instructions” refers to the instructions that are customarily included in the market packaging of a therapeutic product and include information regarding indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings relating to the use of such therapeutic product.

[0098] With respect to the reference polypeptide sequence used herein, the term “percent (%) amino acid sequence identity” is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the reference polypeptide sequence, after the sequences have been aligned, gaps introduced where necessary to achieve maximum percent sequence identity, and no conservative substitutions have been considered as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the scope of the art of this field using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes of this specification, the percentage value of amino acid sequence identity is generated using the ALIGN-2 sequence comparison computer program. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office (Washington DC, 20559) and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or can be compiled from source code. The ALIGN-2 program needs to be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0099] In situations where ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity percentage of a given amino acid sequence B to or with respect to a given amino acid sequence A (or, alternatively, a given amino acid sequence A that has or contains a specific amino acid sequence identity percentage to or with respect to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y In the formula, X is the number of amino acid residues scored as identical in the alignment of A and B by the sequence alignment program ALIGN-2, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding section.

[0100] As used herein, the term “pharmaceutical preparation” refers to a preparation that is in a form that enables the biological activity of the active ingredient contained herein, and that does not contain any additional toxic ingredients that would be unacceptable to the subject to which the preparation is administered.

[0101] As used herein, the term “pharmaceutically acceptable carrier” refers to a component in a pharmaceutical formulation other than the active ingredient that is non-toxic to the target. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0102] As used herein, the terms “purified” and “isolated” refer to antibodies or nucleotide sequences according to the present invention, and mean that the indicated molecules are present in the substantial absence of other biomacromolecules of the same kind. As used herein, the term “purified” means preferably that at least 75% by weight, more preferably at least 85% by weight, even more preferably 95% by weight, and most preferably at least 98% by weight of biomacromolecules of the same kind are present. An “isolated” nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that substantially does not contain other nucleic acid molecules that do not encode the polypeptide, although the molecule may contain several additional bases or parts that do not adversely affect the basic characteristics of the composition.

[0103] As used herein, the term “recombinant antibody” refers to an antibody expressed by a recombinant host cell containing a nucleic acid encoding the antibody (e.g., a chimeric antibody, a humanized antibody, or a human antibody, or its antigen-binding fragment). Examples of “host cells” for producing recombinant antibodies include: (1) mammalian cells, e.g., Chinese hamster ovary (CHO), COS, myeloma cells (including Y0 and NS0 cells), baby hamster kidney (BHK), Hela cells, and Vero cells; (2) insect cells, e.g., sf9, sf21, and Tn5; (3) plant cells, e.g., plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, e.g., those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or Aspergillus (e.g., Aspergillus niger); and (5) bacterial cells, e.g., Escherichia coli cells or Bacillus subtilis cells.

[0104] As used herein, the term “single-stranded Fv” (“scFv”) refers to covalently bonded V H ::V L It is a heterodimer, which typically encodes a gene linked by a peptide-encoded linker, V H and V LIt is expressed from a gene fusion containing [the specified gene]. "dsFv" is V stabilized by a disulfide bond. H ::V L It is a heterodimer. Divalent and polyvalent antibody fragments can be spontaneously formed by the association of monovalent scFv or generated by ligating monovalent scFv with a peptide linker (e.g., divalent sc(Fv)2).

[0105] The term "therapeutic dose" of the antibody in this invention means an amount of antibody sufficient to treat the cancer in question with a reasonable benefit-risk ratio applicable to any medical treatment. However, it will be understood that the total daily dose of the antibody and composition of this invention is to be determined by the attending physician within the bounds of sound medical judgment. The level of a specific therapeutic dose for any particular patient will depend on a variety of factors, including the disorder being treated and its severity, the activity of the specific antibody used, the specific composition used, the patient's age, weight, overall health, sex, and diet, the timing of administration, route of administration, and excretion rate of the specific antibody used, the duration of treatment, drugs used in combination with or concurrently with the specific antibody used, and similar factors known in the medical field. For example, it is known in the art to start administration of a compound at a level lower than the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0106] As used herein, the terms “treatment,” “to treat,” or “to treat” refer to a clinical intervention in an attempt to alter the natural course of an individual being treated, which may be carried out either for prevention or in the course of clinicopathology. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, reduction of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, remission or mitigation of the disease state, and improvement of remission or prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or to slow the progression of disease.

[0107] As used herein, the term “tumor” refers to all tumor cell proliferation and growth, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive as used herein.

[0108] As used herein, the terms “variable region” or “variable domain” refer to domains in the heavy or light chain of an antibody that are involved in the binding of the antibody to an antigen. (V) H and V L The domains generally have similar structures, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). H or V L The domain may be sufficient to confer antigen-binding specificity. Furthermore, the V from the antibody that binds to the antigen... H or V L Using the domain, antibodies that bind to specific antigens are isolated, and each is a complementary V L or V H You can screen domain libraries. For example, see Portolano et al., J.Immunol., vol.150, pp.880-887, 1993 and Clarkson et al., Nature, vol.352, pp.624-628, 1991.

[0109] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. This term includes vectors as self-replicating nucleic acid structures, as well as vectors integrated into the genome of a host cell into which they are introduced. Certain vectors can induce the expression of nucleic acids to which they are operably ligated. Such vectors are referred to herein as “expression vectors.” [Modes for carrying out the invention]

[0110] For illustrative purposes, the principles of the present invention are described by reference to various exemplary embodiments. While certain embodiments of the present invention are specifically described herein, those skilled in the art will readily understand that the same principles are equally applicable to and can be used in other systems and methods. Before describing in detail the embodiments disclosed herein, it should be understood that the present invention is not limited in its application to any specific embodiment shown. In addition, the terminology used herein is for illustrative purposes only, not limiting purposes. Furthermore, while certain methods are described by reference to steps presented herein in a particular order, in many cases these steps can be performed in any order as can be understood by those skilled in the art, and therefore novel methods are not limited to a particular arrangement of the steps disclosed herein.

[0111] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context clearly indicates otherwise. Furthermore, the terms “a” (or “an”), “one or more,” and “at least one” may be used interchangeably herein. The terms “comprising,” “including,” “having,” and “constructed from” may also be used interchangeably.

[0112] Unless otherwise indicated, all numbers used herein and in the claims, such as quantities, molecular weights, percentages, ratios, and reaction conditions, should be understood to be modified by the term "approximately" in all cases, regardless of whether the term "approximately" is present or not. Therefore, unless otherwise indicated, the numerical parameters described herein and in the claims are approximations that may vary depending on the desired properties to be obtained by this disclosure. Each numerical parameter should be interpreted, at least in light of the reported number of significant figures and by applying common rounding techniques, not as an attempt to limit the application of the doctrine of equivalents to the claims. Although the numerical ranges and parameters described in the broad scope of this disclosure are approximations, the numbers shown in specific examples are reported as accurately as possible. However, each number inherently contains certain errors that inevitably result from the standard deviation found in the respective test measurements.

[0113] It should be understood that each component, compound, substituent, or parameter disclosed herein is disclosed for use alone or in combination with one or more other components, compounds, substituents, or parameters disclosed herein.

[0114] Furthermore, each quantity / value or range of each component, compound, substituent, or parameter disclosed herein should be interpreted as being disclosed in combination with any other quantity / value or range of each component, compound, substituent, or parameter disclosed herein. Therefore, for the purposes of this description, any combination of two or more quantities / values ​​or ranges of each component, compound, substituent, or parameter disclosed herein should also be understood as being disclosed in combination with each other.

[0115] It is further understood that each lower limit of each range disclosed herein should be interpreted as being disclosed in combination with each upper limit of each range disclosed herein for the same component, compound, substituent, or parameter. Thus, a disclosure of two ranges should be interpreted as a disclosure of four ranges derived by combining each lower limit of each range with each upper limit of each range. A disclosure of three ranges should be interpreted as a disclosure of nine ranges derived by combining each lower limit of each range with each upper limit of each range, and so on. Furthermore, any specific amount / value of a component, compound, substituent, or parameter disclosed in a description or example should be interpreted as a disclosure of either a lower or upper limit of a range, and thus can be combined with any other lower or upper limit or specific amount / value of the same component, compound, substituent, or parameter disclosed elsewhere in this application to form a range for that component, compound, substituent, or parameter.

[0116] A. Isolated polypeptide In one embodiment, the present invention provides an isolated polypeptide that specifically binds to the HER2 protein, wherein the polypeptide comprises a heavy chain variable region having three complementarity-determining regions, the regions having sequences H1, H2, and H3. The H1 sequence is GFX1IKDTYIH (sequence number 1), The H2 sequence is X2IX3PTX4X5YX6X7YADSVKG (sequence number 2), The H3 sequence is WGGDGFYX8MDY (sequence number 3), In the formula, X1 is N or W, X2 is R or K, X3 is Y, K or D, X4 is N or A, X5 is G or K, X6 is T or D, X7 is R or E, and X8 is A or E, representing a heavy-chain variable region. A light chain variable region comprising three complementarity-determining regions having sequences L1, L2, and L3, The L1 sequence is RASQDVNTX9VA (sequence number 4), The L2 sequence is SASFLYS (sequence number 5), The L3 array is QQX 10 YTTPPT (sequence number 6) In the formula, X9 is A or D, and X 10 However, it includes a light chain variable region which is H, D, or E, As a premise, if X1 to X8 are N, R, Y, N, G, T, R, and A respectively, then X9 is not A, but X 10 But it's not H.

[0117] In a particular aspect of this embodiment, the H1 sequence may be GFWIKDTYIH (sequence number 7) or GFNIKDTYIH (sequence number 50), the H2 sequence may be any one of KIYPTNGYTRYADSVKG (sequence number 8), RIKPTNGYTRYADSVKG (sequence number 9), RIDPTNGYTRYADSVKG (sequence number 10), RIYPTAGYTRYADSVKG (sequence number 11), RIYPTNKYTRYADSVKG (sequence number 12), RIYPTNGYDRYADSVKG (sequence number 13), RIYPTNGYTEYADSVKG (sequence number 14), and RIYPTNGYTRYADSVKG (sequence number 49), and the H3 sequence may be WGGDGFYEMDY (sequence number 15) or WGGDGFYAMDY (sequence number 51). The L1 sequence may be RASQDVNTDVA (sequence number 16) or RASQDVNTAVA (sequence number 52). The L2 sequence is SASFLYS (sequence number 5). The L3 sequence may be QQDYTTPPT (sequence number 17), QQEYTTPPT (sequence number 18), or QQHYTTPPT (sequence number 53).

[0118] In certain embodiments of the present invention, the anti-HER2 isolated polypeptide may be selected from any of the following anti-HER2 isolated polypeptides, each comprising a specific combination of the six CDRs H1, H2, H3, L1, L2, and L3 shown below. [Table 1] JPEG0007856312000002.jpg232170JPEG0007856312000003.jpg218170JPEG0007856312000004.jpg22617 0JPEG0007856312000005.jpg215170JPEG0007856312000006.jpg221170JPEG0007856312000007.jpg37170

[0119] A preferred isolated polypeptide can be selected from isolated polypeptides, including specific combinations of each of the six CDRs shown below. [Table 2]

[0120] In another aspect, the disclosure provides an isolated polypeptide that specifically binds to the HER2 protein, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs. 19-28, and the light chain variable region has an amino acid sequence selected from SEQ ID NOs. 29-32.

[0121] Another embodiment provides an isolated polypeptide that specifically binds to the HER2 protein, the polypeptide comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs. 33 and 19-28, and the light chain variable region has an amino acid sequence selected from SEQ ID NOs. 30-32.

[0122] In yet another aspect, the disclosure provides an isolated polypeptide that specifically binds to the HER2 protein, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs: 34-39, and the light chain variable region has an amino acid sequence selected from SEQ ID NOs: 40-48.

[0123] In certain embodiments, the Disclosure provides an isolated polypeptide that specifically binds to the HER2 protein, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 35, and the light chain variable region has an amino acid sequence selected from SEQ ID NOs: 41-48.

[0124] In one embodiment, the disclosure provides an isolated polypeptide that specifically binds to the HER2 protein, the polypeptide comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having the amino acid sequence of SEQ ID NO: 36, and the light chain variable region having an amino acid sequence selected from SEQ ID NOs: 41-48.

[0125] In one embodiment, the disclosure provides an isolated polypeptide that specifically binds to the HER2 protein, the polypeptide comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having the amino acid sequence of SEQ ID NO: 37, and the light chain variable region having an amino acid sequence selected from SEQ ID NOs: 41-48.

[0126] In another embodiment, the Disclosure provides an isolated polypeptide that specifically binds to the HER2 protein, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 38, and the light chain variable region has an amino acid sequence selected from SEQ ID NOs: 41-48.

[0127] In another aspect, the disclosure provides an isolated polypeptide that specifically binds to the HER2 protein, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 39, and the light chain variable region has an amino acid sequence selected from SEQ ID NOs: 41-48.

[0128] In certain embodiments of the present invention, the anti-HER2 isolated polypeptide may be selected from any of the following anti-HER2 isolated polypeptides, each comprising a combination of heavy chain variable regions and light chain variable regions as shown below. [Table 3] JPEG0007856312000010.jpg225170JPEG0007856312000011.jpg228170JPEG0007856312000012.jpg221170 JPEG0007856312000013.jpg226170JPEG0007856312000014.jpg226170JPEG0007856312000015.jpg162170 [Table 4]

[0129] B. Anti-HER2 antibody In another embodiment, the present invention relates to an anti-HER2 antibody or antibody fragment comprising the isolated polypeptide described above.

[0130] Antibodies or antibody fragments may have a higher binding affinity to the HER2 protein at the pH of the tumor microenvironment compared to the pH in the non-tumor microenvironment. The pH in the tumor microenvironment may be in the range of 5.0 to 7.0, while the pH in the non-tumor microenvironment may be in the range of 7.2 to 7.8.

[0131] In another embodiment, the present invention relates to an antibody or antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity-determining regions, the regions having sequences H1, H2, and H3, The H1 sequence is GFX1IKDTYIH (sequence number 1), The H2 sequence is X2IX3PTX4X5YX6X7YADSVKG (sequence number 2), The H3 sequence is WGGDGFYX8MDY (sequence number 3), In the formula, X1 is N or W, X2 is R or K, X3 is Y, K or D, X4 is N or A, X5 is G or K, X6 is T or D, X7 is R or E, and X8 is A or E. The light chain variable region includes three complementarity-determining regions having sequences L1, L2, and L3. The L1 sequence is RASQDVNTX9VA (sequence number 4), The L2 sequence is SASFLYS (sequence number 5), The L3 array is QQX 10 YTTPPT (sequence number 6) In the formula, X9 is A or D, and X 10 However, it is either H, D, or E. As a premise, if X1 to X8 are N, R, Y, N, G, T, R, and A respectively, then X9 is not A and X10 is not H.

[0132] In a particular aspect of this embodiment, the H1 sequence may be GFWIKDTYIH (sequence number 7) or GFNIKDTYIH (sequence number 50), the H2 sequence may be KIYPTNGYTRYADSVKG (sequence number 8), RIKPTNGYTRYADSVKG (sequence number 9), RIDPTNGYTRYADSVKG (sequence number 10), RIYPTAGYTRYADSVKG (sequence number 11), RIYPTNKYTRYADSVKG (sequence number 12), RIYPTNGYDRYADSVKG (sequence number 13), RIYPTNGYTEYADSVKG (sequence number 14), or RIYPTNGYTRYADSVKG (sequence number 49), and the H3 sequence may be WGGDGFYEMDY (sequence number 15) or WGGDGFYAMDY (sequence number 51).

[0133] Furthermore, in a particular aspect of this embodiment, the L1 sequence may be RASQDVNTDVA (sequence number 16) or RASQDVNTAVA (sequence number 52), the L2 sequence may be SASFLYS (sequence number 5), and the L3 sequence may be QQDYTTPPT (sequence number 17), QQEYTTPPT (sequence number 18), or QQHYTTPPT (sequence number 53).

[0134] In certain embodiments, the anti-HER2 antibody and antibody fragment of the present invention comprises a combination of the six CDRs listed above for an isolated polypeptide. A preferred anti-HER2 antibody and antibody fragment of the present invention comprises a preferred combination of the six CDRs listed above for an isolated polypeptide.

[0135] In certain embodiments, the Disclosure provides an antibody or antibody fragment that specifically binds to the HER2 protein, the antibody or antibody fragment comprising a heavy chain variable region which may be any one of SEQ ID NOs: 19-28 and 33, and a light chain variable region which may be any one of SEQ ID NOs: 29-32.

[0136] In certain embodiments of the anti-HER2 antibody or antibody fragment, the heavy chain variable region may be one of SEQ ID NOs. 33 and 19-28, and the light chain variable region may be one of SEQ ID NOs. 30-32.

[0137] In each of the aforementioned embodiments of this aspect of the antibody or antibody fragment, the heavy chain variable region may be any one of SEQ ID NOs: 35 to 39, and the light chain variable region may be any one of SEQ ID NOs: 41 to 48.

[0138] In one embodiment, the present disclosure provides an antibody or antibody fragment that specifically binds to the HER2 protein, the antibody or antibody fragment comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence selected from SEQ ID NOs. 35 to 39, and the light chain variable region has an amino acid sequence selected from SEQ ID NOs. 41 to 48.

[0139] In certain embodiments, the Disclosure provides an antibody or antibody fragment that specifically binds to the HER2 protein, the antibody or antibody fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having the amino acid sequence of SEQ ID NO: 35, and the light chain variable region having an amino acid sequence selected from SEQ ID NOs: 41-48.

[0140] In certain embodiments, the Disclosure provides an antibody or antibody fragment that specifically binds to the HER2 protein, the antibody or antibody fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having the amino acid sequence of SEQ ID NO: 36, and the light chain variable region having an amino acid sequence selected from SEQ ID NOs: 41-48.

[0141] In certain embodiments, the Disclosure provides an antibody or antibody fragment that specifically binds to the HER2 protein, the antibody or antibody fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having the amino acid sequence of SEQ ID NO: 37, and the light chain variable region having an amino acid sequence selected from SEQ ID NOs: 41-48.

[0142] In another particular embodiment, the Disclosure provides an antibody or antibody fragment that specifically binds to the HER2 protein, the antibody or antibody fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having the amino acid sequence of SEQ ID NO: 38, and the light chain variable region having an amino acid sequence selected from SEQ ID NOs: 41-48.

[0143] In another particular embodiment, the Disclosure provides an antibody or antibody fragment that specifically binds to the HER2 protein, the antibody or antibody fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region having the amino acid sequence of SEQ ID NO: 39, and the light chain variable region having an amino acid sequence selected from SEQ ID NOs: 41-48.

[0144] In certain embodiments, the anti-HER2 antibodies and antibody fragments of the present invention include a combination of heavy chain variable regions and light chain variable regions as shown in the list above for an isolated polypeptide. Preferred anti-HER2 antibodies and antibody fragments of the present invention include a preferred combination of heavy chain variable regions and light chain variable regions as shown in the list above for an isolated polypeptide.

[0145] The antibody or antibody fragment in this embodiment may also have a higher binding affinity to the HER2 protein at the pH in the tumor microenvironment compared to the different pH levels that occur in the non-tumor microenvironment. The pH in the tumor microenvironment may be in the range of 5.0 to 7.0, while the pH in the non-tumor microenvironment may be in the range of 7.2 to 7.8.

[0146] An antibody or antibody fragment of this embodiment may have a ratio of binding affinity to the HER2 protein at pH in the tumor microenvironment to binding affinity to the HER2 protein at different pH in the non-tumor microenvironment of at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

[0147] Figure 1 shows an exemplary alignment of the light chain variable regions of the present invention, with complementarity determination regions L1, L2, and L3 enclosed in frames. Figure 2 shows an exemplary alignment of the heavy chain variable regions of the present invention, with complementarity determination regions H1, H2, and H3 enclosed in frames.

[0148] The heavy-chain variable region and light-chain variable region of the present invention were each obtained from a parent antibody using the method disclosed in U.S. Patent No. 8,709,755. This method for generating the heavy-chain variable region and light-chain variable region, as well as the method for generating the antibody and antibody fragment, is disclosed in U.S. Patent No. 8,709,755, which is incorporated herein by reference.

[0149] The amino acid sequences of the light chain variable region in Figure 1 are shown in SEQ ID NOs. 29-32. The amino acid sequences of the heavy chain variable region in Figure 2 are shown in SEQ ID NOs. 19, 20, and 33.

[0150] In one embodiment, the antibody or antibody fragment comprises a light chain variable region and a heavy chain variable region having any pair of sequences selected from the following: SEQ ID NOs: 30 and 33, SEQ ID NOs: 31 and 33, SEQ ID NOs: 32 and 33, SEQ ID NOs: 29 and 19, SEQ ID NOs: 29 and 20, SEQ ID NOs: 30 and 21, SEQ ID NOs: 30 and 22, SEQ ID NOs: 30 and 23, SEQ ID NOs: 30 and 24, SEQ ID NOs: 30 and 25, SEQ ID NOs: 30 and 26, SEQ ID NOs: 30 and 27, and SEQ ID NOs: 30 and 28.

[0151] Antibodies and antibody fragments containing these heavy chain and light chain variable regions can specifically bind to the HER2 protein, particularly human HER2 protein. Antibodies or antibody fragments containing a combination of one of these heavy chain variable regions and one of these light chain variable regions have been found to have a higher binding affinity to the HER2 protein at the pH of the tumor microenvironment (e.g., pH 5.0–7.0) than at the pH of the non-tumor microenvironment (e.g., pH 7.2–7.8). As a result, anti-HER2 antibodies or antibody fragments have a higher binding affinity to the HER2 protein in the tumor microenvironment compared to their binding affinity to the HER2 protein in a typical normal tissue microenvironment.

[0152] Therefore, the anti-HER2 antibody or antibody fragment of the present invention is expected to exhibit reduced side effects compared to unconditionally active anti-HER2 antibodies, due to reduced binding affinity to the HER2 protein in the normal tissue microenvironment. The anti-HER2 antibody or antibody fragment of the present invention is also expected to have efficacy equivalent to or higher than monoclonal anti-HER2 antibodies known in the art. Several examples of anti-HER2 antibodies that exhibited virtually no side effects and efficacy equivalent to or higher than isotype control antibodies are demonstrated in in vivo studies in the BALB / c mouse model in Example 7 below. This combination of features, due to reduced side effects, allows for the use of higher doses of these anti-HER2 antibodies or antibody fragments, which can provide a more effective therapeutic option.

[0153] In addition to polypeptides and antibodies or antibody fragments having the heavy-chain and light-chain variable regions described, the present invention also includes variants of these polypeptides, antibodies and antibody fragments that can specifically bind to the HER2 protein, particularly the human HER2 protein. In some embodiments, these variants have different H1, H2, H3, L1, L2, or L3 sequences. In other embodiments, portions of the amino acid sequences of the heavy-chain and light-chain variable regions outside the complementarity-determining regions can be mutated according to the substitution, insertion, and deletion principles considered in this application to provide these variants. In further embodiments, the constant regions can be modified to provide these variants. In even further embodiments, two or all of these regions may be modified to provide these variants.

[0154] The processes described herein guide the induction of these variants. Variants of the heavy chain variable region and light chain variable region can be prepared by introducing appropriate modifications to the nucleotide sequences encoding the heavy chain variable region and light chain variable region, or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions of residues within the amino acid sequences of the heavy chain variable region and light chain variable region, and / or substitutions of residues. Any combination of deletions, insertions, and substitutions can be performed to arrive at the antibodies or antibody fragments of the present invention, provided that they possess the desired characteristics, such as antigen binding to the human HER2 protein and conditional activity, based on a change in pH from the tumor microenvironment to a normal tissue environment.

[0155] C. Substitution, insertion, and deletion variants In certain embodiments, antibody or antibody fragment variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include CDRs and framework regions (FRs). Conservative substitutions are shown in Table 1 under the heading "Conservative Substitutions." More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions," and with respect to classes of amino acid side chains, further discussion follows. Amino acid substitutions may be introduced into the antibody or antibody fragment of interest, and the product may be screened for desired activities, such as retention / improvement of antigen binding, conditional activity, and / or decreased immunogenicity. [Table 5]

[0156] Amino acids can be grouped according to their general side-chain characteristics: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu (4) Basicity: His, Lys, Arg (5) Residues that affect chain orientation: Gly, Pro (6) Aromatic: Trp, Tyr, Phe

[0157] Non-conservative substitution involves swapping one member of one of these classes with one of another.

[0158] One type of substitution variant involves substituting residues in one or more complementarity-determining regions of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, the selected resulting variant will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, improved conditional activity or selectivity, reduced immunogenicity) compared to the parent antibody, and / or substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-mature antibodies that can be produced using phage display-based affinity maturation techniques, such as those described herein.

[0159] For example, modifications (e.g., substitutions) may be made in the CDR to improve antibody affinity. Such modifications can be made in the CDR "hotspots," i.e., residues encoded by codons that are frequently mutated during the somatic cell maturation process (see, e.g., Chowdhury, Methods Mol. Biol., vol.207, pp.179-196, 2008), and / or in the SDR(a-CDR), resulting in the obtained variant V H or V L The binding affinity is then tested. Affinity maturation by constructing a secondary library and then re-selecting from it is described, for example, in Hoogenboom et al. in Methods in Molecular Biology, vol.178, pp.1-37, 2001. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-specific mutagenesis). A secondary library is then constructed. The library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity involves a CDR-specific approach in which several CDR residues (e.g., 4-6 residues per trial) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often targeted.

[0160] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such modifications do not substantially reduce the ability of the antibody or antibody fragment to bind to the HER2 antigen. For example, conservative modifications that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be made within a CDR. Such modifications may be in a "hot spot" of the CDR or outside the SDR. Variant V provided above H and V L In certain embodiments of the sequence, each CDR is either unmodified or contains one, two, or three or more amino acid substitutions.

[0161] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is "alanine scanning mutagenesis," described by Cunningham and Wells, Science, vol. 244, pp. 1081-1085, 1989. In this method, target residues or groups of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction between the antibody or antibody fragment and the antigen is affected. Further substitutions may be introduced at amino acid positions that are functionally sensitive to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex identifies contact points between the antibody or antibody fragment and the antigen. Such contact residues and adjacent residues may be targeted as candidates for substitution or excluded. Variants may be screened to determine whether they possess the desired properties.

[0162] Amino acid sequence insertions include amino-terminal and / or carboxyl-terminal fusions (ranging in chain length from 1 residue to polypeptides of 100 or more residues), as well as intrasequence insertions of single or multiple amino acid residues. An example of terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibodies include the fusion of an enzyme (e.g., ADEPT) or polypeptide that increases the serum half-life of the antibody to the N-terminus or C-terminus.

[0163] Modification of the amino acid sequence of antibodies described herein is intended. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. H and V L Only the CDR inside, simply human antibody V H and V L When humanized antibodies are produced by transplanting them into FR tissue, their antigen-binding activity is known to be reduced compared to the antigen-binding activity of the original antibody derived from a non-human animal. This is true not only for CDR but also for FR tissue. H and V L Several amino acid residues are thought to be directly or indirectly related to antigen-binding activity. Therefore, these amino acid residues are considered to be related to the V of human antibodies. H and V L Substitution with different amino acid residues derived from FR would reduce binding activity. To solve this problem, in antibodies transplanted with human CDR, the V of human antibodies H and V L Within the amino acid sequence of the FR, it is necessary to attempt to identify amino acid residues that are directly related to antibody binding, amino acid residues that interact with amino acid residues of the CDR, or amino acid residues that maintain the three-dimensional structure of the antibody and are directly related to antigen binding. The reduced antigen-binding activity can be increased by substituting the identified amino acids with amino acid residues from the original antibody derived from a non-human animal.

[0164] Modifications and alterations are made to the structure and encoding DNA sequence of the antibody of the present invention, and a functional molecule encoding an antibody that still possesses the desired characteristics can be obtained.

[0165] When modifying amino acid sequences, the hydrophilicity of amino acids may be considered. The importance of hydrophilic amino acid indicators in conferring the biological function of interactions to proteins is generally understood in the art. It is accepted that the relative hydrophilicity of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction between the protein and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, and antigens. Each amino acid is assigned a hydrophilic index based on its hydrophobic and charge characteristics, and these are isoleucine (+4.5), valine (+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine / cystine (+2.5), methionine (+1.9), alanine (+1.8), glycine (-0.4), threonine (-0.7), serine (-0.8), tryptophan (-0.9), tyrosine (-1.3), proline (-1.6), histidine (-3.2), glutamic acid (-3.5), glutamine (-3.5), aspartic acid (-3.5), asparagine (-3.5), lysine (-3.9), and arginine (-4.5).

[0166] The present invention also includes functionally conserved variants of the antibodies and antibody fragments of the present invention.

[0167] Two amino acid sequences are "substantially homologous" or "substantially similar" if more than 80%, or more than 85%, preferably more than 90%, more preferably more than 95%, or more than 98% of their amino acids are identical. In some embodiments, at least 90% or more than 95% of the amino acids are similar (functionally identical) throughout the entire length of the sequences. Preferably, similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pile-up program or a sequence comparison algorithm such as BLAST or FASTA.

[0168] For example, certain amino acids can be substituted in a protein structure with other amino acids without any expected significant loss of activity (see, for example, Table 1 above). Since the ability and properties of protein interactions define the biological functional activity of a protein, specific amino acid substitutions can be made within the protein sequence, and naturally within the DNA-encoded sequence, and yet proteins with similar properties can be obtained. Therefore, it is intended that various modifications can be made to the sequences of antibodies or antibody fragments of the present invention, or the corresponding DNA sequences encoding such antibodies or antibody fragments, without significantly impairing their biological activity.

[0169] In the field of the art, it is known that certain amino acids may be substituted with other amino acids having similar hydrophilicity indices or scores, and that this still results in proteins with similar biological activity, i.e., proteins that are still equivalent in biological function.

[0170] As outlined above, amino acid substitutions can be based on the relative similarities of the substituents on the amino acid side chains, such as their hydrophobicity, hydrophilicity, charge, and size. Exemplary substitutions considering the various characteristics mentioned above are well known to those skilled in the art and include substitutions using the following pairs: arginine and lysine, glutamate and aspartate, serine and threonine, glutamine and asparagine, as well as valine, leucine, and isoleucine.

[0171] D. Glycosylated variant In certain embodiments, the anti-HER2 antibody or antibody fragment provided herein is modified to increase or decrease the degree to which the antibody or antibody fragment is glycosylated. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by modifying the amino acid sequence so that one or more glycosylation sites are generated or removed.

[0172] If an antibody contains an Fc region, the carbohydrate bound to it can be modified. Natural antibodies produced by mammalian cells typically contain branched or bibranched oligosaccharides, generally bound to Asn297 of the CH2 domain of the Fc region by an N-bond. See, for example, Wright et al. TIBTECH, vol.15, pp.26-32, 1997. Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to GlcNAc in the "stem" of the bibranched oligosaccharide structure. In some embodiments, modification of the oligosaccharide in the antibody of the present invention can be performed to produce antibody variants with specific improved properties.

[0173] In one embodiment, an antibody variant is provided having a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans at Asn297 for the sum of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, as measured by MALDI-TOF mass spectrometry, as described in WO2008 / 077546, for example. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of the Fc region residue), although Asn297 may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., at positions 294 to 300, due to slight sequence variations in the antibody. Such fucosylated variants may have improved ADCC function. For example, see U.S. Patent Publication No. US2003 / 0157108 (Presta, L.) and U.S. Patent Publication No. US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include U.S.2003 / 0157108, WO2000 / 61739, WO2001 / 29246, U.S.2003 / 0115614, U.S.2002 / 0164328, U.S.2004 / 0093621, and U.S.2004 / 01321. References include 40, US2004 / 0110704, US2004 / 0110282, US2004 / 0109865, WO2003 / 085119, WO2003 / 084570, WO2005 / 035586, WO2005 / 035778, WO2005 / 053742, WO2002 / 031140, Okazaki et al. J.Mol.Biol., vol.336, pp.1239-1249, 2004, and Yamane-Ohnuki et al. Biotech.Bioeng., vol.87, pp.614-622, 2004.Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys., vol.249, pp.533-545, 1986, U.S. Patent Publication No. US2003 / 0157108A, and WO2004 / 056312A1 (especially Example 11)), as well as knockout cell lines such as α-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng., vol.87, pp.614-622, 2004; Kanda, Y. et al. Biotechnol. Bioeng., vol.94, pp.680-688, 2006, and WO2003 / 085107).

[0174] Antibody variants containing branched oligosaccharides are also provided, for example, in which the branched oligosaccharide bound to the Fc region of the antibody is bifurcated by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, WO2003 / 011878, U.S. Patent No. 6,602,684, and U.S.2005 / 0123546. Also provided are antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, WO1997 / 30087, WO1998 / 58964, and WO1999 / 22764.

[0175] E.Fc region variant In certain embodiments, an Fc region variant can be generated by introducing one or more amino acid modifications into the Fc region of an anti-HER2 antibody or antibody fragment provided herein. The Fc region variant may include a human Fc region sequence (e.g., the Fc region of human IgG1, IgG2, IgG3, or IgG4) containing amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0176] In certain embodiments, the present invention envisions antibody variants possessing some, but not all, effector functions, which are desirable candidates for applications where the half-life of the antibody in vivo is important, but specific effector functions (such as ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxic assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that the antibody lacks FcγR binding (and therefore is likely to lack ADCC activity) but retains FcRn binding ability. NK cells, which are primary cells that mediate ADCC, express only FcγRIII, while mononuclear cells express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol., vol. 9, pp. 457-492, 1991. Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent No. 5,500,362 (see also Hellstrom et al., Proc. Nat'l Acad. Sci. USA, vol. 83, pp. 7059-7063, 1986) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA, vol. 82, pp. 1499-1502, 1985, and U.S. Patent No. 5,821,337 (see also Bruggemann et al., J. Exp. Med., vol. 166, pp. 1351-1361, 1987). Alternatively, non-radioactive assays can be used (see, for example, the ACTI® non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, Calif.) and the CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells.Alternatively or additionally, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, such as those disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA, vol. 95, pp. 652-656, 1998. A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To evaluate complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J.Immunol.Methods, vol.202, pp.163-171, 1996; Cragg, MS et al., Blood, vol.101, pp.1045-1052, 2003; and Cragg, MS, and MJ Glennie, Blood, vol.103, pp.2738-2743, 2004). Furthermore, FcRn binding and in vivo clearance / half-life determination can be performed using methods known in the art (see, e.g., Petkova, S B et al., Int'l.Immunol., vol.18, pp.1759-1769, 2006).

[0177] Examples of antibody or antibody fragment variants having reduced effector function include those having one or more substitutions of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant (U.S. Patent No. 7,332,581), which has alanine substitutions at residues 265 and 297, and Fc variants having substitutions at two or more amino acid positions 265, 269, 270, 297, and 327.

[0178] Specific antibody variants with improved or reduced binding to FcR have been described (see, for example, U.S. Patent No. 6,737,056, WO2004 / 056312, and Shields et al., J. Biol. Chem., vol.9, pp.6591-6604, 2001).

[0179] In certain embodiments, the antibody variant includes an Fc region having one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 of the Fc region (residue EU numbering).

[0180] In some embodiments, modifications are made to the Fc region, resulting in modified (i.e., improved or reduced) C1q binding and / or complement-dependent cell injury (CDC). See, for example, U.S. Patent No. 6,194,551, WO99 / 51642, and Idusogie et al. J.Immunol., vol.164, pp.4178-4184, 2000.

[0181] Antibodies with improved binding to the neonatal Fc receptor (FcRn) (Guyer et al., J. Immunol., vol. 117, pp. 587-593, 1976 and Kim et al., J. Immunol., vol. 24, p. 249, 1994) involved in the transfer of maternal IgG to the fetus and increased half-life are described in US2005 / 0014934. These antibodies contain an Fc region having one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include variants having substitutions at one or more of the residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 of the Fc region (e.g., substitution of residue 434 of the Fc region, U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan & Winter, Nature, vol. 322, pp. 738-740, 1988, U.S. Patent Nos. 5,648,260, 5,624,821, and WO94 / 29351.

[0182] F. Cysteine-Modified Antibody Variants In certain embodiments, it may be desirable to generate cysteine-modified antibodies, e.g., “thioMAbs,” in which one or more residues of an anti-HER2 antibody or antibody fragment are replaced with cysteine residues. In certain embodiments, the residue substitutions occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are positioned at accessible sites of the antibody, and the antibody can be conjugated to other moieties such as drug moieties or linker-drug moieties, as further described herein, to generate an immunoconjugate. In certain embodiments, any one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) of the light chain, A118 (EU numbering) of the heavy chain, and 5400 (EU numbering) of the heavy chain Fc region. Cysteine-modified antibodies can be generated, for example, as described in U.S. Patent No. 7,521,541.

[0183] G. Antibody derivatives In certain embodiments, the anti-HER2 antibodies or antibody fragments provided herein may be further modified to include additional non-proteinogenic moieties known and readily available in the art. Suitable moieties for derivatization of antibodies or antibody fragments include, but are not limited to, water-soluble polymers. Non-limited examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), as well as dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolipropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers bound to the antibody or antibody fragment may vary, and if two or more polymers are bound, they may be the same or different molecules. In general, the number and / or types of polymers used in derivatization can be determined, but are not limited, based on considerations including the specific properties or functions of the antibody or antibody fragment to be improved, and whether the derivative will be used therapeutically under given conditions.

[0184] In another embodiment, a conjugate of an antibody or antibody fragment that can be selectively heated by exposure to radiation and a non-proteinaceous moiety is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA, vol. 102, pp. 11600-11605, 2005). The radiation can be of any wavelength, including wavelengths that do not damage normal cells but heat the non-proteinaceous moiety to a temperature at which cells in proximity to the antibody-non-proteinaceous moiety die.

[0185] The anti-HER2 antibody or antibody fragment of the present invention, or variants thereof, has a higher binding affinity for the HER2 protein under conditions in the tumor microenvironment than under conditions in the non-tumor microenvironment. The conditions in the tumor microenvironment and the non-tumor microenvironment are both pH. Thus, the anti-HER2 antibody or antibody fragment of the present invention can selectively bind to the HER2 protein at a pH of about 5.0 to 7.0 or 5.0 to 6.8, but has a lower binding affinity for the HER2 protein at a pH of about 7.2 to 7.8 encountered in the normal non-tumor microenvironment. As shown in Examples 3 to 6, the anti-HER2 antibody or antibody fragment has a higher binding affinity for the HER2 protein at pH 6.0 than at pH 7.4.

[0186] In certain embodiments, the anti-HER2 antibody or antibody fragment of the present invention is, under conditions in the tumor microenvironment, about 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, or 10 -8 M to 10 -13 M, or 10 -9 M to 10 -13It has a dissociation constant (Kd) with the HER2 protein M). In one embodiment, the ratio of the Kd of the antibody or antibody fragment with the HER2 protein under tumor microenvironment conditions to the Kd under the same conditions in the non-tumor microenvironment is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

[0187] In one embodiment, Kd is measured by a radiolabeled antigen-binding assay (RIA) performed on the antibody of interest and its Fab version using the following assay: The solution binding affinity of Fab to the antigen is measured by Fab being subjected to a titration series of unlabeled antigens at the lowest concentration. 125 I) The bound antigen is measured by equilibrating with a labeled antigen and then capturing it using a plate coated with anti-Fab antibody (see, for example, Chen et al., J.Mol.Biol.293:865-881 (1999)). To establish the assay conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [ 125Mix the [I]-antigen with the serially diluted Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, although the incubation may continue for a longer time (e.g., about 65 hours) to ensure reaching equilibrium. Thereafter, transfer the mixture to a capture plate and incubate at room temperature (e.g., for 1 hour). Then remove the solution and wash the plate 8 times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20 (trademark), Packard) and count the plate for 10 minutes on a TOPCOUNT (trademark) gamma counter (Packard). Select the concentration of each Fab that gives no more than 20% of the maximum binding for use in the competitive binding assay.

[0188] According to another embodiment, the Kd is measured at about 10 response units (RU) at 25 °C using a surface plasmon resonance assay with a BIACORE (registered trademark)-2000 or BIACORE (registered trademark)-3000 (BIAcore, Inc., Piscataway, N.J.) using an immobilized antigen CM5 chip. Briefly, according to the supplier's instructions, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen is diluted to 5 μg / ml (about 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to achieve an immobilized protein of approximately 10 response units (RU). After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, a 2-fold serial dilution of the Fab (0.78 nM to 500 nM) is injected at 25 °C at a flow rate of about 25 μl / min into PBS containing 0.05% polysorbate 20 (TWEEN-20 (trademark)) surfactant (PBST). Association rate (k on) and dissociation rate (k off The equilibrium dissociation constant (Kd) is calculated using a simple one-to-one Langmuir coupled model (BIACORE® evaluation software version 3.2) by simultaneously fitting the association sensorgram and dissociation sensorgram. off / k on Calculate as a ratio. For example, see Chen et al., J.Mol.Biol.293:865-881(1999). The above surface plasmon resonance assay yields an on-rate of 10 6 M -1 s -1 If it exceeds [value], the on-rate can be determined by increasing the antigen concentration, as measured by a spectrophotometer such as an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic) equipped with a stop flow or an 8000 series SLM-AMINCO® spectrophotometer (ThermoSpectronic) equipped with a stirring cuvette, using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C.

[0189] The anti-HER2 antibody of the present invention may be a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, an anti-HER2 antibody fragment, such as Fv, Fab, Fab', Fab'-SH, scFv, diabody, triabody, tetrabody, or F(ab')2 fragment formed from the antibody fragment, and a polyspecific antibody are used. In another embodiment, the antibody is a full-length antibody, such as an intact IgG antibody, or another antibody class or isotype as defined herein. For an overview of specific antibody fragments, see Hudson et al. Nat. Med., vol. 9, pp. 129-134, 2003. For an overview of the ScFv fragment, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), as well as WO93 / 16185, and U.S. Patents Nos. 5,571,894 and 5,587,458. For a discussion of the Fab and F(ab')2 fragments, which contain salvage receptor-binding epitope residues and have increased in vivo half-lives, see U.S. Patent No. 5,869,046.

[0190] The diabodies of the present invention may be divalent or bispecific. For example, see EP404,097, WO1993 / 01161, Hudson et al., Nat.Med.9:129-134(2003), and Hollinger et al., Proc.Natl.Acad.Sci.USA,vol.90,pp.6444-6448,1993. Examples of triabodies and tetrabodies are also described in Hudson et al., Nat.Med.,vol.9,pp.129-134,2003.

[0191] In some embodiments, the present invention comprises a single-domain antibody fragment comprising all or part of the heavy chain variable domain, or all or part of the light chain variable domain of the antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see Domantis, Inc., Waltham, Mass., e.g., U.S. Patent No. 6,248,516B1).

[0192] Antibody fragments can be prepared by a variety of techniques, but are not limited to, the protein digestion of intact antibodies, as well as production by recombinant host cells (e.g., Escherichia coli or phages) as described herein.

[0193] In some embodiments, the anti-HER2 antibody of the present invention may be a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and in Morrison et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp. 6851-6855, (1984). In one example, a chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In further examples, a chimeric antibody is a "class-switched" antibody in which the class or subclass of the antibody is modified compared to the class or subclass of the parent antibody. A chimeric antibody includes its antigen-binding fragment.

[0194] In certain embodiments, the chimeric antibody of the present invention is a humanized antibody. Typically, such a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains, where the CDR (or a portion thereof) is derived from the non-human antibody and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody may also optionally contain at least a portion of the human constant region. In some embodiments, several FR residues of the humanized antibody are replaced with corresponding residues derived from the non-human antibody (e.g., the antibody from which the CDR residue is derived) to restore or improve the specificity or affinity of the antibody, for example.

[0195] Humanized antibodies and methods for producing them are outlined, for example, in Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008, and further, for example, Riechmann et al., Nature, vol. 332, pp. 323-329, 1988, Queen et al., Proc. Nat'l Acad. Sci. USA, vol. 86, pp. 10029-10033, 1989, U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409, Kashmiri et al. This is described in al., Methods, vol.36, pp.25-34, 2005 (SDR(a-CDR) grafts), Padlan, Mol.Immunol., vol.28, pp.489-498, 1991 ("resurfacing" is described), Dall'Acqua et al., Methods, vol.36, pp.43-60, 2005 ("FR shuffling" is described), as well as Osbourn et al., Methods, vol.36, pp.61-68, 2005 and Klimka et al., Br.J.Cancer, vol.83, pp.252-260, 2000 ("guided selection" approach to FR shuffling is described).

[0196] Human framework regions that may be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol., vol. 151, p. 2296, 1993), framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, vol. 89, p. 4285, 1992 and Presta et al. J. Immunol., vol. 151, p. 2623, 1993), human maturation (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008), and framework regions derived from screening of FR libraries (e.g., Baca et al. See also al., J. Biol. Chem., vol. 272, pp. 10678-10684, 1997 and Rosok et al., J. Biol. Chem., vol. 271, pp. 22611-22618, 1996.

[0197] multispecific antibody This disclosure provides a multispecific antibody comprising at least one binding site for a cell antigen and at least one binding site for a tumor-reactive lymphocyte antigen. The multispecific antibody binds to at least one of the cell antigen and tumor-reactive lymphocyte antigen with higher activity, affinity, and / or binding activity under first physiological conditions and under second physiological conditions.

[0198] In some embodiments, the first physiological condition is an abnormal condition, and the second physiological condition is a normal physiological condition. For example, the abnormal condition may be the conditions of a tumor microenvironment. The polyspecific antibodies of the present invention may be referred to as conditionally active polyspecific antibodies.

[0199] In some embodiments, conditionally active polyspecific antibodies are virtually inactive under normal physiological conditions but active under abnormal conditions, and optionally have a higher level of activity than the conditionally active polyspecific antibody under normal physiological conditions, or the parental antibody from which it is derived. In other embodiments, conditionally active polyspecific antibodies are virtually inactive at pH 7.2–7.8 but active at lower pH 5.0–7.0. In some cases, conditionally active polyspecific antibodies are reversibly or irreversibly inactivated under normal physiological conditions. In another example, conditionally active polyspecific antibodies may be more or less active in highly oxygenated blood, such as after passing through the lungs or in lower pH environments found in the tumor microenvironment. Conditionally active polyspecific antibodies can be used as drugs, therapeutic agents, or diagnostic agents.

[0200] While not limited to theory, the multispecific antibodies of the present invention bind to both target cells and tumor-reactive lymphocytes, thereby bringing target cells closer to tumor-reactive lymphocytes. This is thought to facilitate the tumor-reactive lymphocytes' attack on target cells, thereby inhibiting, damaging, or destroying them. The therapeutic effect of inhibiting or eliminating tumor cells may be achieved by using the multispecific antibodies of the present invention to deliver reactive lymphocytes to tumor cells in order to inhibit, destroy, and eliminate tumor cells from a target.

[0201] The first and second physiological conditions are different numerical values ​​of the same condition, which can be selected from temperature, pH, osmotic pressure, gravimetric osmolality, oxidative stress, oxygen concentration, and electrolyte concentration. For example, the first physiological condition may be an acidic pH in the tumor microenvironment within the range of 5.2–7.0, 5.8–7.0, or 6.0–6.8. The second physiological condition may be a normal physiological pH in the subject's blood within the range of 7.2–7.8 or 7.2–7.6.

[0202] In some embodiments, the first physiological condition is a lower oxygen concentration in the tumor microenvironment, and the second physiological condition is a normal physiological oxygen concentration in the blood of the subject. In some embodiments, the conditionally active polyspecific antibody is virtually inactive under normal physiological conditions but active under abnormal conditions, and optionally has a higher level of activity than the activity of the conditionally active polyspecific antibody under normal physiological conditions, or the activity of the parent antibody from which it is derived, under normal physiological conditions. In another embodiment, the conditionally active polyspecific antibody is virtually inactive at pH 7.2–7.8 but active at lower pH 5.0–7.0. In some cases, the conditionally active polyspecific antibody is reversibly or irreversibly inactivated under normal physiological conditions. In another example, the conditionally active polyspecific antibody may be more or less active in highly oxygenated blood, such as after passing through the lungs or in a lower pH environment found in the tumor microenvironment. The conditionally active polyspecific antibody may be used as a drug, therapeutic agent, or diagnostic agent.

[0203] In some embodiments, the binding of a multispecific antibody to a cell antigen and / or tumor-reactive lymphocyte antigen is reversible. This means that the multispecific antibody can bind to the cell antigen and / or tumor-reactive lymphocyte antigen, and then the two can separate. The separated multispecific antibody can then re-bind to the cell antigen and / or tumor-reactive lymphocyte antigen.

[0204] In some embodiments, the cell antigen may be a cell surface antigen or an internal antigen of the cell. Cells may be targeted by tumor-reactive lymphocytes for inhibition, damage, destruction, or killing. Cells may be referred to as target cells. Thus, cells may be targeted in treatment with the multispecific antibodies of the present invention. Specifically, cells may be targeted for removal for the treatment of certain diseases or conditions.

[0205] In some embodiments, the cell antigen is an antigen that preferentially associates with the target cell but is less affected by other cell types. In this way, the multispecific antibody of the present invention can preferentially interact with the target cell. The target cell can be a cancer cell. Examples of cancer cell-specific antigens include CD3 and HER2.

[0206] In one embodiment, the target cancer cell is a breast cancer cell, and in this case, the breast cancer cell-specific antigen can be HER2 (human epidermal growth factor receptor 2).

[0207] The multispecific antibody binds to at least one cell-specific antigen and a reactive lymphocyte antigen, and the affinity in the first physiological condition is increased compared to the affinity in the second physiological condition. In some embodiments, the multispecific antibody binds to at least one of the cell-specific antigen and the reactive lymphocyte antigen, and the affinity in the first physiological condition is increased compared to the affinity in the second physiological condition. For example, the multispecific antibody can bind to the cell-specific antigen with an increased binding affinity in the first physiological condition compared to the binding affinity in the second physiological condition, while still being able to bind to the reactive lymphocyte antigen with non-conditional activity. In another example, the multispecific antibody binds to the reactive lymphocyte antigen, and the binding affinity in the first physiological condition is increased compared to the binding affinity in the second physiological condition, while still being able to bind to the cell-specific antigen with non-conditional activity. In some embodiments, the multispecific antibody binds to both the cell-specific antigen and the reactive lymphocyte antigen, and the binding activity in the first physiological condition is higher compared to the binding activity in the second physiological condition.

[0208] The structure / format of a bispecific antibody may be one of the structures / formats described in Brinkmann and Kontermann, “The making of bispecific antibodies,” MABs, vol.9, pp.182-212, 2017, or as described in Orcutt et al., Protein Engineering, Design & Selection, 23(4):221-228 (2010). Specifically, Figure 2 by Brinkmann and Kontermann describes 19 different structures / formats of bispecific antibodies. These structures / formats include: (1) bispecific antibody conjugates, (2) hybrid bispecific IgG2, (3) "variable domain only" bispecific antibody molecules, (4) CH1 / CL fusion proteins, (5) Fab fusion proteins, (6) non-immunoglobulin fusion proteins, (7) Fc-modified IgG, (8) added and Fc-modified IgG, (9) modified Fc and CH3 fusion proteins, (10) added IgG-HC fusions, (11) added IgG-LC fusions, (12) added IgG-HC&LC fusions, (13) Fc fusions, (14) CH3 fusions, (15) IgE / IgM CH2 fusions, (16) F(ab')2 fusions, (17) CH1 / CL fusion proteins, (18) modified IgG, and (19) non-immunoglobulin fusions. Similarly, Orcutt describes a bispecific antibody (bsAb) format in which a disulfide-stabilized scFv fuses to the C-terminus of the IgG light chain to produce an IgG-scFv bifunctional antibody. The structure of a fully assembled bsAB showing the heavy chain, light chain, and N-terminus and C-terminus is shown in Figure 1 of Orcutt's work.

[0209] In certain embodiments, the polyspecific antibody may be a bivalent scFv-Fc heterodimer or a tetravalent homodimer "butterfly," as shown in Figure 12. In these two structures, the reactive lymphocyte antigen is not limited to CD3, shown only as a representative of tumor-reactive lymphocyte antigens. The polyspecific antibody in Figure 12 has a first binding site to a cellular antigen (Ag) linked to a first heavy chain constant region (e.g., IgG), and a second binding site to a reactive lymphocyte antigen (e.g., CD3) linked to a second heavy chain constant region (e.g., IgG). The two heavy chains are manipulated so that they can form only heterodimers, for example by using a knob-in-hole technique. The first and second binding sites are scFv antibodies that bind to the cellular antigen and the reactive lymphocyte antigen, respectively. Either or both of the first and second binding sites have conditionally active binding activity to their respective antigens.

[0210] The multispecific antibody in Figure 12 may have a full-length IgG antibody that binds to a cell-specific antigen (Ag) and an scFv antibody that binds to a reactive lymphocyte antigen (e.g., CD3). The scFv antibody is ligated to the C-terminus of the light chain of the IgG antibody via a linker. The linker is a short alanine linker (Ala). n Serine Linker (Ser) n The linker can be hydrophilic or glycine-serine rich. The heavy chain of the IgG antibody pairs with the light chain of the IgG antibody linked to the scFv antibody, thus forming half of the homodimer. This polyspecific antibody has a "butterfly" structure.

[0211] In some embodiments, the polyspecific antibody comprises an IgG antibody or fragment thereof that binds to tumor-reactive lymphocyte antigens, and a single-chain antibody that binds to tumor cell antigens, similarly forming the “butterfly” configuration shown in Figure 12. The single-chain antibody may also be an scFv antibody. The scFv antibody may be conjugated to the C-terminus of the IgG antibody via a linker as described herein.

[0212] The binding sites of the polyspecific antibodies of the present invention each include a light chain variable region and a heavy chain variable region. The light chain variable region and the heavy chain variable region may be in a single-chain antibody format or in a double-chain format formed by the pairing of the light chain and the heavy chain. In a binding site having conditional activity, either one of the light chain variable region and the heavy chain variable region may be conditionally active, or both may be conditionally active.

[0213] In some embodiments, the anti-HER2 antibody of the present invention is a polyspecific antibody, such as a bispecific antibody. A polyspecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In certain embodiments, one binding specificity is to the HER2 protein and the other is to another antigen. In certain embodiments, a bispecific antibody may bind to two different epitopes of the HER2 protein. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing the HER2 protein. Bispecific antibodies may be prepared as full-length antibodies or antibody fragments.

[0214] In some embodiments, the polyspecific antibody or antibody fragment is a heavy chain variable region comprising three anti-HER2 complementarity determining regions, H1, H2, and H3. The H1 sequence is GFX1IKDTYIH (sequence number 1), The H2 sequence is X2IX3PTX4X5YX6X7YADSVKG (sequence number 2), The H3 sequence is WGGDGFYX8MDY (sequence number 3), In the formula, X1 is N or W, X2 is R or K, X3 is Y, K or D, X4 is N or A, X5 is G or K, X6 is T or D, X7 is R or E, and X8 is A or E, representing a heavy-chain variable region. It comprises a light chain variable region containing three anti-HER2 complementarity-determining regions having sequences L1, L2, and L3, and six anti-CD3 complementarity-determining regions, L4, L5, L6, L7, L8, and L9, The L1 sequence is RASQDVNTX9VA (sequence number 4), The L2 sequence is SASFLYS (sequence number 5), The L3 array is QQX 10 YTTPPT (sequence number 6) In the formula, X9 is A or D, and X 10 However, it is either H, D, or E. As a premise, if X1 to X8 are N, R, Y, N, G, T, R, and A respectively, then X9 is not A, but X 10 However, not H The L4 sequence is GFTFNTYAMN (sequence number 54), The L5 sequence is RIRSKYNNYATYYADSVKD (sequence number 55), L6 array, HX 11 NTE 12 NSKVSWFX 13 Y (sequence number 70), L7 array, RSSX 14 GAVTTSNYDN (Sequence ID 71) The L8 sequence is GTNKRAP (sequence number 58), The L9 sequence is ALWYSNLWV (sequence number 59), In the formula, X 11 However, it is G, S, A, or T, and X 12 However, it is G or P, and X 13 However, it is A or Q, and X 14 However, it is either T or A.

[0215] In a particular embodiment, the polyspecific antibody or antibody fragment is a heavy chain variable region comprising three anti-HER2 complementarity determining regions, H1, H2, and H3, The H1 sequence is sequence number 50. The H2 sequence is sequence number 49, sequence number 9, sequence number 12, or sequence number 13. The H3 sequence is sequence number 51. It comprises a light chain variable region containing three anti-HER2 complementarity-determining regions L1, L2, and L3, and six anti-CD3 complementarity-determining regions L4, L5, L6, L7, L8, and L9, The L4 sequence is GFTFNTYAMN (sequence number 54), The L5 sequence is RIRSKYNNYATYYADSVKD (sequence number 55), L6 array, HX 11 NTE 12 NSKVSWFX 13 Y (sequence number 70), L7 array, RSSX 14 GAVTTSNYDN (Sequence ID 71) The L8 sequence is GTNKRAP (sequence number 58), The L9 sequence is ALWYSNLWV (sequence number 59), In the formula, X 11 However, it is G, S, A, or T, and X 12 However, it is G or P, and X 13 However, it is A or Q, and X 14 However, it is either T or A.

[0216] In another embodiment of the polyspecific antibody or antibody fragment, the L6 sequence is one of sequence numbers 56 and 60-67, and the L7 sequence is sequence number 57, 68, or 69.

[0217] In certain embodiments of the present invention, the polyspecific antibody or antibody fragment is a bispecific antibody that binds to HER2 and CD3. Such a polyspecific antibody or antibody fragment may be selected from any of the following combinations of heavy chain variable regions and light chain variable regions shown below. [Table 6] JPEG0007856312000019.jpg233170JPEG0007856312000020.jpg237170JPEG0007856312000021.jpg157170

[0218] Preferred bispecific antibodies are those having the combination of heavy chain variable regions and light chain variable regions listed in Table 10 below.

[0219] In another specific embodiment of the present invention, the polyspecific antibody is a bispecific antibody that binds to Her2 and CD3, comprising a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises H1, H2, and H3 sequences, each of which may be selected from any of the following combinations shown below. The light chain variable region comprises L1, L2, L3, L4, L5, L6, L7, L8, and L9 sequences, each of which may be selected from any of the following combinations shown below.

[0220] In certain embodiments of the present invention, the polyspecific antibody is a bispecific antibody that can be selected from any of the following antibodies, each containing a specific combination of the 12 CDRs H1, H2, H3, L1, L2, L3, L4, L5, L6, L7, L8, and L9 shown below. [Table 7] JPEG0007856312000023.jpg245170JPEG0007856312000024.jpg222170JPEG0007856312000025.jpg249170JPEG0007856 312000026.jpg255170JPEG0007856312000027.jpg227170JPEG0007856312000028.jpg247170JPEG0007856312000029.j pg230170JPEG0007856312000030.jpg238170JPEG0007856312000031.jpg238170JPEG0007856312000032.jpg249170JPE G0007856312000033.jpg242170JPEG0007856312000034.jpg241170JPEG0007856312000035.jpg238170JPEG00078563120 00036.jpg243170JPEG0007856312000037.jpg228170JPEG0007856312000038.jpg228170JPEG0007856312000039.jpg23 8170JPEG0007856312000040.jpg238170JPEG0007856312000041.jpg225170JPEG0007856312000042.jpg251170JPEG000 7856312000043.jpg232170JPEG0007856312000044.jpg238170JPEG0007856312000045.jpg228170JPEG00078563120000 46.jpg245170JPEG0007856312000047.jpg238170JPEG0007856312000048.jpg233170JPEG0007856312000049.jpg213170

[0221] Preferred bispecific antibodies or antibody fragments that bind to the Her2 protein and CD3 protein may be selected from any of the following antibodies or antibody fragments, each containing a specific combination of the 12 CDRs H1, H2, H3, L1, L2, L3, L4, L5, L6, L7, L8, and L9 shown below. Table 8 JPEG0007856312000051.jpg237170JPEG0007856312000052.jpg236170JPEG0007856312000053.jpg245170JPEG0007856312000054.jpg168170

[0222] Techniques for producing multispecific antibodies include, but are not limited to, the recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature, vol.305, pp.537-540, 1983, WO93 / 08829, and Traunecker et al., EMBO J. vol.10, pp.3655-3659, 1991), and "knob-in-hole" engineering (see, for example, U.S. Patent No. 5,731,168). Furthermore, polyspecific antibodies can be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimer molecules (WO2009 / 089004A1), crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, vol. 229, pp. 81-83, 1985), producing bispecific antibodies using leucine zippers (see, e.g., Kostelny et al., J. Immunol., vol. 148, pp. 1547-1553, 1992), or using "diabody" technology to produce bispecific antibody fragments (see, e.g., Hollinger et al.) They can also be prepared by using single-stranded Fv(scFv) dimers (see, for example, Gruber et al., J.Immunol., vol.152, pp.5368-5374, 1994), and by preparing tripspecific antibodies (see, for example, Tutt et al., J.Immunol., vol.147, pp.60-69, 1991).

[0223] In one embodiment, the bispecific antibody comprises the antibody or antibody fragment of this disclosure against HER2 and a second antibody or antibody fragment against a tumor-reactive lymphocyte antigen. In another embodiment, the tumor-reactive lymphocyte antigen is CD3.

[0224] Modified antibodies having three or more functional antigen-binding sites, including "Octopus antibody," are also included herein (see, for example, US2006 / 0025576A1).

[0225] The anti-HER2 antibody or antibody fragment of the present invention can be produced using the recombinant method and composition described in detail in US2016 / 0017040.

[0226] The physical / chemical properties and / or biological activity of the anti-HER2 antibody or antibody fragment of the present invention can be tested and measured by various assays known in the art. Some of these assays are described in U.S. Patent No. 8,853,369.

[0227] H. immunoconjugate In another embodiment, the present invention also provides an immunoconjugate comprising an anti-HER2 antibody or antibody fragment conjugated to one or more cytotoxic agents such as chemotherapeutic agents or chemotherapeutic drugs, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, plant, or animal-derived enzyme-active toxins, or fragments thereof), and radioisotopes.

[0228] In one embodiment, the immune conjugate is an antibody-drug conjugate (ADC) in which an antibody or antibody fragment is conjugated to one or more drugs, and is not limited to mytansinoids (see U.S. Patent Nos. 5,208,020, 5,416,064, and European Patent No. EP0425235B1), auristatins such as monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483) See U.S. Patent Nos. 5,780,588 and 7,498,298), drastatin, calicheamycin or its derivatives (see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296, Hinman See et al., Cancer Res., vol.53, pp.3336-3342, 1993, and Lode et al., Cancer Res., vol.58, pp.2925-2928, 1998), anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem., vol.13, pp.477-523, 2006, Jeffrey et al., Bioorganic & Med. Chem. Letters, vol.16, pp.358-362, 2006, Torgov et al., Bioconj. Chem., vol.16, pp.717-721, 2005, Nagy et al., Proc. Natl. Acad. Sci. USA, vol.97, pp.829-834, 2000, Dubowchik et al.,Bioorg.& Med.Chem.Letters,vol.12,vol.1529-1532,2002, King et al.,J.Med.Chem.,vol.45,pp.Examples include methotrexate, vindesine, taxanes (see U.S. Patent Nos. 4336-4343, 2002, and U.S. Patent No. 6,630,579), trichothecenes, and CC1065.

[0229] In another embodiment, the immunoconjugate includes, but is not limited to, the antibodies or antibody fragments described herein that are conjugated to an enzymatically active toxin or a fragment thereof, diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modesine A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana protein (PAPI, PAPII, and PAP-S), bitter melon (Momordica charantia) inhibitor, curcin, crotin, soapwort (Sapaonaria officinalis) inhibitor, geronin, maitogerin, restrictosin, phenomycin, enomycin, and trichothecene.

[0230] In another embodiment, the immunoconjugate comprises an antibody or antibody fragment described herein conjugated to a radioactive atom, forming a radioconjugate. Various radioisotopes are available for the production of the radioconjugate. For example, At 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Pb 212Examples include radioactive isotopes of , and Lu. When a radioactive conjugate is used for detection, it may include radioactive atoms for scintigraphy studies, e.g., tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, and iron.

[0231] In some embodiments, the immunoconjugate comprises a radioagent which may be selected from α-emitters, β-emitters, and γ-emitters. An example of an α-emitter is: 211 At, 210 Bi, 212 Bi, 211 Bi, 223 Ra, 224 Ra, 225 Ac, and 227 It is Th. An example of a β-emitter is, 67 Cu, 90 Y, 131 I, 153 Sm, 166 Ho, and 186 It is Re. An example of a γ-emitting material is, 60 Co, 137 Ce, 55 Fe, 54 Mg, 203 Hg, and 133 It is Ba. In certain embodiments, the immunoconjugate may contain a highly radioactive atom. Zirconium-89 may be conjugated with various metal chelating agents and, for example, conjugated to an antibody for PET imaging (WO2011 / 056983).

[0232] Radiolabeling or other labeling may be incorporated into the immunoconjugate by known methods. For example, peptides may be biosynthesized or chemosynthesized using suitable amino acid precursors containing, for example, one or more fluorine-19 atoms instead of one or more hydrogen atoms. In some embodiments, Tc 99 , I 123 Re 186 Re 188, and In 111 Labels such as can be bound via cysteine ​​residues in the antibody. In some embodiments, yttrium-90 can be bound via lysine residues in the antibody. In some embodiments, iodine-123 can be incorporated using the IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun., vol. 80, pp. 49-57, 1978). "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes certain other methods.

[0233] Conjugates of antibodies / antibody fragments with cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethylHCl adipimidoate), active esters (e.g., disaxinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science, vol.238, pp.1098-, 1987. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO94 / 11026. The linker may be a “cleavable linker” that facilitates the release of cytotoxic drugs into cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers may be used (Chari et al., Cancer Res., vol.52, pp.127-131, 1992, U.S. Patent No. 5,208,020).

[0234] Examples of immunoconjugates include, but are not limited to, those prepared with crosslinking reagents, including, commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0235] Exemplary embodiments of an ADC include an antibody or antibody fragment (Ab) that targets tumor cells, a drug moiety (D), and a linker moiety (L) that binds Ab to D. In some embodiments, the antibody is bound to the linker moiety (L) via one or more amino acid residues, such as lysine and / or cysteine.

[0236] An example ADC is Ab-(LD) p The ADC has formula I, where p is 1 to about 20. In some embodiments, the number of drug moieties that can be conjugated to the antibody is limited by the number of free cysteine ​​residues. In some embodiments, free cysteine ​​residues are introduced into the antibody amino acid sequence by the method described herein. Exemplary ADCs of formula I include, but are not limited to, antibodies having 1, 2, 3, or 4 modified cysteine ​​amino acids (Lyon et al., Methods in Enzym., vol. 502, pp. 123-138, 2012). In some embodiments, one or more free cysteine ​​residues are already present in the antibody without modification, in which case the existing free cysteine ​​residues can be used to conjugate the antibody to the drug. In some embodiments, the antibody is exposed to reducing conditions prior to antibody conjugation to generate one or more free cysteine ​​residues.

[0237] A linker is used to conjugate a portion to an antibody to form an immunoconjugate such as an ADC. Suitable linkers are described in WO2017 / 180842. Several drug portions that can be conjugated to antibodies are also described in WO2017 / 180842. Drug portions also include compounds having nucleolytic activity (e.g., ribonucleases or DNA endonucleases).

[0238] In certain embodiments, the immunoconjugate may include an antibody conjugated to a prodrug-activating enzyme. In some such embodiments, the prodrug-activating enzyme converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO81 / 01145) into an active drug such as an anticancer agent. Such immunoconjugates are useful in antibody-dependent enzyme-mediated prodrug therapy ("adept") in some embodiments. Enzymes that can be conjugated to an antibody include, but are not limited to, alkaline phosphatases useful for converting phosphate-containing prodrugs into free drugs, arylsulfatases useful for converting sulfate-containing prodrugs into free drugs, cytosine deaminases useful for converting harmless 5-fluorocytosine into the anticancer drug 5-fluorouracil, serratia proteases, pyrolysis, subtilisin, carboxypeptidases and proteases useful for converting peptide-containing prodrugs such as cathepsins (cathepsin B and L, etc.) into free drugs, and D-amino acids. Examples include D-alanyl carboxypeptidase, β-galactosidase, and neuraminidase, which are useful for converting substituent-containing prodrugs; carbohydrate-cleaving enzymes, useful for converting glycosylated prodrugs into free drugs; β-lactamases, useful for converting β-lactam-derivative drugs into free drugs; and penicillin amidases, such as penicillin V amidase and penicillin G amidase, which are useful for converting drugs derivatized with amine nitrogen having a phenoxyacetyl group or a phenylacetyl group into free drugs. In some embodiments, the enzymes may be covalently bound to an antibody by recombinant DNA techniques well known in the art. See, for example, Neuberger et al., Nature, vol.312, pp.604-608, 1984.

[0239] The drug load in a conjugate is represented by p, which is the average number of drug moieties per antibody. The drug load can range from 1 to 20 drug moieties per antibody. The conjugate of the present invention may have a range of 1 to 20 drug moieties. The average number of drug moieties per antibody used in the preparation of the conjugate from the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assay, and HPLC.

[0240] For some antibody-drug conjugates (ADCs), the drug load may be limited by the number of binding sites on the antibody. For example, if the binding is cysteinethiol, as in the specific exemplary embodiments described above, the antibody may have only one or more cysteinethiol groups, or only one or more sufficiently reactive thiol groups to which the linker can bind. In certain embodiments, a higher drug load, e.g., p>5, may cause aggregation, insolubility, toxicity, or loss of cell permeability in certain antibody-drug conjugates. In certain embodiments, the average drug load of an ADC is in the range of 1 to about 8, about 2 to about 6, or about 3 to about 5. In fact, for certain ADCs, the optimal ratio of drug portion per antibody may be less than 8 and may be about 2 to about 5 (U.S. Patent No. 7,498,298).

[0241] In certain embodiments, during the conjugation reaction, less than the theoretical maximum value of the drug moiety is conjugated to the antibody. The antibody may contain lysine residues that do not react with the drug-linker intermediate or linker reagent, for example, as considered below. Generally, antibodies do not contain many free and reactive cysteinethiol groups that can be linked to the drug moiety. In fact, most cysteinethiol residues in antibodies exist as disulfide crosslinks. In certain embodiments, the antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteinethiol groups under partially or entirely reducing conditions. In certain embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophiles such as lysine or cysteine.

[0242] The ADC load (drug / antibody ratio) can be adjusted in different ways, for example, by (i) limiting the molar excess of the drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the reaction time or temperature of the conjugation, and (iii) partial or limited reduction conditions for cysteinethiol modification.

[0243] I. Methods and compositions for diagnosis and detection In certain embodiments, any of the anti-HER2 antibodies or antibody fragments provided herein may be used to detect the presence of the HER2 protein in a biological sample, either quantitatively or qualitatively. In certain embodiments, the biological sample may include cells or tissues such as those of the breast, pancreas, esophagus, lungs, and / or brain.

[0244] A further aspect of the present invention relates to an anti-HER2 antibody or antibody fragment of the present invention for diagnosing and / or monitoring cancer or another disease in which HER2 protein expression levels are increased or decreased from normal physiological levels at at least one location in the body.

[0245] In one embodiment, the antibody or antibody fragment of the present invention may be labeled with a detectable molecule or substance, such as a fluorescent molecule, radioactive molecule, or any other label known in the art, as described above. For example, the antibody or antibody fragment of the present invention may be labeled with a radioactive molecule. For example, suitable radioactive molecules include, but are not limited to, 123 I, 124 I, 111 In, 186 Re, and 188 Examples of radioactive atoms used in scintigraphy studies include Re. Furthermore, the antibodies or antibody fragments of the present invention may also be labeled with spin labeling for nuclear magnetic resonance (NMR) imaging, such as iodine-123, iodine-131, indium-I11, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. After administration of the antibody, the distribution of the radiolabeled antibody within the patient is detected. Any suitable known method can be used. Some non-limiting examples include computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI), fluorescence, chemiluminescence, and ultrasound.

[0246] The antibodies or antibody fragments of the present invention may be useful in the diagnosis and staging of cancers and diseases associated with the overexpression of the HER2 protein. Cancers associated with the expression or overexpression of the HER2 protein include, but are not limited to, breast cancer, ovarian cancer, bladder cancer, gallbladder cancer, extrahepatic or intrahepatic cholangiocarcinoma, salivary duct cancer, esophageal cancer, gastric junction cancer and gastric adenocarcinoma and gastrointestinal stromal tumor, colon cancer, lung cancer including non-small cell and small cell lung cancer, pancreatic cancer such as pancreatic adenocarcinoma, penile cancer, pituitary cancer, prostate cancer, sarcomas including soft tissue sarcoma, peritoneal sarcoma and retroperitoneal sarcoma, solitary fibroma, thymic cancer, thyroid cancer, cervical cancer, testicular cancer, endometrial cancer, glioblastoma such as glioblastoma multiforme, glioma, oligodendroglioma, head and neck cancer, hepatocellular carcinoma, small intestine cancer, malignant melanoma, neuroendocrine tumors, or other cancers that express or overexpress the HER2 protein. HER2 is typically overexpressed in epithelial malignancies and cancers originating from mesenchymal, neuroendocrine, central nervous system, and kidney tissues; therefore, these types of cancers can be treated using the antibodies or antibody fragments of the present invention. Information on various forms of HER2 expression in cancer can be found, for example, in “HER2 expression status in diverse cancers: review of results from 37,992 patients,” Yan, Min et al., Cancer Metastasis Rev., (2015) 34:157-164. A disease associated with HER2 expression or overexpression is vulvar Paget's disease.

[0247] The antibodies or antibody fragments of the present invention may be useful for diagnosing non-cancerous diseases characterized by increased or decreased expression of the HER2 protein. Typically, such diagnostic methods involve the use of biological samples obtained from patients. Biological samples encompass a variety of sample types obtained from subjects that can be used in diagnostic or monitoring assays. Examples of biological samples include, but are not limited to, blood and other fluid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom, and their offspring. For example, biological samples include cells obtained from tissue samples collected from individuals suspected of having cancer associated with overexpression of the HER2 protein. Biological samples encompass clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0248] In one embodiment, the present invention includes a method for diagnosing cancer associated with the expression or overexpression of the HER2 protein in a subject by detecting the HER2 protein on cells derived from the subject using the antibody of the present invention. This method is 1) The step of contacting a biological sample with the antibody or antibody fragment according to the present invention under conditions suitable for the antibody or antibody fragment to form a complex with cells in the biological sample expressing the HER2 protein, (b) A step of detecting and / or quantifying the complex, wherein the detection of the complex indicates a cancer associated with overexpression of the HER2 protein.

[0249] To monitor cancer progression, the method according to the present invention may be repeated at different time points to determine whether antibody binding to the sample increases or decreases, from which it can be determined whether the cancer has progressed, regressed, or stabilized.

[0250] Another embodiment of the present invention is a method for diagnosing diseases associated with the expression or overexpression of the HER2 protein. Examples of such diseases include the cancers and vulvar Paget's disease mentioned above.

[0251] In one embodiment, an anti-HER2 antibody or antibody fragment is provided for use in a diagnostic or detection method. In a further embodiment, a method for detecting the presence of HER2 protein in a biological sample is provided. In a further embodiment, a method for quantifying the amount of HER2 protein in a biological sample is provided. In a particular embodiment, the method includes contacting a biological sample with the anti-HER2 antibody or antibody fragment described herein under conditions that allow binding of the anti-HER2 antibody or antibody fragment to HER2 protein, and detecting whether a complex is formed between the anti-HER2 antibody or antibody fragment and the HER2 protein. Such a method may be performed in vitro or in vivo. In one embodiment, such a method may be used to select a subject eligible for therapy. In some embodiments, the treatment includes administering the anti-HER2 antibody or antibody fragment to the subject.

[0252] In certain embodiments, labeled anti-HER2 antibodies or antibody fragments are used. Labels include, but are not limited to, directly detectable labels or parts (e.g., fluorescent labels, chromogenic labels, electron density labels, chemiluminescent labels, and radioactive labels), as well as indirectly detectable parts such as enzymes or ligands (e.g., by enzymatic reactions or molecular interactions). Exemplary labels include, but are not limited to, radioactive isotopes. 32 P, 14 C, 125 I, 3 H, and 131I) Other examples include fluorophores (rare earth chelates, or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.), luciferases (e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456)), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase, xanthine oxidase, etc.), coupled enzymes that oxidize the pigment precursor using hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, bacteriphage labeling, and stable free radicals.

[0253] J. Pharmaceutical preparations Anti-HER2 antibodies or antibody fragments possess antiproliferative activity. Furthermore, once conjugated with cytotoxic agents, these antibodies or antibody fragments can further reduce tumor size and exhibit reduced toxicity. Therefore, anti-HER2 antibodies, their fragments, or immunoconjugates may be useful for treating proliferative disorders associated with HER2 protein expression. Antibodies, fragments, or immunoconjugates can be used alone or in combination with any suitable agent or other conventional treatment.

[0254] Anti-HER2 antibodies or antibody fragments can be used to treat diseases associated with the expression, overexpression, or activation of the HER2 protein. There are no particular restrictions on the types of cancer or tissues that can be treated other than those related to HER2 protein expression.

[0255] Anti-HER2 antibodies or antibody fragments are potential activators of the innate immune response and can therefore be used in immunotherapy. Furthermore, the anti-HER2 antibodies or antibody fragments of the present invention can be used as adjuvants for immunity, such as vaccines, and as anti-infective agents.

[0256] In each embodiment of the therapeutic methods described herein, an anti-HER2 antibody, antibody fragment, or immunoconjugate of an anti-HER2 antibody or antibody fragment may be delivered in a manner consistent with conventional methods relating to the management of the disease or disorder for which treatment is required. In accordance with the disclosure herein, an effective amount of the antibody, antibody fragment, or immunoconjugate is administered to a subject requiring such treatment for a sufficient time and under conditions to prevent or treat the disease or disorder. Accordingly, one aspect of the present invention relates to a method for treating a disease related to the expression of the HER2 protein, comprising administering a therapeutically effective amount of the antibody, antibody fragment, or immunoconjugate of the present invention to a subject requiring such treatment.

[0257] For administration, anti-HER2 antibodies, antibody fragments, or immunoconjugates may be formulated as pharmaceutical compositions. Pharmaceutical compositions containing anti-HER2 antibodies, antibody fragments, or immunoconjugates can be formulated according to known methods for preparing pharmaceutical compositions. In such methods, the therapeutic molecule is typically combined with a mixture, solution, or composition containing a pharmaceutically acceptable carrier.

[0258] A pharmaceutically acceptable carrier is a substance that is tolerable by the recipient patient. Sterile phosphate-buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable pharmaceutically acceptable carriers are well known to those skilled in the art (see, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995)). The formulation may further contain one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, etc.

[0259] The form, route of administration, dosage, and regimen of a pharmaceutical composition naturally depend on the condition being treated, the severity of the disease, the patient's age, weight, and sex, etc. These considerations can be evaluated by those skilled in the art to formulate a suitable pharmaceutical composition. The pharmaceutical compositions of the present invention can be formulated for topical administration, oral administration, parenteral administration, intranasal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intraocular administration, etc.

[0260] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. An example vehicle may be an isotonic sterile saline solution (such as monosodium or disodium phosphate, sodium, potassium, calcium, or magnesium chloride, or a mixture of such salts), or a dry, particularly lyophilized, composition that enables the formation of an injectable solution, for example, when sterile water or saline solution is added.

[0261] In some embodiments, it is sometimes also known as a "stabilizer". etc. Tonicing agents are present to adjust or maintain the tonicity of a liquid in a composition. When used with large charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can reduce the potential for intermolecular and intramolecular interactions by interacting with the charged groups of amino acid side chains. etc. The tensor may be present in any amount of the pharmaceutical composition, preferably 0.1 to 25% by weight, preferably 1 to 5% by weight. etc. Examples of tensing agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0262] Further excipients include agents that can function as one or more of the following: (1) volume extenders, (2) dissolution accelerators, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to the container wall. Such excipients include polyhydric sugar alcohols (listed above), amino acids (e.g., alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine), organic sugars or sugar alcohols (e.g., sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinitose, myonititol, galactose, galactitol, glycerol, cyclitol (e.g., inositol), polyethylene glycol), Examples include sulfur-containing reducing agents (e.g., urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-mothioglycerol, and sodium thiosulfate), low molecular weight proteins (e.g., human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins), hydrophilic polymers (e.g., polyvinylpyrrolidone), monosaccharides (e.g., xylose, mannose, fructose, glucose), disaccharides (e.g., lactose, maltose, sucrose), trisaccharides (e.g., raffinose), and polysaccharides (e.g., dextrin or dextran).

[0263] Nonionic surfactants or detergents (also known as "wetting agents") can be used to help solubilize the therapeutic agent and protect the therapeutic protein from aggregation induced by agitation, which also allows the formulation to be exposed to shear surface loading without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant may be present in a concentration range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0264] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxomers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Usable anionic detergents include sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0265] The dosage used for administration can be adapted as a function of various parameters, such as the mode of administration, the associated pathology, and / or the desired duration of treatment.

[0266] To prepare pharmaceutical compositions, an effective amount of antibody or antibody fragments can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Suitable dosage forms for injection include sterile aqueous solutions or dispersions, formulations containing sesame oil, peanut oil, or propylene glycol aqueous solutions, and sterile powders for the immediate preparation of sterile, injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi.

[0267] Solutions of active compounds, either as free bases or pharmacologically acceptable salts, can be prepared in water, preferably mixed with a surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth.

[0268] Anti-HER2 antibodies or antibody fragments can be formulated into neutral or salt-formulated compositions. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein), which are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids such as acetic acid, oxalic acid, tartaric acid, and mandelic acid. Salts formed with free carboxyl groups may be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide) and organic bases such as isopropylamine, trimethylamine, histidine, and procaine.

[0269] The carrier may also be a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained by maintaining the required particle size, for example, by using a coating such as lecithin, and by using a surfactant. Prevention of microbial action can be brought about by various antimicrobial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. Sustained absorption of the injectable composition can be brought about by using absorption-delaying agents in the composition, for example, aluminum monostearate and gelatin.

[0270] Sterile injection solutions are prepared by incorporating the required amount of the active compound into a suitable solvent containing, if necessary, one or more components other than those listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating various sterilizing active ingredients into a sterile vehicle and contain a basic dispersion medium and other components from those listed above as needed. In the case of sterilizing powders for preparing sterilizing injection solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, from which powders of the active ingredient and any additional desired components are obtained from a pre-filtered sterilized solution.

[0271] Furthermore, the preparation of larger or higher-concentration solutions for direct injection is also being considered. By using dimethyl sulfoxide (DMSO) as the solvent, it is anticipated that very rapid penetration will be achieved, allowing for the delivery of high concentrations of the active agent to small tumor areas.

[0272] During formulation, the solution is administered in a form compatible with the drug formulation and in a therapeutically effective amount. The formulation can be easily administered in various dosage forms, such as the injectable solutions described above, but drug-releasing capsules can also be used.

[0273] For parenteral administration in aqueous solution, the solution may be buffered appropriately as needed, and the liquid diluent may first be isotonicized with sufficient saline or glucose. Aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art. For example, the dose may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion solution, or injected into the proposed injection site (see, for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Several variations in dosage may arise depending on the condition of the subject being treated. In any case, the person responsible for administration will determine the appropriate dose for each individual subject.

[0274] In addition to compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable dosage forms include, for example, tablets or other solids for oral administration, sustained-release capsules, and any other dosage forms currently in use.

[0275] In certain embodiments, the use of liposomes and / or nanoparticles is intended to introduce antibodies or antibody fragments into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art.

[0276] Nanocapsules can generally capture compounds in a stable and reproducible manner. To avoid side effects due to intracellular polymer overload, such ultrafine particles (around 0.1 μm in size) are generally designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are intended for use in this invention.

[0277] Liposomes are dispersed in an aqueous medium and are formed from phospholipids that spontaneously form multilayer concentric bilayer vesicles (also called multilayer vesicles, or MLVs). MLVs generally have a diameter of 25 nm to 4 μm. Sonication of MLVs creates small monolayer vesicles (SUVs) with a diameter ranging from 200 to 500 Å, containing an aqueous solution within their core. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0278] Pharmaceutical formulations containing anti-HER2 antibodies or antibody fragments as described herein may be prepared by mixing such antibodies or antibody fragments of desired purity in the form of lyophilized formulations or aqueous solutions with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the doses and concentrations used, and are not limited to, but include buffers (such as phosphoric acid, citrate, and other organic acids), antioxidants (including ascorbic acid and methionine), preservatives (such as octadecyldimethylbenzylammonium chloride), hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl, or benzyl alcohol, alkylparabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), and low molecular weight polypeptides (about 1 Examples include less than 0 residues, proteins (such as serum albumin, gelatin, or immunoglobulins), hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine), monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrin), chelating agents (such as EDTA), sugars (such as sucrose, mannitol, trehalose, or sorbitol), salt-forming counterions (such as sodium), metal complexes (e.g., Zn protein complexes), and / or nonionic surfactants (such as polyethylene glycol (PEG)).

[0279] Exemplary pharmaceutically acceptable carriers herein may include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Specific exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968. In one embodiment, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0280] An example of a lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Examples of aqueous antibody preparations are described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter of which contains histidine-acetate buffer.

[0281] The formulations described herein may also contain more than one active ingredient for the indication being treated, as needed. Preferably, a single formulation may be composed of ingredients having complementary activity that does not adversely affect each other. For example, in addition to the anti-CTLA4 antibody, antibody fragment, or immunoconjugate of the present invention, it may be desirable to provide an EGFR antagonist (such as erlotinib), an anti-angiogenic agent (such as a VEGF antagonist, which may be an anti-VEGF antibody), or a chemotherapeutic agent (such as a taxoid or platinum agent). Such active ingredients are preferably present in combination in amounts effective for the intended purpose.

[0282] In one embodiment, the anti-HER2 antibody, antibody fragment, or immunoconjugate of the present invention is combined in a formulation with another antibody or antibody fragment against an antigen selected from CTLA4, PD1, PD-L1, AXL, ROR2, CD3, EpCAM, B7-H3, ROR1, SFRP4, and WNT proteins (including WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16). The combination may be in the form of two distinct molecules, namely, the anti-HER2 antibody, antibody fragment, or immunoconjugate of the present invention and another antibody or antibody fragment. Alternatively, the combination may also be in the form of a single molecule, having binding affinity to both the HER2 protein and the other antigen, and thus forming a multispecific (e.g., bispecific) antibody.

[0283] The active ingredient may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization. For example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules may be used in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0284] Sustained-release preparations can be prepared. A suitable example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing an antibody or antibody fragment, the matrix of which may be in the form of a molded article, such as a film or microcapsules.

[0285] Preparations used for in vivo administration are generally sterile. Sterilization can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0286] K. Treatment methods and compositions Any anti-HER2 antibody or antibody fragment provided herein can be used in a therapeutic method. In one embodiment, an anti-HER2 antibody or antibody fragment is provided for use as a drug. In a further embodiment, an anti-HER2 antibody or antibody fragment is provided for use in treating cancer. A list of cancers that have been found to express or overexpress HER2, which is a preferred target of the therapeutic method, is provided above.

[0287] In certain embodiments, an anti-HER2 antibody or antibody fragment is provided for use in a therapeutic method. In certain embodiments, the present invention provides an anti-HER2 antibody or antibody fragment for use in a method of treating an individual having cancer, comprising administering a therapeutically effective amount of the anti-HER2 antibody or antibody fragment to the individual. In such one embodiment, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent (e.g., those described below) to the individual. In further embodiments, the present invention provides an anti-HER2 antibody or antibody fragment for use in inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting the secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), inhibiting tumor vascular systems (e.g., intratumor vascular systems or tumor-associated vascular systems), and / or inhibiting tumor stromal function, as well as a method of treating these conditions using an anti-HER2 antibody or antibody fragment, comprising administering an effective anti-HER2 antibody or antibody fragment to treat the condition. The “individual” in any of the embodiments of the present invention is preferably a human.

[0288] In further embodiments, the present invention provides the use of anti-HER2 antibodies or antibody fragments in the manufacture or preparation of a drug. In one embodiment, the drug is for the treatment of any of the cancers or diseases described above. The drug is for use in a method of treating cancer, which comprises administering a therapeutically effective amount of the drug to an individual having cancer. In such one embodiment, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent (e.g., one described below) to the individual. In further embodiments, the drug is for inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., tumor-associated macrophage-derived), tumor vascular structure (e.g., intratumor vascular structure or tumor-associated vascular structure), and / or tumor stromal function.

[0289] In further embodiments, the present invention provides a method for treating cancer. In one embodiment, the method comprises administering a therapeutically effective dose of an anti-HER2 antibody or antibody fragment to an individual having such cancer. In such one embodiment, the method further comprises administering a therapeutically effective dose of at least one additional therapeutic agent (described below) to the individual. The “individual” in any of the above embodiments may be a human.

[0290] In further embodiments, the present invention provides methods for inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., tumor-associated macrophages), tumor vascular structures (e.g., intratumor vascular structures or tumor-associated vascular structures), and / or tumor stromal function in an organism. In one embodiment, the method involves administering a therapeutically effective dose of an anti-HER2 antibody or antibody fragment to an organism to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce fragments of inflammatory cytokines (e.g., tumor-associated macrophages), inhibit the development of tumor vascular structures (e.g., intratumor vascular structures or tumor-associated vascular structures), and / or tumor stromal function.

[0291] In a further embodiment, the present invention provides a pharmaceutical formulation comprising, for example, one of the anti-HER2 antibodies or antibody fragments provided herein for use in any of the therapeutic methods described above, and at least one additional therapeutic agent as described below.

[0292] In each of the therapies described above and in each of the therapies, the antibody or antibody fragment of the present invention may be used alone, as an immune conjugate, or in combination with other agents during treatment. For example, the antibody of the present invention may be administered concurrently with at least one additional therapeutic agent. In certain embodiments, the additional therapeutic agent is an anti-angiogenic agent. In certain embodiments, the additional therapeutic agent is a VEGF antagonist (in some embodiments, an anti-VEGF antibody, e.g., bevacizumab). In certain embodiments, the additional therapeutic agent is an EGFR antagonist (in some embodiments, erlotinib). In certain embodiments, the additional therapeutic agent is a chemotherapeutic agent and / or a cell division inhibitor. In certain embodiments, the additional therapeutic agent is a taxoid (e.g., paclitaxel) and / or a platinum agent (e.g., carboplatinum). In certain embodiments, the additional therapeutic agent is an agent that enhances the patient's immune system or immune response.

[0293] Such combination therapies described above include combined administration (where two or more therapeutic agents are contained in the same or separate formulations) as well as individual administrations, in which case the administration of antibodies or antibody fragments may occur before, simultaneously with, and / or after the administration of additional therapeutic agents and / or adjuvants. Antibodies or antibody fragments may also be used in combination with radiotherapy.

[0294] Anti-HER2 antibodies or antibody fragments may be formulated, administered, and given in a manner consistent with good medical practice. Factors to consider in this context include the disorder being treated, the mammal being treated, the individual patient's clinical condition, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to the physician. Antibodies or antibody fragments may be formulated, optionally but not necessarily, with one or more drugs currently used to prevent or treat the disorder in question. The effective dose of such other drugs depends on the amount of antibody or antibody fragment present in the formulation, the type of disorder or treatment, and other factors considered above. These are generally used in the same doses and routes of administration described herein, or at approximately 1–99% of the doses described herein, or in any dose and route deemed empirically / clinically appropriate.

[0295] For the prevention or treatment of a disease, the appropriate dose of an antibody or antibody fragment (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the type of antibody or antibody fragment, the severity and course of the disease, whether the antibody or antibody fragment is administered for preventive or therapeutic purposes, previous therapies, the patient's medical history and response to the antibody or antibody fragment, and the discretion of the attending physician. The antibody or antibody fragment is preferably administered to the patient in a single dose or over a series of treatments. Depending on the type and severity of the disease, approximately 1 μg of antibody or antibody fragment / kg of patient weight to 40 mg of antibody or antibody fragment / kg of patient weight may be the initial candidate dose for administration to the patient, whether by one or more separate doses or by continuous infusions. A typical daily dose may range from approximately 1 μg of antibody or antibody fragment / kg of patient weight to 100 mg of antibody or antibody fragment / kg of patient weight or more, depending on the factors mentioned above. In the case of repeated administrations over several days or more, treatment will generally continue, depending on the condition, until the desired suppression of disease symptoms occurs. Such doses may be administered intermittently, for example, weekly or every three weeks (e.g., the patient receives approximately 2 to 20 doses of antibody or antibody fragments, or, for example, approximately 6 doses). One or more lower doses may be administered following an initial higher dose. However, other dosing regimens may be useful. The progression of this therapy can be easily monitored by conventional techniques and assays.

[0296] When administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor or another antibody or antibody fragment, or as an immune conjugate, the dosage of the antibody or antibody fragment is the same. Furthermore, polypeptides with anti-HER2 activity are administered in the same amount as the antibody or antibody fragment.

[0297] The amount of antibody or antibody fragment in a single dose of a pharmaceutical formulation remains the same when administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor, as an immune conjugate, or in combination with another antibody or antibody fragment against another antigen disclosed herein. Furthermore, polypeptides having anti-HER2 activity will be included in a single dose of the pharmaceutical formulation in the same amount as the antibody or antibody fragment.

[0298] In one embodiment, an anti-HER2 antibody or antibody fragment may be conjugated to an immune checkpoint inhibitor molecule or may form part of a bispecific antibody with an immune checkpoint inhibitor. The combination may be an anti-HER2 antibody or antibody fragment disclosed herein and an immune checkpoint inhibitor molecule administered as a separate molecule or as a bispecific antibody. Such a bispecific antibody has binding activity to the HER2 protein and a second binding activity to immune checkpoints.

[0299] Immune checkpoints can be selected from CTLA4, LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, and GITR (Zahavi and Weiner, International Journal of Molecular Sciences, vol.20, 158, 2019). Further immune checkpoints include B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS (Manni et al., Immune checkpoint blockade and its combination therapy with small-molecule inhibitors for cancer treatment, Bbacan, https: / / doi.org / 10.1016 / j.bbcan.2018.12.002, 2018).

[0300] The immune checkpoint is preferably CTLA4, PD-1, or PD-L1.

[0301] It should be understood that any of the above formulations or treatment methods may be carried out using the antibody fragment or immunoconjugate of the present invention, either in place of or in addition to the anti-HER2 antibody.

[0302] Enhancement of the host's immune function to fight tumors can be used in conjunction with the methods of the present invention. Conventional methods include (i) enhancement of APCs, for example, (a) injecting the tumor with DNA encoding an alloantigen of exogenous MHC, or (b) transfection of biopsy tumor cells with genes that increase the probability of tumor immune antigen recognition (e.g., immunostimulatory cytokines, GM-CSF, costimulatory molecules B7.1, B7.2), or (iii) adoptive cell immunotherapy, or treatment with activated tumor-specific T cells. Adoptive cell immunotherapy involves isolating tumor-infiltrating host T lymphocytes, for example, expanding the population in vitro through stimulation by IL-2, the tumor, or both. Furthermore, the isolated, dysfunctional T cells can also be activated by in vitro application of an anti-PD-L1 antibody. The thus activated T cells may then be re-administered to the host. One or more of these methods can be used in combination with the administration of the antibodies, antibody fragments, or immune conjugates of the present invention.

[0303] Traditional therapies for cancer include: (i) radiotherapy (e.g., radiotherapy, X-ray therapy, irradiation), or the use of ionizing radiation to kill cancer cells and shrink tumors; radiotherapy can be administered via external beam radiotherapy (EBRT) or via internal close-range radiotherapy; (ii) chemotherapy, or the application of cytotoxic drugs that generally affect rapidly dividing cells; (iii) targeted therapy, or drugs that specifically affect dysregulated proteins in cancer cells (e.g., tyrosine kinase inhibitors imatinib, gefitinib; monoclonal antibodies, photodynamic therapy); (iv) immunotherapy, or enhancing the host immune response (e.g., vaccines); (v) hormone therapy, or hormone blockade (e.g., if the tumor is hormone-sensitive); (vi) angiogenesis inhibitors, or blocking the formation and growth of blood vessels; and (vii) palliative care, or treatment aimed at improving the quality of care to reduce pain, nausea, vomiting, diarrhea, and bleeding. Analgesics such as morphine and oxycodone, and antiemetics such as ondansetron and aprepitant, can enable more aggressive treatment regimens.

[0304] In cancer treatment, any of the aforementioned conventional therapies for cancer immunotherapy may be administered before, after, or concurrently with the administration of anti-HER2 antibodies or antibody fragments. In addition, anti-HER2 antibodies or antibody fragments may be administered before, after, or concurrently with conventional cancer therapies such as the administration of tumor-binding antibodies (e.g., monoclonal antibodies, toxin-conjugated monoclonal antibodies) and / or chemotherapeutic agents.

[0305] L. Products and Kits In another aspect of the present invention, a product is provided comprising an anti-HER2 antibody or antibody fragment and other materials useful for the treatment, prevention, and / or diagnosis of the disorders described above. The product comprises a container and a label or accompanying document attached to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous infusion bags, etc. Containers may be formed from a variety of materials such as glass or plastic. The container may hold the composition, either by itself or in combination with another composition effective for the treatment, prevention, and / or diagnosis of the condition, and may have a sterile access port (for example, the container may be an intravenous infusion bag or a vial with a stopper that can be punctured by a subcutaneous needle). At least one activator in the composition is the antibody or antibody fragment of the present invention. The label or accompanying document indicates that the composition is used to treat a selected condition. Furthermore, the product may comprise (a) a first container (containing the composition, which comprises an antibody or antibody fragment), and (b) a second container (containing the composition, which comprises a further cytotoxic agent or other therapeutic agent). The product in this embodiment of the present invention may further comprise a document indicating that the composition can be used to treat a condition. Alternatively or additionally, the product may further comprise a second (or third) container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0306] It should be understood that any of the above products may contain the immunoconjugate of the present invention in place of, or in addition to, an anti-HER2 antibody or antibody fragment.

[0307] Finally, the present invention also provides a kit comprising at least one antibody or antibody fragment of the present invention. Kits comprising the polypeptide, antibody or antibody fragment, or antibody-drug conjugate of the present invention are useful in the detection (increase or decrease) of HER2 protein expression, or in therapeutic or diagnostic assays. The kits of the present invention may comprise antibodies coupled to a solid support, such as a tissue culture plate or beads (e.g., Sepharose beads). Kits can be provided comprising antibodies for in vitro detection and quantification of the HER2 protein, for example, in ELISA or Western blotting. Such antibodies useful for detection may be provided with labeling, such as fluorescent or radiolabeling.

[0308] The kit further includes instructions for their use. In some embodiments, the instructions include instructions required by the U.S. Food and Drug Administration for in vitro diagnostic kits. In some embodiments, the kit further includes instructions for diagnosing the presence or absence of HER2 protein in a sample based on the presence or absence of the HER2 protein in the sample. In some embodiments, the kit includes one or more antibodies or antibody fragments. In other embodiments, the kit further includes one or more enzymes, enzyme inhibitors or enzyme activators. In yet another embodiment, the kit further includes one or more chromatographic compounds. In yet another embodiment, the kit further includes one or more compounds used to prepare a sample for a spectroscopic assay. In a further embodiment, the kit further includes comparative reference material for interpreting the presence or absence of HER2 protein according to the intensity, color spectrum, or other physical attributes of the indicator.

[0309] The following examples illustrate, but are not limited to, the anti-HER2 antibodies of this disclosure. Various other suitable modifications and adaptations of conditions and parameters commonly encountered in the art and apparent to those skilled in the art are within the scope of this disclosure. [Examples]

[0310] Example 1: Binding activity of a humanized, conditionally active anti-HER2 antibody to the human HER2 protein. The binding activity of conditionally active anti-HER2 antibodies to the human HER2 protein was measured by ELISA using a benchmark antibody as a control. The benchmark antibody is indicated by "BM". For each conditionally active antibody, one of the heavy chain (HC) and light chain (LC) is designated in each figure. Unspecified heavy or light chains are the heavy or light chains of the benchmark antibody. The Y-axis represents the optical density (OD) at 450 nm. The X-axis represents the antibody concentration with a starting concentration of 300 ng / mL (log ng / mL). The results are shown in Figures 3A-3E.

[0311] The pH affinity ELISA assay was performed using the following protocol. pH affinity ELISA assay 1) Coat the ELISA plate with 1 μg / mL recombinant human HER2 antigen in 100 μL of carbonate-bicarbonate coating buffer. 2) Cover the plate with sealing film and incubate overnight at 4°C. 3) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 4) Wash the wells twice by dispensing 200 μL of pH 6.0 or pH 7.4 ELISA incubation buffer into each well, and aspirate the contents completely. 5) Add 200 μL of pH 6.0 or pH 7.4 ELISA incubation buffer to each well. Cover the plate with sealing film and place it in a plate shaker set to 50 rpm at room temperature for 60 minutes. 6) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 7) Serially dilute the antibody in ELISA incubation buffer at pH 6.0 or pH 7.4, starting with a 3-fold dilution at 300 ng / mL. 8) Add 100 μL / well of diluted antibody to the plate. 9) Cover the plate with sealing film and place it in a plate shaker set to 50 rpm at room temperature for 60 minutes. 10) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 11) Wash the wells three times by dispensing 200 μL of ELISA washing buffer at pH 6.0 or pH 7.4 into each well, and aspirate the contents completely. 12) Dilute the HRP secondary antibody to 1:2500 in ELISA incubation buffer at pH 6.0 or pH 7.4. 13) Add 100 μL of HRP secondary antibody, diluted in ELISA incubation buffer at pH 6.0 or pH 7.4, to each well. 14) Cover the plate with sealing film and place it in a plate shaker set to 50 rpm for 60 minutes at room temperature. 15) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 16) Wash the wells three times by dispensing 200 μL of ELISA washing buffer at pH 6.0 or pH 7.4 into each well, and aspirate the contents completely. 17) Dispense 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution into all wells of the plate. Incubate at room temperature for approximately 2 minutes and 15 seconds or 2 minutes. 18) Add 50 μL of 1N hydrochloric acid (HCl) per well to all wells of the plate. Read the plate at 450 nm using a PerkinElmer, EnSpire 2300 Multilabel Reader.

[0312] Example 2: Binding activity of conditionally active anti-HER2 antibody The binding activity of the same HER2 benchmark antibody and CAB antibody to the human HER2 protein at various pH values ​​was determined by pH range ELISA assay. The benchmark antibody is indicated by "BM". For each conditionally active antibody, the heavy chain (HC) and light chain (LC) are designated in Figure 4. Unspecified heavy or light chains are the heavy or light chains of the benchmark antibody. The Y axis is the optical density (OD) at 450 nm. The antibodies were diluted to 10 ng / mL in ELISA incubation buffers at various pH ranges from pH 5.0 to pH 7.4. The X axis shows the pH of the incubation buffer and wash buffer (pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4). The results are shown in Figure 4.

[0313] A pH range ELISA assay was performed using the following protocol. pH range ELISA assay 1) Coat the ELISA plate with 1 μg / mL recombinant human HER2 antigen in 100 μL of carbonate-bicarbonate coating buffer. 2) Cover the plate with sealing film and incubate overnight at 4°C. 3) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 4) Wash the wells twice by dispensing 200 μL of incubation buffer of various pH levels into each well, and aspirate the contents completely. 5) Add 200 μL of incubation buffers of various pH levels (pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4) to each well. Cover the plate with sealing film and place it in a plate shaker (set to 200 rpm) at room temperature for 60 minutes. 6) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 7) The test substance is sequentially diluted to 10 ng / mL in incubation buffers of various pH levels (pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4). 8) Add 100 μL / well of diluted test material to the plate. 9) Cover the plate with sealing film and place it in a plate shaker (set to 200 rpm) at room temperature for 60 minutes. 10) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 11) Wash the wells three times by dispensing 200 μL of washing buffer of various pH values ​​(pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4) into each well, and aspirate the contents completely. 12) Dilute the HRP secondary antibody to 1:2500 in incubation buffers of various pH levels (pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4). 13) Add 100 μL of horseradish peroxidase (HRP) secondary antibody, diluted in incubation buffers of various pH levels (pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4), to each well. 14) Cover the plate with sealing film and place it in a plate shaker (set to 200 rpm) at room temperature for 60 minutes. 15) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 16) Wash the wells three times by dispensing 200 μL of washing buffer of various pH values ​​(pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4) into each well, and then aspirate the contents completely. 17) Dispense 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution into all wells of the plate. Incubate at room temperature for 3 minutes. 18) Add 50 μL of 1N hydrochloric acid (HCl) per well to all wells of the plate. Read the plate at 450 nm using a PerkinElmer EnSpire 2300 Multilabel Reader.

[0314] Example 3: Binding activity of conditionally active anti-HER2 antibody as measured by FACS Conditionally active anti-HER2 antibodies were analyzed for binding to the HER2 protein using the benchmark antibody BM as a control. The binding activity of these anti-HER2 antibodies to the HER2 protein expressed on SKBR3 cancer cells was measured by fluorescence-activated cell sorting (FACS) at two different pH values, 6.0 and 7.4 (ATCC, catalog number HTB30). Different concentrations of antibody (10 μg / mL, 3.3 μg / mL, 1.1 μg / mL, and 0.37 μg / mL) were used. The antibody was first diluted to 10 μg / mL in FACS buffer at pH 6.0 or pH 7.4, and then serially diluted 3-fold in FACS buffer at pH 6.0 or pH 7.4. SKBR3 cells (ATCC, catalog number HTB30) were maintained in SKBR3 culture medium (McCoy's + 10% FBS). Cells were routinely subcultured twice weekly. Cells were harvested during the exponential growth phase and counted for plating.

[0315] The median fluorescence intensity (MFI) of Alexa Fluor 488 (AF488) in cell singlets was plotted using GraphPad Prism software version 7.03. Conditionally active anti-HER2 antibodies consistently showed higher binding activity to the HER2 protein expressed in SKBR3 cells at pH 6.0 (blue) than at pH 7.4 (orange). See Figure 5. Y-axis: MFI. X-axis: Test antibody at different concentrations. MFI sub BK: True fluorescence is obtained by subtracting the median fluorescence intensity of the secondary antibody-only sample from the median fluorescence intensity of the test antibody, thereby subtracting background fluorescence.

[0316] The test protocol used is shown below. Cell staining using test antibodies 1) Following the vendor's instructions, fill the T-75 flask and culture medium with 3 × 10 6 Seed individual cells. 2) On the day of the FACS analysis, remove and discard the culture medium. 3) Briefly rinse the cell layer with PBS solution. 4) Add 1.5 mL of Detachin solution to each T-75 flask. Wait until the cell layer is dispersed. 5) Add 4.5 mL of culture medium to the corresponding cell line and resuspend the cells by gentle pipetting. 6) Pool the cells and transfer the cell suspension to a 50 mL conical tube. 7) After counting the cells using trypan blue staining, centrifuge at 1500 rpm for 5 minutes at 4°C. 8) Wash the cells once with phosphate-buffered saline (PBS). 9) Add 3.5 × 10⁶ to FACS buffer at pH 6.0 or pH 7.4 6 Resuspend the cells in cells / mL. 10) Place 3.5 × 10¹⁶ units in 100 μL of pH 6.0 or pH 7.4 FACS buffer in a 96-well U-bottom plate. 5 Aliquot the cells. 11) Centrifuge the cells and discard the buffer solution. 12) Serially dilute the antibody in FACS buffer at pH 6.0 or pH 7.4, starting with a 3-fold dilution at 10 μg / mL. 13) Add 100 μL / well of diluted antibody to the cells, gently mix the wells, and incubate on ice for 1 hour with shaking (200 rpm). 14) Centrifuge the cells at 1500 rpm for 5 minutes at 4°C. Wash the cells twice with 150 μL of pH 6.0 or pH 7.4 washing buffer. 15) Dilute the goat anti-human IgG AF488 antibody to 1:300 with FACS buffer at pH 6.0 or pH 7.4. 16) Add 100 μL of the diluted antibody from the above steps to the cells, protect from light, and incubate on ice for 45 minutes with shaking (200 rpm). 17) Pellet the cells and wash them three times with 150 μL of pH 6.0 or pH 7.4 washing buffer. 18) Fix the cells with 4% paraformaldehyde diluted in 1×PBS for 10 minutes at room temperature, then wash the cells with 1×PBS. 19) Resuspend the cells in 100 μL of 1XPBS. 20) Analyze cells using a NovoCyte flow cytometer with Ex488nm / Em530nm. Collect at least 5,000 singlet cells at each data point.

[0317] Example 4: Binding activity of conditionally active anti-HER2 antibody to human HER2 protein The binding activity of conditionally active anti-HER2 antibodies to the human HER2 protein was measured by ELISA using a benchmark antibody as a control. The benchmark antibody is indicated by "BM". For each conditionally active antibody, the heavy chain (HC) is specified in Figures 6A-6B. Each antibody tested had a light chain LC-A032D. The antibodies were first diluted to 100 ng / mL in ELISA incubation buffer at pH 6.0 or pH 7.4. Then, the 100 ng / mL antibody was serially diluted 3-fold in ELISA incubation buffer at pH 6.0 or pH 7.4.

[0318] The results are shown in Figures 6A-6B. The Y-axis represents the optical density (OD) at 450 nm. The X-axis represents the antibody concentration (log ng / mL) with a starting concentration of 100 ng / mL.

[0319] Example 5 - Binding activity of HER2 antibody to cynoHER2 protein at pH 6.0 and pH 7.4 as determined by pH affinity ELISA assay. The binding activity of conditionally active anti-HER2 antibodies to the cynoHER2 protein was measured by ELISA using a benchmark antibody as a control. The benchmark antibody is indicated by "BM". For each conditionally active antibody, the heavy chain (HC) is specified in Figures 6A-6B. Each antibody tested had a light chain LC-A032D. The antibodies were first diluted to 100 ng / mL in ELISA incubation buffer at pH 6.0 or pH 7.4. Then, the 100 ng / mL antibody was serially diluted 3-fold in ELISA incubation buffer at pH 6.0 or pH 7.4.

[0320] The results are shown in Figures 7A-7B. The Y-axis represents the optical density (OD) at 450 nm. The X-axis represents the antibody concentration (log ng / mL) with a starting concentration of 100 ng / mL.

[0321] The pH affinity ELISA assays in Examples 4-5 were performed using the following protocol. pH affinity ELISA assays used in Examples 4-5 1) Coat the ELISA plate with 1 μg / mL of recombinant human or cynoHER2 antigen in 100 μL of carbonate-bicarbonate coating buffer (see legend in the figure for species information). 2) Cover the plate with sealing film and incubate overnight at 4°C. 3) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 4) Wash the wells twice by dispensing 200 μL of pH 6.0 or pH 7.4 ELISA incubation buffer into each well, and aspirate the contents completely. 5) Add 200 μL of pH 6.0 or pH 7.4 ELISA incubation buffer to each well. Cover the plate with sealing film and place it in a plate shaker set to 50 rpm at room temperature for 60 minutes. 6) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 7) Serially dilute the antibody in ELISA incubation buffer at pH 6.0 or pH 7.4, starting with a 3-fold dilution at 100 ng / mL. 8) Add 100 μL / well of diluted antibody to the plate. 9) Cover the plate with sealing film and place it in a plate shaker set to 50 rpm at room temperature for 60 minutes. 10) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 11) Wash the wells three times by dispensing 200 μL of ELISA washing buffer at pH 6.0 or pH 7.4 into each well, and aspirate the contents completely. 12) Dilute the HRP secondary antibody to 1:2500 in ELISA incubation buffer at pH 6.0 or pH 7.4. 13) Add 100 μL of HRP secondary antibody, diluted in ELISA incubation buffer at pH 6.0 or pH 7.4, to each well. 14) Cover the plate with sealing film and place it in a plate shaker set to 50 rpm for 60 minutes at room temperature. 15) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 16) Wash the wells three times by dispensing 200 μL of ELISA washing buffer at pH 6.0 or pH 7.4 into each well, and aspirate the contents completely. 17) Dispense 50 μL of TMB substrate solution into each well of the plate. Incubate at room temperature for approximately 2 minutes and 15 seconds or 2 minutes. 18) Add 50 μL of 1N HCl per well to all wells of the plate. Read the plate at 450 nm using a PerkinElmer, EnSpire 2300 Multilabel Reader.

[0322] Example 6: Binding activity of conditionally active anti-HER2 antibody to human HER2 protein The binding activity of HER2 benchmark antibodies and CAB antibodies to the human HER2 protein at various pH values ​​was determined by pH range ELISA assays. Benchmark antibodies are indicated by "BM". For each conditionally active antibody, the heavy chain (HC) is specified in Figure 8. Each antibody tested had a light chain LC-A032D. The Y axis represents optical density (OD) at 450 nm. Antibodies were diluted to 100 ng / mL in ELISA incubation buffers at various pH ranges from pH 5.0 to pH 7.4. The X axis represents the pH of the incubation buffer and wash buffer (pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4).

[0323] The average OD values ​​for each pH were plotted against the pH of the buffer using GraphPad Prism 5.03. Curve fitting was performed using the software's built-in 4-parameter model. Binding activity at pH 6.0 was set to 100%. The results are shown in Figure 8.

[0324] The inflection point of the pH curve (50% binding activity) is equal to the parameter EC50 of the compatibility equation. The pH inflection points are shown in Table 2 below. [Table 9]

[0325] A pH range ELISA assay was performed using the following protocol. pH range ELISA assay 1) Coat the ELISA plate with 1 μg / mL recombinant human HER2 antigen in 100 μL of carbonate-bicarbonate coating buffer. 2) Cover the plate with sealing film and incubate overnight at 4°C. 3) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 4) Wash the wells twice by dispensing 200 μL of incubation buffer of various pH levels into each well, and aspirate the contents completely. 5) Add 200 μL of incubation buffers of various pH levels (pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4) to each well. Cover the plate with sealing film and place it in a plate shaker (set to 200 rpm) at room temperature for 60 minutes. 6) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 7) The test substance is sequentially diluted to 100 ng / mL in incubation buffers of various pH levels (pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4). 8) Add 100 μL / well of diluted test material to the plate. 9) Cover the plate with sealing film and place it in a plate shaker (set to 200 rpm) at room temperature for 60 minutes. 10) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 11) Wash the wells three times by dispensing 200 μL of washing buffer of various pH values ​​(pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4) into each well, and aspirate the contents completely. 12) Dilute the horseradish peroxidase (HRP) secondary antibody to 1:2500 in incubation buffers of various pH values ​​(pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4). 13) Add 100 μL of HRP secondary antibody, diluted in incubation buffers of various pH levels (pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4), to each well. 14) Cover the plate with sealing film and place it in a plate shaker (set to 200 rpm) at room temperature for 60 minutes. 15) Decant the plate and gently tap it against a stack of paper towels to remove any remaining liquid. 16) Wash the wells three times by dispensing 200 μL of washing buffer of various pH values ​​(pH 5.0, 5.5, 6.0, 6.5, 7.0, and 7.4) into each well, and then aspirate the contents completely. 17) Dispense 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution into all wells of the plate. Incubate at room temperature for 3 minutes. 18) Add 50 μL of 1N hydrochloric acid (HCl) per well to all wells of the plate. Read the plate at 450 nm using a PerkinElmer EnSpire 2300 Multilabel Reader.

[0326] Example 7 - Evaluation of the in vivo efficacy of a conditionally active antibody in a subcutaneous xBT474 CDX model in BALB / c nude mice. In vivo testing of the antibody was performed in BALB / c nude mice as described below. [Table 10] Note: aN: The number of animals per group. b. The dosage was adjusted to 10 μl / g body weight. material animal Species: House mouse Category: BALB / c nude Age: 6-8 weeks Sex: Female Weight: 18-22g Number of animals: 64 mice + spares Laboratory equipment and reagents Equipment Equipment name: Centrifugal separator Supplier: Eppendorf Device type: 5424R Equipment name: CO2 incubator Supplier: Thermo Fisher Equipment type: Heracell 240i Equipment name: Balance Supplier: Changzhou Keyuan Electronic Instrument Co., Ltd. Device type: JA20002 Instrument name: Digimatic caliper Supplier: MITUTOYO / ABSLUTE Device type: CD-6”ASX reagent Product identification: Phosphate-buffered saline (PBS) Manufacturer: Hyclone Catalog number: SH30256.01 Lot number: AD2158027 Product identification: Hybri-Care Manufacturer: Gibco Catalog number: ATCC46-X Lot number: 80719180 Product Identification: Penicillin / Streptomycin Manufacturer: HyClone Catalog number: 15240-062 Lot number: 1989506 Product Identification: Trypsin-EDTA Manufacturer: Gibco Catalog number: 25200-072 Lot number: 2001888 Product identification: Fetal bovine serum Manufacturer: Hyclone Catalog number: SV30087.03 Lot number: RBC35932

[0327] Experimental methods and procedures cell culture xBT474 tumor cells (ATCC® HTB-20®) were maintained in vitro at 37°C with 5% CO2 in air as monolayer cultures in Hybri-Care medium supplemented with 1.5 g / l sodium bicarbonate, 10% heat-inactivated fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells that had grown during the exponential growth phase were collected and counted for tumor inoculation.

[0328] Tumor inoculation and animal grouping Each mouse was given xBT474 tumor cells (10 × 10) in 0.2 ml of PBS for tumor development. 6 Three days prior to subcutaneous cell inoculation of Matrigel (1:1) into the right flank, a 0.36 mg 17-β-estradiol pellet was administered. The procedure was performed on patients with an average tumor size of approximately 207 mm. 3 When the tumor volume reached a certain level, the study was initiated on day 16 after tumor inoculation. Animals were assigned to groups according to tumor volume using an Excel-based stratified randomization program. Each group consisted of 8 tumor-bearing mice. The test substance was administered according to the experimental design shown in Table 3.

[0329] In vivo efficacy evaluation of conditionally active antibodies in a subcutaneous xBT474 CDX model in BALB / c nude mice. In vivo testing of the antibody was performed in BALB / c nude mice as described below. Table 3. Tumor size was measured in two dimensions twice a week using calipers and calculated using the following formula: Tumor volume (TV) = 0.5axb 2 (a and b are the longest and shortest diameters of the tumor, respectively). The tumor size was then used to calculate the T / C ratio, tumor growth inhibition (TGI), and relative tumor volume (RTV) values. The T / C value (percent) is an indicator of antitumor efficacy, where T and C are the mean volumes of the treatment group and control group on a given day, respectively. The TGI for each treatment group was calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100;T i V is the mean tumor volume of the treatment group on a given day, T0 is the mean tumor volume of the treatment group on day 0, and V i is, T i V0 is the mean tumor volume of the vehicle control group on the same day, and V0 is the mean tumor volume of the vehicle group on day 0. Individual RTVs were calculated by dividing the tumor volume on a particular day by its volume on day 0. The RTV values ​​for each mouse were calculated individually and then used to calculate the average RTV for the group.

[0330] statistical analysis The mean tumor volume and standard error for each group at different time points were calculated (Table 4). Based on data obtained on days 23 and 27 after the start of treatment, a statistical analysis of the difference in tumor volume between the groups was performed. [Table 11]

[0331] One-way ANOVA was performed to compare mean tumor volume and RTV between groups. A significant F-statistic was obtained, and group comparisons were performed using the Games-Howell test. All data were analyzed using IBM® SPSS Statistics® software (version 17.0). A p<0.05 value was considered statistically significant.

[0332] Mortality, morbidity, and weight gain or loss Animal body weight was regularly monitored as an indicator of toxicity. During this study, the group did not exhibit significant weight loss (≥10%). See Figure 9A. No deaths or morbidities were observed. Therefore, no apparent toxicity was observed with the current dosing regimen in association with antibody administration to BALB / c nude mice with tumors.

[0333] The changes in body weight and relative body weight for the different groups are shown in Figures 9A and 9B, respectively. Tumor growth inhibition is shown in Tables 5-6 below. The numbering of substitutions referenced in Tables 5-6 is based on the BAP-130 benchmark antibody in Figure 2. [Table 12] Note: a. Mean ± standard error b. Tumor growth inhibition is calculated by dividing the group-average tumor volume of the treatment group by the group-average tumor volume of the vehicle control group (T / C). c. p-value calculated based on tumor size on day 23. d. p-value calculated based on RTV on day 23. [Table 13] Note: a. Mean ± standard error b. Tumor growth inhibition is calculated by dividing the group-average tumor volume of the treatment group by the group-average tumor volume of the vehicle control group (T / C). c. p-value calculated based on tumor size on day 27. d. p-value calculated based on RTV on day 27.

[0334] The tumor growth curve is shown in Figure 9C.

[0335] The average tumor size in the vehicle-treated group was 1,685 mm on day 23 after the start of treatment. 3The RTV reached (RTV = 8.08 ± 0.51). All tested antibodies at a dose level of 3 mg / kg (BA-130-00-01, BA-130-03-02, BA-130-03-05, BA-130-03-06, BA-130-03-07, BA-130-03-08) showed dramatic antitumor activity, resulting in complete remission in most mice treated within 16–27 days (T / C < 1%, TGI > 114%, p < 0.001, PG-D23, Figure 9C). The difference in tumor volume between the TA group and the isotype group was also significant (T / C < 1%, TGI > 118%, p < 0.001, PG-D27).

[0336] B12 (isotype control antibody) slightly delayed tumor growth, but this result was not statistically significant compared to the vehicle group (T / C=73%, TGI=31%, p=0.387, PG-D23).

[0337] No severe weight loss or death / morbidity events were observed throughout the study. Therefore, no apparent toxicity was observed in association with antibody administration.

[0338] All procedures related to the handling, care, and treatment of animals in the study were carried out in accordance with the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and the guidelines approved by the WuXi AppTec Animal Experimentation Committee (IACUC). At routine monitoring points, animals were checked daily for normal behavior such as mobility, food and water consumption (simply by observation), weight gain / loss (weight was measured twice a week), eye / hair matting, and any other abnormal effects of tumor growth and any effects of treatment as described in the protocol. Deaths and observed clinical signs were recorded for each animal in each group.

[0339] Example 8 - A polyspecific antibody that binds to CD3 and HER2 We constructed multispecific antibodies that bind to CD3 and HER2. One multispecific antibody provided a butterfly configuration WT-HER2×WT-CD3 using an unconditionally active binding site (scFv antibody) for CD3 (WT-CD3) paired with an unconditionally active binding site (IgG antibody) for HER2 (WT-HER2) (Figures 12 and 13A-13D). Similarly, a second multispecific antibody formed a butterfly configuration WT-HER2×CAB-CD3 using an unconditionally active binding site (IgG antibody) for HER2 (WT-HER2) paired with a conditionally active binding site (scFv antibody) for CD3 (CAB-CD3) (Figures 12 and 13A-13D). The third polyspecific antibody formed a butterfly configuration CAB-HER2 × CAB-CD3 by using a conditionally active binding site (IgG antibody) for HER2 (CAB-HER2) paired with a conditionally active (scFv antibody) for CD3 (CAB-CD3) (Figures 12 and 13A-13D).

[0340] Using an ELISA assay, bispecific antibodies were assayed for affinity to CD3 and HER2 at pH 6.0 and pH 7.4, respectively (Figures 13A-13D). These three polyspecific antibodies were compared to isotype × WT CD3. The ELISA assay described in this application used the following protocol: 1. The day before ELISA, 96-well plates were coated with 100 μl of 0.5 μg / ml recombinant CD3 or HER2 in ELISA coating buffer overnight at 4°C. 2. Dilute the sample in the ELISA assay buffer. 3. The buffer was wiped off the antigen-coated plate and the plate was blot-dried on a paper towel. A plate containing 4,200 μl of ELISA assay buffer was blocked at room temperature for 1 hour. Add 5,100 μl of diluted sample to each well. 6. Incubate the plate at room temperature for 1 hour. 7. Prepare the secondary antibody in the screening buffer according to the plate layout. 8. Wipe off the buffer from the plate and blot dry with a paper towel. 9. Wash the plate a total of three times with ELISA washing buffer. Add 1 ug / ml of human HER2 fused to mouse IgG Fc in 10,100 μl of ELISA assay buffer to each well. 11. Incubate the plate at room temperature for 1 hour. 12. Wipe off the buffer solution from the plate and blot dry with a paper towel. 13. Wash the plate a total of three times with ELISA washing buffer. 14. Wipe off the buffer solution from the plate and dry the blot with a paper towel. Add 15,100 μl of anti-mouse HRP secondary antibody diluted to 1:2500 to each well. 16. Incubate the plate at room temperature for 1 hour. 17. Wipe off the buffer solution from the plate and blot dry with a paper towel. 18. Wash the plate a total of three times with ELISA washing buffer. 19. Wipe off the buffer solution from the plate and dry the blot with a paper towel. 20. Add 50 μl of 3,3',5,5'-tetramethylbenzidine (TMB) substrate according to the plate layout. Stop the generation with 21.50 μl of 1N HCl. 22. Use a plate reader to read at OD450nm.

[0341] WT / CAB HER2 × CAB CD3 butterfly dual-specificity pH sandwich ELISA assay Tables 7 and 8 show the binding activity of WT HER2×WT CD3, WT HER2×CAB CD3-BF45, and CAB HER2-24-06×CAB CD3-BF19 bispecific antibodies at various pH values, as determined by pH sandwich ELISA assays. [Table 14] [Table 15]

[0342] WT / CAB HER2 × CAB CD3 Butterfly Dual-Specificity pH Range ELISA Assay Table 9 shows the binding activity of WT HER2×WT CD3, WT HER2×CAB CD3-BF45, and CAB HER2-24-06×CAB CD3-BF-19 bispecific antibodies at various pH values, as determined by pH range ELISA assays. [Table 16]

[0343] Example 9 - A polyspecific antibody that binds to CD3 and HER2 In this example, a polyspecific antibody that binds to CD3 and HER2 is constructed, which includes heavy and light chains as shown in Table 10 below. The polyspecific antibody was prepared as described in Example 8 and named as follows. [Table 17] JPEG0007856312000064.jpg224170JPEG0007856312000065.jpg72170

[0344] Example 10 - Surface Plasmon Resonance (SPR) Assay SPR analysis. The binding kinetics of anti-CTLA4 antibody were measured by surface plasmon resonance on a flat amine sensor tip using an SPR2 / 4 instrument (Sierra Sensors, Hamburg, Germany). The SPR sensor contained four flow cells (FC1-FC4), each of which could be addressed individually or in groups. huHER2-His was immobilized on FC2, cynoHER2-His on FC3, and huCD on FC4. No protein was immobilized on FC1 (control surface). All injections were performed at a flow rate of 25 μL / min and 25°C. The sensor surface was activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (200 mM / 50 mM) for 480 seconds. The surface was inactivated by injecting human HER2-His (0.5 μg / mL in 10 mM NaAc, pH 5.5) for 480 seconds and then injecting 1 M ethanolamine-HCl for 480 seconds. CynoHER2-His and huCD3 were immobilized using the same conditions as described for huHER2-His. The control surface was activated and deactivated using the same conditions, but without protein injection. PBST buffer (PBS pH 7.4 containing 0.05% TWEEN20) was used as the electrophoresis buffer for surface preparation. The electrophoresis solution was switched to PBST with 30 mM sodium bicarbonate, and the pH was adjusted as shown in the figure before injecting the analytes. The apparatus was equilibrated in the electrophoresis solution for 1 hour before injecting the first analyte. 100 μL of analytes diluted in the corresponding electrophoretic solutions (25 nM, 10 nM, 5 nM, 2.5 nM, 1.25 nM, 0.625 nM, and 0.0 nM) were injected into overflow cells 1-4. The off-rate was measured for 360 seconds. After each cycle of interaction analysis, the tip surface was regenerated by injecting 6 μL of 10 mM glycine (pH 2.0). Flow cell 1, which did not contain immobilized protein, was used as a reference surface for subtraction. Furthermore, data with only buffer as the analyte (0 nM analyte) was subtracted from each electrophoresis.Using the provided analytical software Analyzer R2 (Sierra Sensors), the double-subtracted data was fitted using a 1:1 coupling model. The molar concentration of the analyte was calculated using a molecular weight of 200 kDa.

[0345] For WT HER2×WT CD3, WT HER2×CAB CD3-BF45, and CAB HER2-24-06×CAB CD3-BF19 with ligands huHER2-His, cyno-HER2-His, and huCD3-His at pH 6.0, pH 6.5, and pH 7.4, the dissociation constant (K) was determined using SPR binding analysis. d The following measurements were taken. The results are shown in Table 11 and Figures 15A to 17I below. [Table 18]

[0346] While many features and advantages of the present invention, along with details of its structure and function, have been described above, it should be understood that this disclosure is illustrative only, and detailed modifications may be made, particularly to matters relating to the shape, size, and arrangement of components within the principles of the present invention, to the full extent indicated by the broad general meaning of the terms expressed in the accompanying claims.

[0347] All documents referenced herein are either incorporated herein in their entirety by reference, or provide alternative disclosures which are particularly relied upon. The applicant does not intend to dedicate the disclosed embodiments to the public, and any disclosed modifications or changes may not be literally included within the claims, so they shall be considered part of the invention under the doctrine of equivalents.

Claims

1. An antibody that binds to the HER2 protein or an antigen-binding antibody fragment thereof, wherein the binding affinity to the HER2 protein at pH 5.0 to 6.8 is higher than the binding affinity to the HER2 protein at pH 7.0 to 7.

6. The antibody or its antigen-binding antibody fragment comprises a heavy chain variable region and a light chain variable region, The heavy chain variable region includes three complementarity-determining regions having sequences H1, H2, and H3. The H1 sequence is GFX １ IKDTYIH (Sequence ID 1) The aforementioned H2 array is X ２ IX ３ PTX ４ X ５ YX ６ X ７ YADSVKG (Sequence ID 2) The H3 sequence is WGGDGFYX ８ MDY (Sequence ID 3) where X １ is N or W, X ２ is R or K, X ３ is Y or K or D, X ４ is N or A, X ５ is G or K, X ６ is T or D, X ７ is R or E, X ８ is A or E, The light chain variable region includes three complementarity-determining regions having sequences L1, L2, and L3. The L1 sequence is RASQDVNTX ９ VA (Sequence ID 4), The L2 sequence is SASFLYS (Sequence ID 5), The L3 array is QQX １０ YTTPPPT (Sequence ID 6), In the formula, X ９ However, it is A or D, and X １０ However, it is H, D, or E, However, X １ ~X ８ However, if they are N, R, Y, N, G, T, R, and A respectively, then X ９ However, not A, but X １０ However, not H X １ If X is W, ２ ~X １０ However, these are not R, Y, N, G, T, R, A, A and H respectively, X ３ If is K or D, then X １ ~X ２ and X ４ ~X １０ However, these are not N, R, N, G, T, R, A, A and H respectively, X ４ If A, then X １ ~X ３ and X ５ ~X １０ However, these are not N, R, Y, G, T, R, A, A and H respectively, X ７ If E, then X １ ~X ６ and X ８ ~X １０ However, these are not N, R, Y, N, G, T, A, A and H respectively, and X ８ If E, then X １ ~X ６ and X ８ ~X １０ However, these are not N, R, Y, N, G, T, R, A and H respectively, The antibody or its antigen-binding antibody fragment, compared to a parent antibody or its antibody fragment where X1 to X10 are N, R, Y, N, G, T, R, A, A, and H respectively, has a single substitution in one of the H1, H2, and H3 sequences in the heavy chain variable region, and up to two substitutions in the L1 and L3 sequences in the light chain variable region. An antibody or an antigen-binding antibody fragment thereof.

2. A bispecific antibody or antigen-binding antibody fragment comprising the antibody or antigen-binding antibody fragment described in Claim 1, wherein the bispecific antibody or antigen-binding antibody fragment further comprises six anti-CD3 complementarity determining regions L4, L5, L6, L7, L8, and L9, The L4 sequence is GFTFNTYAMN (sequence number 54), The L5 sequence is RIRSKYNNYATYYADSVKD (sequence number 55), The L6 array is HX １１ NFX １２ NSKVSWFX １３ Y (Sequence ID 70) The L7 array is RSSX １４ It is GAVTTTSNYDN (Sequence ID 71), The L8 sequence is GTNKRAP (sequence number 58), The L9 sequence is ALWYSNLWV (sequence number 59), In the formula, X １１ However, it is G, S, A, or T, and X １２ However, it is G or P, and X １３ However, it is A or Q, and X １４ However, it is either T or A, The binding affinity to CD3 at pH 5.0–6.8 is higher than the binding affinity to CD3 at pH 7.0–7.

6. The antibody or its antigen-binding antibody fragment, compared to a parent antibody or antibody fragment where X1 to X14 are N, R, Y, N, G, T, R, A, A, H, G, G, A, and T respectively, has a single substitution in one of the H1, H2, and H3 sequences of the heavy chain variable region, and up to three substitutions in the L1, L3-L4, and L6-L7 sequences of the light chain variable region. The antibody according to claim 1 or the antigen-binding antibody fragment thereof.

3. The antibody or antigen-binding antibody fragment thereof, or a bispecific antibody or antigen-binding antibody fragment thereof, according to claim 1 or 2, wherein the H1 sequence is GFWIKDTYIH (SEQ ID NO: 7) or GFNIKDTYIH (SEQ ID NO: 50).

4. The aforementioned H2 sequence is KIYPTNGYTRYADSVKG (Sequence No. 8) RIKPTNGYTRYADSVKG (Sequence No. 9) RIDPTNGYTRYADSVKG (Sequence No. 10) RIYPTAGYTRYADSVKG (Sequence No. 11) RIYPTNKYTRYADSVKG (Sequence No. 12) RIYPTNGYDRYADSVKG (Sequence No. 13) RIYPTNGYTEYADSVKG (Sequence ID 14) and An antibody or antigen-binding antibody fragment thereof, or a bispecific antibody or antigen-binding antibody fragment thereof, selected from the group consisting of RIYPTNGYTRYADSVKG (Sequence ID 49), according to claim 1 or 2.

5. The antibody or antigen-binding antibody fragment thereof, or a bispecific antibody or antigen-binding antibody fragment thereof, according to claim 1 or 2, wherein the H3 sequence is WGGDGFYEMDY (SEQ ID NO: 15) or WGGDGFYAMDY (SEQ ID NO: 51).

6. The antibody or antigen-binding antibody fragment thereof, or a bispecific antibody or antigen-binding antibody fragment thereof, according to claim 1 or 2, wherein the L1 sequence is RASQDVNTDVA (SEQ ID NO: 16) or RASQDVNTAVA (SEQ ID NO: 52).

7. The antibody or antigen-binding antibody fragment thereof, or a bispecific antibody or antigen-binding antibody fragment thereof, according to claim 1 or 2, wherein the L3 sequence is QQDYTTPPPT (SEQ ID NO: 17), QQEYTTPPPT (SEQ ID NO: 18), or QQHYTTPPPT (SEQ ID NO: 53).

8. The bispecific antibody or antigen-binding antibody fragment according to claim 2, wherein the L6 sequence is one of sequence numbers 56 and 60-67, and the L7 sequence is sequence number 57, 68, or 69.

9. The antibody or antigen-binding antibody fragment thereof according to claim 1 or 2, or the bispecific antibody or antigen-binding antibody fragment thereof, wherein the heavy chain variable region is EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAKIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 19) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTEYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 20) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIKPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 21) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIDPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 22) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTAGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 23) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNKYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 24) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYDRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 25) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYEMDYWGQGTLVTVSS (Sequence No. 26) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVES (Sequence ID 27), or It is one of the sequences EVQLVESGGGLLVQPGGSLRLSCAASGFWIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRRWGGDGFYAMDYWGQGTLVTVSS (Sequence ID 28), The aforementioned light chain variable region is DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Sequence ID 29) DIQMTQSPSSLSASVGDRVTITCRASQDVNTDVAWYQQKPGKAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Sequence ID 30) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQDYTTPPTFGQGTKVEIK (Sequence ID 31) or It is one of the sequences DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQEYTTPPTFGQGTKVEIK (Sequence ID 32), However, if the heavy chain variable region is sequence number 20, 21, 22, 23, 26, 27, or 28, the light chain variable region is not sequence number 29. The aforementioned antibody or its antigen-binding antibody fragment, or a bispecific antibody or its antigen-binding antibody fragment.

10. The heavy chain variable region is EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 33) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAKIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 19) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTEYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 20) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIKPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 21) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIDPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 22) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTAGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 23) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNKYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 24) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYDRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 25) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYEMDYWGQGTLVTVSS (Sequence No. 26) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVES (Sequence ID 27), or It is one of the sequences EVQLVESGGGLLVQPGGSLRLSCAASGFWIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRRWGGDGFYAMDYWGQGTLVTVSS (Sequence ID 28), The aforementioned light chain variable region is DIQMTQSPSSLSASVGDRVTITCRASQDVNTDVAWYQQKPGKAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Sequence ID 30) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQDYTTPPTFGQGTKVEIK (Sequence ID 31) or An antibody or antigen-binding antibody fragment thereof, or a bispecific antibody or antigen-binding antibody fragment thereof, which is one of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQEYTTPPTFGQGTKVEIK (Sequence ID 32), according to claim 1 or 2.

11. The heavy chain variable region and the light chain variable region are EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAKIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRRWGGDGFYAMDYWGQGTLVTVSS (Sequence ID 19) and An antibody or antigen-binding antibody fragment thereof, or a bispecific antibody or antigen-binding antibody fragment thereof, selected from DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAAPKLLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Sequence ID 29), according to claim 9.

12. The aforementioned heavy chain variable region is EVQLVESGGGLLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRRWGGDGFYAMDYWGQGTLVTVSS (Sequence ID 33), The aforementioned light chain variable region is DIQMTQSPSSLSASVGDRVTITCRASQDVNTDVAAWYQQKPGKAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Sequence ID 30), DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQDYTTPPTFGQGTKVEIK (Sequence ID 31), or An antibody or antigen-binding antibody fragment thereof according to claim 10, or a bispecific antibody or antigen-binding antibody fragment thereof, which is one of DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAAPKLLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQEYTTPPTFGQGTKVEIK (Sequence ID 32).

13. The aforementioned light chain variable region is DIQMTQSPSSLSASVGDRVTITCRASQDVNTDVAWYQQKPGKAPKLLLIYSASFLYSGVPPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK (Sequence ID 30) The aforementioned heavy chain variable region is EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 33), EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAKIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 19) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTEYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 20) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIKPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 21) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIDPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 22) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTAGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 23) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNKYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 24) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYDRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS (Sequence No. 25) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYEMDYWGQGTLVTVSS (Sequence No. 26) EVQLVESGGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVES (Sequence ID 27), or An antibody or antigen-binding antibody fragment thereof, or a bispecific antibody or antigen-binding antibody fragment thereof, which is one of EVQLVESGGGLLVQPGGSLRLSCAASGFWIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISAADTSKNTAYLQMNSLRAEDTAVYYCSRWGGGDGFYAMDYWGQGTLVTVSS (Sequence ID 28), according to claim 9.

14. The aforementioned heavy chain variable region is EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIKPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 35), EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNKYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 36), EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYDRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 37), EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYEMDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 38), or EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVT VESASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPA It is one of the sequences PELLGGGPSVFLFPPKPKDTLMISRTPEPEVTCVVVDVSSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 39), The aforementioned light chain variable region is DIQMTQSPSSLSASVGDRVTITCRASQDVNTDVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSRSGGGGEVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHSNFGNSKVSWFQYWGQGTLVTVSSGGGSGGGSGGGSGGGQAVVTQEPSLTVSPGGGTVTLTCRSSSTGAVTTTSNYDNWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLSR (Sequence ID 41), DIQMTQSPSSLSASVGDRVTITCRASQDVNTDVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSRSGGGGEVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSKVSWFQYWGQGTLVTVSSGGGSGGGSGGGSGGGQAVVTQEPSLTVSPGGGTVTLTCRSSSTGAVTTTSNYDNWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLSR (Sequence ID 42), DIQMTQSPSSLSASVGDRVTITCRASQDVNTDVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSRSGGGGEVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFPPNSKVSWFQYWGQGTLVTVSSGGGSGGGSGGGSGGGQAVVTQEPSLTVSPGGGTVTLTCRSSSTGAVTTTSNYDNWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLSR (Sequence ID 43), DIQMTQSPSSLSASVGDRVTITCRASQDVNTDVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSRSGGGGEVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHANFGNSKVSWFAYWGQGTLVTVSSGGGSGGGSGGGSGGGQAVVTQEPSLTVSPGGGTVTLTCRSSSTGAVTTTSNYDNWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLSR (Sequence ID 44), DIQMTQSPSSLSASVGDRVTITCRASQDVNTDVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSRSGGGGEVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFPPNSKVSWFAYWGQGTLVTVSSGGGSGGGSGGGSGGQAVVTQEPSLTVSPGGGTVTLTCRSSSTGAVTTTSNYDNWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLSR (Sequence ID 45), DIQMTQSPSSLSASVGDRVTITCRASQDVNTDVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSRSGGGGEVQLVESGGGLVQPGGSLR LSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHTNFGNSKVSWFAYWGQGTLVTVSSGGGSGGGSGGGSGGQAVVTQEPSLTVSPGGGTVTLTCRSSSTGAVTTTSNYDNWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLSR (Sequence ID 46), DIQMTQSPSSLSASVGDRVTITCRASQDVNTDVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSRSGGGGEVQLVESGGGLVQPGGSLRL SCAASSGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHSNFGNSKVSWFAYWGQGTLVTVSSGGGSGGGSGGGSGGQAVVTQEPSLTVSPGGGTVTLTCRSSAGAVTTTSNYDNWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLSR (Sequence ID 47), or DIQMTQSPSSLSASVGDRVTITCRASQDVNTDVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRS GTDFTLISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGECSRSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVAR An antibody or antigen-binding antibody fragment thereof, or a bispecific antibody or antigen-binding antibody fragment thereof, which is one of IRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHTNFGNSKVSWFAYWGQGTLVVTVSSSGGGSGGGSGGGSGGGSGGQAVVTQEPSLTVSPGGGTVTLTCRSSAGAVTTTSNYDNWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEADYYCALWYSNLWVFGGGTKLTVLSR (Sequence ID 48), according to claim 1 or 2.

15. The antibody or antigen-binding antibody fragment thereof, or a bispecific antibody or antigen-binding antibody fragment thereof, according to claim 14, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 35, and the light chain variable region has one of the amino acid sequences of SEQ ID NOs: 41 to 48.

16. The antibody or its antigen-binding antibody fragment or bispecific antibody or its antigen-binding antibody fragment according to claim 14, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 36, and the light chain variable region has any one of the amino acid sequences of SEQ ID NOs: 41 to 48.

17. The antibody or its antigen-binding antibody fragment or bispecific antibody or its antigen-binding antibody fragment according to claim 14, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 37, and the light chain variable region has any one of the amino acid sequences of SEQ ID NOs: 41 to 48.

18. The antibody or its antigen-binding antibody fragment or bispecific antibody or its antigen-binding antibody fragment according to claim 14, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 38, and the light chain variable region has any one of the amino acid sequences of SEQ ID NOs: 41 to 48.

19. The antibody or its antigen-binding antibody fragment or bispecific antibody or its antigen-binding antibody fragment according to claim 14, wherein the heavy chain variable region has the amino acid sequence of SEQ ID NO: 39, and the light chain variable region has any one of the amino acid sequences of SEQ ID NOs: 41 to 48.

20. An antibody or its antigen-binding antibody fragment or a bispecific antibody or its antigen-binding antibody fragment, having a ratio of at least 1.5:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, or at least 10:1 between the binding affinity to the HER2 protein at pH 6.0 in the tumor microenvironment and the binding affinity to the HER2 protein at pH 7.4 in the non-tumor microenvironment.

21. An immunoconjugate comprising an antibody or an antigen-binding antibody fragment thereof according to any one of claims 1 to 20, or a bispecific antibody or an antigen-binding antibody fragment thereof.

22. The immune conjugate according to claim 21, wherein the immune conjugate comprises at least one agent selected from chemotherapeutic agents, radioactive atoms, cell division inhibitors, and cytotoxic agents.

23. The immunoconjugate according to claim 22, comprising at least two of the aforementioned agents.

24. The immunoconjugate according to claim 22 or 23, wherein the at least one agent is a radioactive agent.

25. The immunoconjugate according to claim 24, wherein the radioactive agent is selected from an α-ejector, a β-ejector, and a γ-ejector.

26. The immunoconjugate according to any one of claims 22 to 25, wherein the antibody or its antigen-binding antibody fragment and the at least one drug are covalently bound to a linker molecule.

27. The immunoconjugate according to any one of claims 22 to 26, wherein one of the at least one agents is selected from mytansinoids, auristatin, drastatin, calicheamicin, pyrrolobenzodiazepines, and anthracyclines.

28. An antibody or an antigen-binding antibody fragment thereof according to any one of claims 1 to 20, or a bispecific antibody or an antigen-binding antibody fragment thereof, A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

29. An immunoconjugate according to any one of claims 21 to 27, A pharmaceutical composition comprising a pharmaceutically acceptable carrier.

30. The pharmaceutical composition according to claim 28 or 29, further comprising an isotonic agent.

31. A pharmaceutical composition according to any one of claims 28 to 30, further comprising an immune checkpoint inhibitor molecule.

32. The pharmaceutical composition according to claim 31, wherein the immune checkpoint inhibitor molecule is an antibody against an immune checkpoint or a fragment of that antibody that binds to an antigen.

33. The pharmaceutical composition according to claim 31 or 32, wherein the immune checkpoint is selected from CTLA4, LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, PD-1, PD-L1, GITR, B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS.

34. The pharmaceutical composition according to any one of claims 31 to 33, wherein the immune checkpoint is CTLA4, PD-1, or PD-L1.

35. A pharmaceutical composition according to any one of claims 28 to 34, further comprising an antibody or antigen-binding antibody fragment against an antigen selected from CTLA4, PD1, PD-L1, AXL, ROR2, CD3, EpCAM, B7-H3, ROR1, SFRP4, and WNT proteins.

36. Use in the manufacture of a pharmaceutical for treating HER2-expressing cancer of an antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 20, a bispecific antibody or antigen-binding antibody fragment thereof, an immunoconjugate according to any one of claims 21 to 27, or a pharmaceutical composition according to any one of claims 28 to 35.

37. The use according to claim 36, wherein the HER2-expressing cancer is selected from breast cancer, ovarian cancer, bladder cancer, gallbladder cancer, extrahepatic or intrahepatic cholangiocarcinoma, salivary duct cancer, gastric cancer, colon cancer, lung cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, soft tissue sarcoma, peritoneal sarcoma, and retroperitoneal sarcoma, solitary fibroma, thymic cancer, thyroid cancer, cervical cancer, endometrial cancer, testicular cancer, endometrial cancer, glioblastoma, glioma, oligodendroglioma, head and neck cancer, hepatocellular carcinoma, small intestinal malignancies, malignant melanoma, and neuroendocrine tumors.

38. A kit for diagnosis or treatment, wherein the kit is An antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 20, or a bispecific antibody or antigen-binding antibody fragment thereof, an immunoconjugate according to any one of claims 21 to 27, or a pharmaceutical composition according to any one of claims 28 to 34, A kit comprising: the antibody or its antigen-binding antibody fragment or a bispecific antibody or its antigen-binding antibody fragment, the immunoconjugate, and / or instructions for using the pharmaceutical composition for diagnosis or treatment.

Images