Conditionally active anti-CD46 antibodies, antibody fragments, their immunoconjugates and their use

Abstract

The present invention provides anti-CD46 antibodies or antibody fragments with reduced or minimal side effects, particularly suitable for therapeutic and diagnostic use in the diagnosis and treatment of cancer. [Solution] An antibody or an antigen-binding antibody fragment that binds to the CD46 antigen is provided, wherein the light chain variable region has three complementarity-determining regions (CDRs) having sequences L1, L2, and L3, and the heavy chain variable region has three complementarity-determining regions (CDRs) having sequences H1, H2, and H3. An immunoconjugate, pharmaceutical composition, and kit containing the antibody and antibody fragment are also provided.

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims priority to U.S. Provisional Application No. 63 / 040913, filed on 18 June 2020, the entire disclosure of which is incorporated herein by reference. Reference to the ASCII text array table. The sequence listing (size 13,000 bytes) submitted with this specification as a text file named "BIAT-1032WO_ST25," created on May 25, 2021, is incorporated herein by reference in its entirety. (Technical field) This disclosure relates to anti-CD46 antibodies, antibody fragments, and immunoconjugates of such antibodies and antibody fragments, as well as the use of antibodies, antibody fragments and immunoconjugates in diagnostic and therapeutic methods. [Background technology]

[0002] CD46 is a membrane cofactor protein, or MCP. It is a widely expressed type I transmembrane protein, but due to alternating splicing and glycosylation of its exons, many isoforms exist. Recently, Karosi et al., Laryngoscope 118:1669-1676 (September 2008), reported the detection of 14 different isoforms. mRNA is transcribed from a single gene located on chromosome 1q32, and through extensive alternative splicing, multiple transcripts encoding various protein isoforms are produced. Of the 14 exons, exons 1-6 are conserved across all CD46 protein isoforms, while exons 7-9 encode a serine-threonine-proline ("STP")-rich region that is variably utilized, seemingly increasing the hypermutability of the protein isoforms. Exons 11 and 12 encode the transmembrane region of CD46, while exons 13 and 14 encode the cytoplasmic end of the protein.

[0003] The longest mRNA transcript, variant A (NM_002389), contains sequences from all 14 exons of the gene. Variable splicing of exons 7, 8, 9, and 13 is thought to generate the majority of the 14 isoforms of CD46. Alternative insertion or exclusion of exon 8 has primarily resulted in the observation of 66kDa and 56kDa protein isoforms. Alternative insertion / exclusion of exon 13 results in alterations to the encoded sequence at the cytoplasmic end of the molecule, and these alterations have been suggested to potentially affect intracellular transport, stability, and the signaling properties of the protein.

[0004] As described by Karosi et al., CD46 mRNA isoform D consists of exons 1-6, 8-12, and 14 of the CD46 gene (corresponding to sequence NM_153826, encoding protein NP_722548), isoform F contains exons 1-6, 9-12, and 14 (corresponding to sequence NM_172353, encoding NP_758863), and isoform J contains exons 1-6, 8, 10-12, and 14 (corresponding to sequence NM_172356, encoding NP_758866). Specifically, the CD46 molecule consists of four N-terminal SCR (short consensus repeat) modules ("Sushi" domains: 4 cysteines in a 1-3,2-4 linked topology). These SCR domains are encoded by the first six exons of the gene. The SCR2, 3, and 4 modules possess C3b / C4b binding and regulatory activity (described later), while the distal sequences of the SCR1 module and SCR4 are not essential for complement regulatory function. The membrane-proximal extracellular sequences, alternately used in exons 7-9 and exon 10, are highly glycosylated, mainly via O-linked glycans.

[0005] For the purposes of this disclosure, the term “CD46” shall be deemed to mean any of the above-mentioned proteins, including their splice variants or immunoreactive fragments, and any nucleic acid sequences encoding such proteins, splice variants, or fragments, unless otherwise indicated by the context.

[0006] CD46 is believed to have many biological functions, many of which are involved in the regulation of the immune system. One of CD46's major immunomodulatory functions is the regulation of complement proteins to protect host cells from damage caused by complement proteins, which is part of the innate immune response in higher eukaryotes. Specifically, CD46 is a cofactor for the factor I cleavage of complement proteins C3b and C4b. CD46 has been shown to activate C3 converters, molecules that cleave C3b into inactive fragments, thereby protecting against inappropriate complement activation. (See Liszewski and Atkinson, Human Genomics, Complement regulator CD46: genetic variants and disease associations (2015) 9:7)

[0007] In addition to its role in innate immunity, CD46 also regulates adaptive immune responses. Signaling via CD46 leads to T cell proliferation and differentiation into a specific class of regulatory T cells called Tr1, characterized by the production of large amounts of the anti-inflammatory cytokine IL-10. Furthermore, high levels of CD46 expression in sperm suggest that CD46 is involved in reproduction, possibly in the fusion of sperm and egg. CD46 also appears to be highly expressed in the placenta, which may help protect the fetus from maternal immune rejection.

[0008] Furthermore, CD46 has been shown to be eccentrically expressed in most normal human cells except red blood cells. For example, it has been reported that CD46 is strongly expressed in epithelial cells, moderately expressed in lymphocytes and endothelium, and weakly expressed in other cells such as osteoclasts, osteocytes, stromal cells, and muscle cells. Because CD46 is widely expressed, many human pathogens have developed strategies to utilize it as a receptor or co-receptor for binding to cells as a precursor to infection. These pathogens include human herpesvirus 6, measles virus, several serotypes of adenovirus, and pathogenic species of the commensal Neisseria family. Certain retroviruses are thought to evade complement-mediated immunity by having a CD46 mimetic on their surface (Stoiber et al, Molecular Immunology 2005; Saifuddin et al, J Gen Virol, 1997).

[0009] In addition to its presence in normal cells, CD46 expression levels may be increased in certain cancers. For example, elevated CD46 expression is associated with breast cancer (Thorsteinsson et al., APMIS 106:869-78 (1998); Hofman et al., Breast Cancer Res. Treat. 32:213-9 (1994)); colorectal cancer (Andrew et al., Cancer Res. 50: 5225-30 (1990); Koretz et al., Br. J. Cancer 68:926-31 (1993); Juhl et al., J. Surg. Oncol. 64:222-30 (1997); Bjorge et al., Cancer Immunol. Immunother. 42:185-92 (1996)); lung cancer (Varsano et al., Clin. Exp. Immunol. 113:173-82 (1998); Varsano et al., Am. J. It has been reported in the following cases: Respir.Cell.Mol.Bioi.19:522-9 (1998); ovarian cancer (Bjorge et al., Int.J. Cancer 70: 14-25 (1997)); renal cancer (Blok et al. Lab.Invest.80:335-44 (2000); Gorter et al., Lab.Invest.74:1039-49 (1996)); pancreatic cancer (Juhl et al., J. Surg.Oncol.64:222-30 (1997)); and prostate cancer (Jarvis et al. J. Allergy Clin.Immunol 99 (NO. I, PART 2):S215 (1997); Liu, Cancer Res. 60:3429-3434 (2000)). See also WO 02 / 18948 and WO 01 / 88537.

[0010] The present invention aims to provide anti-CD46 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-CD46 antibodies or antibody fragments may have higher binding activity or affinity to CD46 in the tumor microenvironment compared to binding activity or affinity to CD46 in the non-tumor microenvironment. These anti-CD46 antibodies or antibody fragments typically have at least equivalent efficacy to known anti-CD46 antibodies or antibody fragments. Furthermore, the anti-CD46 antibodies or antibody fragments of the present invention may exhibit reduced side effects compared to monoclonal anti-CD46 antibodies known in the art, as a result of having relatively low binding activity or affinity to CD46 in the non-tumor microenvironment present in normal tissue. These advantages provide more selective targeting of CD46 expressed in tumors, and as a result of the selectivity of the antibodies against CD46 present in the tumor microenvironment, it may be possible to use higher doses of these anti-CD46 antibodies or antibody fragments, thereby enabling more effective therapeutic treatment without a corresponding increase in undesirable side effects.

[0011] (Summary of the invention) In one embodiment, the present invention provides an isolated polypeptide that specifically binds to human CD46. The isolated polypeptide is a light chain variable region having three complementarity-determining regions (CDRs) having sequences L1, L2, and L3, The L1 sequence is RAX1QX2IX3NYLN (sequence number 1), The L2 sequence is YTSSLX4X5 (sequence number 2), The L3 sequence is the light chain variable region QQYIKLPWT (SEQ ID NO: 3), and A heavy chain variable region having three complementarity-determining regions (CDRs) having sequences H1, H2, and H3, The H1 sequence is GGSVSSYDIS (sequence number 8), The H2 sequence is VIWTDGGTNYNSAFMS (sequence number 9), The H3 sequence is VYDGYPWFAY (sequence number 10), which is a heavy chain variable region. Includes, In the formula, X1 is S or L, X2 is G or W, X3 is S or A, X4 is H or F, and X5 is S or E, provided that X1, X2, X3, X4, and X5 cannot be S, G, S, H, and S at the same time.

[0012] The polypeptide described above may have an L1 sequence selected from the amino acid sequences RASQGISNYLN (SEQ ID NO: 5), RASQWISNYLN (SEQ ID NO: 12), RASQGIANYLN (SEQ ID NO: 15), and RALQGISNYLN (SEQ ID NO: 22). The polypeptide described above may have an L2 sequence selected from the amino acid sequences YTSSLHS (SEQ ID NO: 6), YTSSLFS (SEQ ID NO: 17), and YTSSLHE (SEQ ID NO: 19). In each of the embodiments described above, one of the L1 and L2 sequences must be other than the wild-type L1 sequence of SEQ ID NO: 5 and the wild-type L2 sequence of SEQ ID NO: 6.

[0013] In one embodiment, the polypeptide comprises a set of six CDRs having the following amino acid sequence: Sequence ID 12, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 15, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 22, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 5, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 5, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 12, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 12, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 15, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 15, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 22, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence IDs 22, 19, 3, 8, 9, and 10.

[0014] In another embodiment, the isolated polypeptide of the present invention comprises a light chain variable region and a heavy chain variable region, each light chain variable region and heavy chain variable region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a pair of amino acid sequences selected from SEQ ID NOs: 11 and 13, SEQ ID NOs: 14 and 13, SEQ ID NOs: 16 and 13, SEQ ID NOs: 18 and 13, SEQ ID NOs: 20 and 13, or SEQ ID NOs: 21 and 13, and the isolated polypeptide specifically binds to the human CD46 protein.

[0015] In another embodiment, the present invention relates to an isolated polypeptide having an amino acid sequence selected from SEQ ID NOs: 11, 13, 14, 16, 18, 20, and 21.

[0016] In another embodiment, the isolated polypeptide of the present invention includes a light chain variable region and a heavy chain variable region having pairs of sequences selected from SEQ ID NOs: 11 and 13, SEQ ID NOs: 14 and 13, SEQ ID NOs: 16 and 13, SEQ ID NOs: 18 and 13, SEQ ID NOs: 20 and 13, and SEQ ID NOs: 21 and 13.

[0017] In another embodiment, the present invention relates to a light chain variable region having three complementarity-determining regions (CDRs) having sequences L1, L2, and L3, The L1 sequence is RAX1QX2IX3NYLN (sequence number 1), The L2 sequence is YTSSLX4X5 (sequence number 2), The L3 sequence is the light chain variable region QQYIKLPWT (SEQ ID NO: 3), and A heavy chain variable region having three complementarity-determining regions (CDRs) having sequences H1, H2, and H3, The H1 sequence is GGSVSSYDIS (sequence number 8), The H2 sequence is VIWTDGGTNYNSAFMS (sequence number 9), The H3 sequence contains a heavy chain variable region called VYDGYPWFAY (sequence number 10). The formula relates to an isolated antibody or fragment thereof, wherein X1 is S or L, X2 is G or W, X3 is S or A, X4 is H or F, and X5 is S or E, provided that X1, X2, X3, X4, and X5 cannot be S, G, S, H, and S at the same time.

[0018] In one embodiment, the antibody or antibody fragment may have an L1 sequence selected from the amino acid sequences RASQGISNYLN (SEQ ID NO: 5), RASQWISNYLN (SEQ ID NO: 12), RASQGIANYLN (SEQ ID NO: 15), and RALQGISNYLN (SEQ ID NO: 22). The antibody or antibody fragment may also have an L2 sequence selected from the amino acid sequences YTSSLHS (SEQ ID NO: 6), YTSSLFS (SEQ ID NO: 17), and YTSSLHE (SEQ ID NO: 19). In each of the above embodiments of the antibody or antibody fragment, one of the L1 and L2 sequences must be other than the wild-type L1 sequence of SEQ ID NO: 5 and the wild-type L2 sequence of SEQ ID NO: 6.

[0019] In another embodiment, the antibody or antibody fragment may include a set of six CDRs selected from the following set of six CDRs. Sequence ID 12, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 15, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 22, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 5, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 5, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 12, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 12, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 15, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 15, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 22, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence IDs 22, 19, 3, 8, 9, and 10.

[0020] In one embodiment, the antibody or antibody fragment of the present invention may include a light chain variable region and a heavy chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a pair of amino acid sequences selected from SEQ ID NOs: 11 and 13, SEQ ID NOs: 14 and 13, SEQ ID NOs: 16 and 13, SEQ ID NOs: 18 and 13, SEQ ID NOs: 20 and 13, or SEQ ID NOs: 21 and 13, and the antibody or antibody fragment specifically binds to the human CD46 protein.

[0021] In one embodiment, the antibody or antibody fragment of the present invention may include a light chain variable region and a heavy chain variable region having a pair of sequences selected from SEQ ID NOs: 11 and 13, SEQ ID NOs: 14 and 13, SEQ ID NOs: 16 and 13, SEQ ID NOs: 18 and 13, SEQ ID NOs: 20 and 13, or SEQ ID NOs: 21 and 13.

[0022] In one embodiment, the antibody or antibody fragment of the present invention competes with any of the above-mentioned antibodies or antibody fragments for binding to human CD46.

[0023] In each of the embodiments described above, the antibody or antibody fragment may have higher binding activity to the CD46 protein at the values ​​of the conditions in the tumor microenvironment compared to different values ​​of the same conditions occurring in the non-tumor microenvironment. In one embodiment, the condition is pH.

[0024] In one embodiment, binding activity is measured by binding affinity. In each of the embodiments described above, the isolated polypeptide, antibody, or antibody fragment may have an antigen-binding activity at pH 6.0 of at least 70% compared to the same antigen-binding activity at pH 6.0 of the parent polypeptide, parent antibody, or antibody fragment, and the antigen-binding activity at pH 7.4 may be less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% compared to the same antigen-binding activity at pH 7.4 of the parent polypeptide, antibody, or antibody fragment. Antigen-binding activity may also be binding to the CD46 protein.

[0025] In each of the embodiments described above, antigen-binding activity may be measured by an ELISA assay.

[0026] In yet another embodiment, the present invention provides an immunoconjugate comprising any of the antibodies or antibody fragments described above. In the immunoconjugate, the antibody or antibody fragment may be conjugated with a drug selected from chemotherapeutic agents, radioactive atoms, cell division inhibitors, and cytotoxic agents.

[0027] In yet another embodiment, the present invention provides a pharmaceutical composition comprising the polypeptide, antibody or antibody fragment, or immunoconjugate of the present invention together with a pharmaceutically acceptable carrier.

[0028] A single dose of the pharmaceutical composition may contain approximately 135 mg, 235 mg, 335 mg, 435 mg, 535 mg, 635 mg, 735 mg, 835 mg, 935 mg, 1035 mg, 1135 mg, 1235 mg, or 1387 mg of polypeptide, antibody or antibody fragment, or immunoconjugate.

[0029] A single dose of the pharmaceutical composition may contain amounts of polypeptides, antibodies, antibody fragments, or immunoconjugates in the range of 135-235 mg, 235-335 mg, 335-435 mg, 435-535 mg, 535-635 mg, 635-735 mg, 735-835 mg, 835-935 mg, 935-1035 mg, 1035-1135 mg, 1135-1235 mg, or 1235-1387 mg.

[0030] 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 LAG3, TIM3, TIGIT, VISTA, BTLA, OX40, CD40, 4-1BB, CTLA4, PD-1, PD-L1, GITR, B7-H3, B7-H4, KIR, A2aR, CD27, CD70, DR3, and ICOS, or the immune checkpoint may be CTLA4, PD-1, or PD-L1.

[0031] Each of the aforementioned embodiments of the pharmaceutical composition may further comprise an antibody or antibody fragment against an antigen selected from PD1, PD-L1, CTLA4, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4, and WNT proteins. The WNT protein can be selected from WNT1, WNT2, WNT2B, WNT3, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, and WNT16.

[0032] In yet another aspect, the present invention provides a kit for diagnosis or treatment comprising any of the polypeptides, antibodies or antibody fragments, immunoconjugates or pharmaceutical compositions of the present invention described above. [Brief explanation of the drawing]

[0033] [Figure 1] Figure 1 shows the binding activity of an exemplary conditionally active anti-CD46 antibody (hereinafter referred to as "CAB ADC") of the present invention, bound to a linker payload, to human CD46 at pH 6.0. This was measured by enzyme-linked immunosorbent assay (ELISA). In Figure 1, BA-133-00-01 is the benchmark (BM) wild-type antibody (hereinafter referred to as "WT ADC") bound to the linker payload.

[0034] [Figure 2] Figure 2 shows the binding activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention, tested in Figure 1, to human CD46 at pH 7.4, as measured by ELISA.

[0035] [Figure 3] Figure 3 shows the binding activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention to cyno-CD46 at pH 6.0, as measured by ELISA.

[0036] [Figure 4] Figure 4 shows the binding activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention, tested in Figure 3, to cyno-CD46 at pH 7.4, as measured by ELISA.

[0037] [Figure 5] Figure 5 shows the binding activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention to human CD46 under pH titration, as measured by ELISA.

[0038] [Figure 6] Figure 6 shows the binding activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention to human CD46-expressing HEK 293 cells at pH 6.0. This activity was measured by fluorescence-activated cell sorting (FACS).

[0039] [Figure 7]Figure 7 shows the binding activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention to human CD46-expressing HEK 293 cells at pH 7.4, as measured by FACS.

[0040] [Figure 8] Figure 8 shows the binding activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention to CD46-expressing Colo205 cells at pH 6.0, as measured by FACS.

[0041] [Figure 9] Figure 9 shows the binding activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention to CD46-expressing Colo205 cells at pH 7.4, as measured by FACS.

[0042] [Figure 10] Figure 10 shows the binding activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention to cyno-CD46-expressing HEK 293 cells at pH 6.0, as measured by FACS.

[0043] [Figure 11] Figure 11 shows the binding activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention to cyno-CD46-expressing HEK 293 cells at pH 7.4, as measured by FACS.

[0044] [Figure 12] Figure 12 shows the cytotoxic activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention against human CD46-expressing HEK293 cells at pH 6.0.

[0045] [Figure 13] Figure 13 shows the cytotoxic activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention against human CD46-expressing HEK293 cells at pH 7.4.

[0046] [Figure 14] Figure 14 shows the cytotoxic activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention against CD46-expressing Colo205 cells at pH 6.0.

[0047] [Figure 15] Figure 15 shows the cytotoxic activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention against CD46-expressing Colo205 cells at pH 7.4.

[0048] [Figure 16] Figure 16 shows the effect of treatment with exemplary anti-CD46 CAB ADCs and WT ADCs of the present invention on tumor volume in tumor xenograft mice.

[0049] [Figure 17] Figure 17 shows the protein sequence of a typical conditionally active antibody of the present invention.

[0050] definition To facilitate understanding of the examples provided herein, certain frequently used terms are defined herein.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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).

[0055] 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 retain some ability to selectively bind to the antigens (e.g., polypeptide antigens) of their source antibodies and can be prepared using methods well known in the art (see, e.g., Harlow and Lane, op. cit.), which are 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.

[0056] 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.

[0057] Antibody molecule Fab' fragments can be obtained by treating the entire antibody molecule with pepsin and then reducing it to produce molecules consisting of intact light chains and portions of heavy chains. Two Fab' fragments are obtained for each antibody molecule treated in this way.

[0058] 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.

[0059] 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.

[0060] 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 multispecific antibodies formed from antibody fragments.

[0061] As used herein, the terms “anti-CD46 antibody,” “CD46 antibody,” and “CD46-binding antibody” refer to an antibody that can bind to CD46 with sufficient affinity so that it is useful as a diagnostic and / or therapeutic agent when the antibody targets CD46. In one embodiment, the degree of binding of the anti-CD46 antibody to unrelated non-CD46 proteins is less than about 10% of the antibody binding to the CD46 protein, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody binding to the CD46 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-CD46 antibody binds to an epitope of CD46 that is conserved among CD46 from different species, for example, the extracellular domain of CD46.

[0062] 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.

[0063] As used herein, the terms “cancer” and “cancerous” 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 and non-Hodgkin lymphomas), 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.

[0064] 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.

[0065] 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).

[0066] 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.

[0067] 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.

[0068] As used herein, the term “conditionally active antibody” refers to an anti-CD46 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 may be virtually inactive at normal body temperature but active at higher temperatures in the tumor microenvironment. In yet another embodiment, a conditionally active antibody may be less active in normal oxygenated blood but more active in the hypoxic environment present in tumors. In yet another embodiment, a conditionally active antibody may be less active at normal physiological pH 7.2–7.8 but more active at the acidic pH 5.8–7.0 or 6.0–6.8 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-CD46 antibodies have different binding affinities to the CD46 protein.

[0069] 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.

[0070] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or blocks cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At 211 、I 131 、I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、Pb 212 、and radioisotopes of 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 nuclease and fragments thereof, antibiotics, toxins such as small molecule toxins or enzyme active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, and various antitumor agents or anticancer agents disclosed hereinafter.

[0071] As used herein, the term "diabody" refers to a small antibody fragment having two antigen-binding sites, which fragment comprises a heavy chain variable domain (V H -V L ) bound to a light chain variable domain (V L ) of the same polypeptide chain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains pair with the complementary domains on the other chain and are forced to generate two antigen-binding sites.

[0072] 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 detectable directly or indirectly based on light absorption, fluorescence, reflection, light scattering, phosphorescence, or luminescence properties; molecules or ions detectable by radioactivity; and molecules or ions detectable by nuclear magnetic resonance or paramagnetism. For example, among molecules detectable indirectly 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.

[0073] As used herein, the term “diagnosis” refers to the measurement of an object’s susceptibility to a disease or disorder, the determination of whether an object is currently suffering from a disease or disorder, the prognosis of an object suffering from a disease or disorder (e.g., identification of pre-metastatic or metastatic cancerous status, stage of cancer, or response of cancer to treatment), and treatment strategy (e.g., monitoring of the object’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 the detection of early-stage cancer.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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 L In general, they appear in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0079] The terms "full-length antibody," "intact antibody," or "whole antibody" refer to the antigen-binding variable region (V). H or V LThis 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.

[0080] 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.

[0081] 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 do not have to have exactly the same nucleic acid content as 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.

[0082] 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.

[0083] 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.

[0084] As used herein, the term “immunoconjugate” refers to an antibody conjugated to one or more heterologous molecules, including, but not limited to, cytotoxic agents.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] As used herein, the term “isolated nucleic acid encoding an anti-CD46 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 molecule in a single vector or separate vectors, and such nucleic acid molecule present in one or more locations within a host cell.

[0089] As used herein, the term “metastasis” refers to all CD46-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 CD46, such as proliferation, migration, anchorage independence, evasion of apoptosis, or secretion of angiogenic factors.

[0090] 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 in the range of 5.0–7.0, or 5.0–6.8, or 5.8–6.8, or 6.2–6.8. On the other hand, the normal physiological pH is in the range of 7.2–7.8. 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 0.3–1°C higher than the 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 sites other than tumors.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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 matches 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] As used herein, the term “single-chain 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).

[0101] 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.

[0102] 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.

[0103] 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.

[0104] 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 domains, antibodies that bind to specific antigens can be isolated, and libraries of complementary VL or VH domains can be screened accordingly. See, for example, Portolano et al., J.Immunol., vol.150, pp.880-887, 1993 and Clarkson et al., Nature, vol.352, pp.624-628, 1991.

[0105] 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)

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] Anti-CD46 antibody The heavy-chain variable region and light-chain variable region of the present invention were obtained from a parent antibody using the methods disclosed in U.S. Patents 8,709,755 and 8,859,467, respectively. These methods for generating the heavy-chain variable region and light-chain variable region, as well as for generating the antibody and antibody fragments, are disclosed in U.S. Patents 8,709,755 and 8,859,467, which are incorporated herein by reference.

[0113] In one embodiment, the present invention provides an isolated polypeptide that specifically binds to human CD46. The isolated polypeptide is a light chain variable region having three complementarity-determining regions (CDRs) having sequences L1, L2, and L3, The L1 sequence is RAX1QX2IX3NYLN (sequence number 1), The L2 sequence is YTSSLX4X5 (sequence number 2), The L3 sequence is the light chain variable region QQYIKLPWT (SEQ ID NO: 3), and A heavy chain variable region having three complementarity-determining regions (CDRs) having sequences H1, H2, and H3, The H1 sequence is GGSVSSYDIS (sequence number 8), The H2 sequence is VIWTDGGTNYNSAFMS (sequence number 9), The H3 sequence contains a heavy chain variable region called VYDGYPWFAY (sequence number 10). In the formula, X1 is S or L, X2 is G or W, X3 is S or A, X4 is H or F, and X5 is S or E, provided that X1, X2, X3, X4, and X5 cannot be S, G, S, H, and S at the same time.

[0114] The polypeptide described above may have an L1 sequence selected from the amino acid sequences RASQGISNYLN (SEQ ID NO: 5), RASQWISNYLN (SEQ ID NO: 12), RASQGIANYLN (SEQ ID NO: 15), and RALQGISNYLN (SEQ ID NO: 22). The polypeptide described above may have an L2 sequence selected from the amino acid sequences YTSSLHS (SEQ ID NO: 6), YTSSLFS (SEQ ID NO: 17), and YTSSLHE (SEQ ID NO: 19). In each of the embodiments described above, one of the L1 and L2 sequences must be other than the wild-type L1 sequence of SEQ ID NO: 5 and the wild-type L2 sequence of SEQ ID NO: 6.

[0115] In one embodiment, the polypeptide comprises a set of six CDRs having the following amino acid sequence: Sequence ID 12, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 15, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 22, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 5, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 5, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 12, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 12, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 15, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 15, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 22, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence IDs 22, 19, 3, 8, 9, and 10.

[0116] In another embodiment, the isolated polypeptide of the present invention comprises a light chain variable region and a heavy chain variable region, each light chain variable region and heavy chain variable region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a pair of amino acid sequences selected from SEQ ID NOs: 11 and 13, SEQ ID NOs: 14 and 13, SEQ ID NOs: 16 and 13, SEQ ID NOs: 18 and 13, SEQ ID NOs: 20 and 13, or SEQ ID NOs: 21 and 13, and the isolated polypeptide specifically binds to the human CD46 protein.

[0117] In another embodiment, the present invention relates to an isolated polypeptide having an amino acid sequence selected from SEQ ID NOs: 11, 13, 14, 16, 18, 20, and 21.

[0118] In another embodiment, the isolated polypeptide of the present invention includes a light chain variable region and a heavy chain variable region having pairs of sequences selected from SEQ ID NOs: 11 and 13, SEQ ID NOs: 14 and 13, SEQ ID NOs: 16 and 13, SEQ ID NOs: 18 and 13, SEQ ID NOs: 20 and 13, and SEQ ID NOs: 21 and 13.

[0119] In another embodiment, the present invention relates to a light chain variable region having three complementarity-determining regions (CDRs) having sequences L1, L2, and L3, The L1 sequence is RAX1QX2IX3NYLN (sequence number 1), The L2 sequence is YTSSLX4X5 (sequence number 2), The L3 sequence is the light chain variable region QQYIKLPWT (SEQ ID NO: 3), and A heavy chain variable region having three complementarity-determining regions (CDRs) having sequences H1, H2, and H3, The H1 sequence is GGSVSSYDIS (sequence number 8), The H2 sequence is VIWTDGGTNYNSAFMS (sequence number 9), The H3 sequence contains a heavy chain variable region called VYDGYPWFAY (sequence number 10). The formula relates to an isolated antibody or fragment thereof, wherein X1 is S or L, X2 is G or W, X3 is S or A, X4 is H or F, and X5 is S or E, provided that X1, X2, X3, X4, and X5 cannot be S, G, S, H, and S at the same time.

[0120] In one embodiment, the antibody or antibody fragment may have an L1 sequence selected from the amino acid sequences RASQGISNYLN (SEQ ID NO: 5), RASQWISNYLN (SEQ ID NO: 12), RASQGIANYLN (SEQ ID NO: 15), and RALQGISNYLN (SEQ ID NO: 22). The antibody or antibody fragment may also have an L2 sequence selected from the amino acid sequences YTSSLHS (SEQ ID NO: 6), YTSSLFS (SEQ ID NO: 17), and YTSSLHE (SEQ ID NO: 19). In each of the above embodiments of the antibody or antibody fragment, one of the L1 and L2 sequences must be other than the wild-type L1 sequence of SEQ ID NO: 5 and the wild-type L2 sequence of SEQ ID NO: 6.

[0121] In another embodiment, the antibody or antibody fragment may include a set of six CDRs selected from the following set of six CDRs. Sequence ID 12, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 15, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 22, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 5, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 5, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 12, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 12, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 15, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 15, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence ID 22, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10, or Sequence IDs 22, 19, 3, 8, 9, and 10.

[0122] In one embodiment, the antibody or antibody fragment of the present invention may include a light chain variable region and a heavy chain variable region, each region independently having at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a pair of amino acid sequences selected from SEQ ID NOs: 11 and 13, SEQ ID NOs: 14 and 13, SEQ ID NOs: 16 and 13, SEQ ID NOs: 18 and 13, SEQ ID NOs: 20 and 13, or SEQ ID NOs: 21 and 13, and the antibody or antibody fragment specifically binds to the human CD46 protein.

[0123] In one embodiment, the antibody or antibody fragment of the present invention may include a light chain variable region and a heavy chain variable region having a pair of sequences selected from SEQ ID NOs: 11 and 13, SEQ ID NOs: 14 and 13, SEQ ID NOs: 16 and 13, SEQ ID NOs: 18 and 13, SEQ ID NOs: 20 and 13, or SEQ ID NOs: 21 and 13.

[0124] In one embodiment, the antibody or antibody fragment of the present invention competes with any of the above-mentioned antibodies or antibody fragments for binding to human CD46.

[0125] In each of the embodiments described above, the antibody or antibody fragment may have higher binding activity to the CD46 protein at the values ​​of the conditions in the tumor microenvironment compared to different values ​​of the same conditions occurring in the non-tumor microenvironment. In one embodiment, the condition is pH. In one embodiment, the binding activity is determined by binding affinity.

[0126] In each of the embodiments described above, the isolated polypeptide, antibody, or antibody fragment may have an antigen-binding activity at pH 6.0 of at least 70% compared to the same antigen-binding activity at pH 6.0 of the parent polypeptide, parent antibody, or antibody fragment, and the antigen-binding activity at pH 7.4 may be less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% compared to the same antigen-binding activity at pH 7.4 of the parent polypeptide, antibody, or antibody fragment. The antigen-binding activity may also be binding to the CD46 protein.

[0127] In each of the embodiments described above, antigen-binding activity may be measured by an ELISA assay.

[0128] Antibodies and antibody fragments containing these heavy-chain and light-chain variable regions can specifically bind to CD46, particularly human CD46. 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 higher binding activity to CD46 at the pH of the tumor microenvironment (e.g., pH 6.0–6.8) than at the pH of the non-tumor microenvironment (e.g., pH 7.0–7.6). As a result, the anti-CD46 antibodies or antibody fragments of the present invention have higher binding activity to CD46 in the tumor microenvironment compared to their binding activity to CD46 in a typical normal (non-tumor) tissue microenvironment.

[0129] Therefore, the anti-CD46 antibody or antibody fragment of the present invention is expected to exhibit reduced side effects compared to unconditionally active anti-CD46 antibodies, due to reduced binding to CD46 in normal tissues such as the non-tumor microenvironment. Furthermore, the anti-CD46 antibody or antibody fragment of the present invention is expected to have efficacy equivalent to monoclonal anti-CD46 antibodies known in the art. This combination of features, due to the reduced side effects, allows for the use of higher doses of these anti-CD46 antibodies or antibody fragments, which can provide a more effective therapeutic option.

[0130] In other embodiments, the amino acid sequences of the heavy-chain variable region and light-chain variable region outside the complementarity-determining region can be mutated according to the substitution, insertion, and deletion principles considered in this application to provide these variants. In further embodiments, the constant region can be modified to provide these variants. In yet another embodiment, the amino acid sequences of both the heavy-chain and light-chain variable regions other than the complementarity-determining region and the constant region may be modified to provide these variants.

[0131] 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 in the amino acid sequences of the heavy chain variable region and light chain variable region, and / or substitutions of residues. The antibody or antibody fragment of the present invention can be achieved by any combination of deletions, insertions, and substitutions, provided that it has the desired properties, such as antigen-binding and / or conditional activity to human CD46.

[0132] 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 substitutions 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 can be introduced into the antibody or antibody fragment of interest, and the product can be screened for desired activity, such as retention / improvement of antigen binding or reduction of immunogenicity. JPEG2026053430000001.jpg160170

[0133] 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

[0134] Non-conservative substitution involves swapping one member of one of these classes with one of another.

[0135] One type of substitution variant involves substituting one or more residues in the complementarity-determining region of a parent antibody (e.g., a humanized antibody or a human antibody). Generally, variants selected for further testing have a modification (e.g., improvement) of certain biological properties (e.g., increased affinity, decreased immunogenicity) relative to the parent antibody, and / or substantially retain certain biological properties of the parent antibody. Exemplary substitution variants are affinity-mature antibodies, which can be readily generated using, for example, phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more CDR residues are mutated, the variant antibody is displayed on a phage, and it is screened for specific biological activity (e.g., binding affinity).

[0136] 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 LThe 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 in particular.

[0137] In some 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 antigen. For example, conservative modifications that do not substantially reduce binding activity or binding affinity (e.g., conservative substitutions provided herein) may be made in 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.

[0138] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as 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.

[0139] Amino acid sequence insertions include amino and / or carboxyl terminus fusions, which are polypeptides containing 1 to 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminus 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.

[0140] Modification of the amino acid sequence of antibodies described herein is intended. For example, it may be desirable to improve the binding, activity, affinity and / or other biological properties of the antibody. H and V L Only the CDR inside, simply human antibody V H and V LWhen 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.

[0141] Modifications and alterations can be made in the structure of the antibody of the present invention and in the encoding DNA sequence, still yielding a functional molecule encoding an antibody with desired characteristics.

[0142] When modifying amino acid sequences, the hydrophilicity of the 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).

[0143] A further object of the present invention also includes function-conserving variants of the antibody of the present invention.

[0144] Two amino acid sequences are "substantially homologous" or "substantially similar" if more than 80%, preferably more than 85%, preferably more than 90% of the amino acids are identical compared to the full length of the shorter sequence, or if about 90%, preferably more than 95%, are similar (functionally identical). 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 one of sequence comparison algorithms such as BLAST or FASTA.

[0145] For example, one amino acid can be substituted for another in a protein structure without any recognizable loss of activity. Since the interaction capacity and properties of a protein determine its biological functional activity, an amino acid substitution can be made in the protein sequence and, of course, in its DNA coding sequence, while nevertheless obtaining a protein with similar properties. Therefore, it is intended that various modifications can be made in the sequence of an antibody or antibody fragment of the present invention, or in the corresponding DNA sequence encoding said antibody or antibody fragment, without significantly impairing their biological activity.

[0146] 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.

[0147] As outlined above, amino acid substitutions are generally based on the relative similarities of amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions considering various of the above characteristics are well known to those skilled in the art, and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0148] Glycosylated variant In certain embodiments, the anti-CD46 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.

[0149] 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 made to produce antibody variants with certain improved properties.

[0150] 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).

[0151] 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.

[0152] 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-CD46 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.

[0153] 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 ensure 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).

[0154] 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.

[0155] Certain antibody variants exhibiting 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).

[0156] 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).

[0157] In some embodiments, modifications resulting in altered (i.e., improved or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), such as those described in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al. J. Immunol., vol. 164, pp. 4178-4184, 2000, are produced in the Fc region.

[0158] Antibodies that improve 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), which is involved in the transfer of maternal IgG to the fetus, and increase its half-life, are described in US2005 / 0014934. These antibodies contain an Fc region with one or more substitutions, improving the binding of the Fc region to FcRn. Such Fc variants include variants having substitutions in one or more of the following residues in the Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (e.g., substitution of residue 434 in 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. Patents No. 5,648,260, 5,624,821, and WO94 / 29351.

[0159] Cysteine-engineered antibody variant In certain embodiments, it may be desirable to produce a cysteine-modified antibody, e.g., "thioMAb," in which one or more residues of an anti-CD46 antibody or antibody fragment are substituted with cysteine ​​residues. In certain embodiments, the residue substitution occurs at an antibody-accessible site. By substituting these residues with cysteine, a reactive thiol group is positioned at an antibody-accessible site, and the antibody can be conjugated to another part, such as a drug moiety or a linker-drug moiety, as further described herein, to produce an immunoconjugate. In certain embodiments, any one or more of the following residues may be substituted 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 produced, for example, as described in U.S. Patent No. 7,521,541.

[0160] antibody derivative In certain embodiments, the anti-CD46 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 polymers may have 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 are not limited but can be determined 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.

[0161] In another embodiment, a conjugate of an antibody or antibody fragment and a non-proteinaceous moiety is provided, which can be selectively heated by exposure to radiation. 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 may be of any wavelength, but is not limited, and includes wavelengths that do not harm normal cells but heat the non-proteinaceous moiety to a temperature that kills cells adjacent to the antibody-non-proteinaceous moiety.

[0162] The anti-CD46 antibody or antibody fragment, or variants thereof, of the present invention exhibit higher binding activity or affinity to CD46 under tumor microenvironment conditions than under non-tumor microenvironment conditions. In one embodiment, the conditions in the tumor microenvironment and the conditions in the non-tumor microenvironment are both pH values. Therefore, the anti-CD46 antibody or antibody fragment of the present invention can selectively bind to the CD46 protein at pH approximately 5.0–6.8, but exhibits lower binding activity or affinity to CD46 at pH approximately 7.2–7.8, which is encountered in a normal non-tumor microenvironment. As shown in Examples 3 and 6, the anti-CD46 antibody or antibody fragment exhibits higher binding activity or affinity to CD46 at pH 6.0 than at pH 7.4.

[0163] In certain embodiments, the anti-CD46 antibody or antibody fragment of the present invention has a dissociation constant (Kd) with CD46 under conditions in the tumor microenvironment of approximately ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -8 M or less, or 10 -8 M~10 -13 M, or 10 -9 M~10 -13M) In one embodiment, the ratio of the Kd of the antibody or antibody fragment with CD46 under non-tumor microenvironmental conditions to the Kd under the same conditions in the 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.

[0164] In another embodiment, the ratio of the binding activity of the antibody or antibody fragment to CD46 under non-tumor microenvironmental conditions to the binding activity under the same conditions in the 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.

[0165] 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. 125I) 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 [ 125 Mix the [I]-antigen with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). Then incubate the Fab of interest overnight, although incubation may be continued for a longer time (e.g., about 65 hours) to ensure equilibrium is reached. Transfer the mixture to a capture plate and incubate at room temperature (e.g., 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μl / well of scintillant (MICROSCINT-20®, Packard) and count the plate with a TOPCOUNT® gamma counter (Packard) for 10 minutes. Select the concentration of each Fab that gives less than 20% of the maximum binding for use in competitive binding assays.

[0166] According to another embodiment, Kd is measured at approximately 10 response units (RUs) at 25°C using a surface plasmon resonance assay with BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) with an immobilized antigen CM5 chip. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RUs) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For dynamic measurement, serially diluted 2-fold dilutions of Fab (0.78 nM to 500 nM) are injected into PBS containing 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at a flow rate of approximately 25 μl / min at 25°C. The 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 -1If 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.

[0167] The anti-CD46 antibody of the present invention may be a chimeric antibody, a humanized antibody, or a human antibody. In one embodiment, an anti-CD46 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 multispecific 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 some antibody fragments, see Hudson et al. Nat. Med., vol. 9, pp. 129-134, 2003. For an overview of the scFv fragment, see, for example, Pluckthuen, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); International Publication No. 93 / 16185; and U.S. Patent 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.

[0168] 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.

[0169] 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).

[0170] Antibody fragments can be produced by various techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (e.g., Escherichia coli or phages), as described herein.

[0171] In some embodiments, the anti-CD46 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.

[0172] 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.

[0173] 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).

[0174] 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.

[0175] In some embodiments, the anti-CD46 antibody of the present invention is a multispecific antibody, for example, a bispecific antibody. A multispecific antibody is a monoclonal antibody having binding specificity to at least two different sites. In some embodiments, one binding specificity is for CD46, and the other is for another antigen. In some embodiments, a bispecific antibody may bind to two different epitopes of CD46. A bispecific antibody can also be used to localize a cytotoxic agent to cells expressing CD46. A bispecific antibody may be prepared as a full-length antibody or as an antibody fragment.

[0176] 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, multispecific 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).

[0177] Modified antibodies having three or more functional antigen-binding sites, including "Octopus antibody," are also included herein (see, for example, US2006 / 0025576A1).

[0178] The anti-CD46 antibody or antibody fragment of the present invention can be produced using the recombinant method and composition described in detail in US2016 / 0017040.

[0179] The physical / chemical properties and / or biological activity of the anti-CD46 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.

[0180] B. Immunoconjugates In another embodiment, the present invention also provides an immunoconjugate comprising an anti-CD46 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.

[0181] 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.

[0182] 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.

[0183] 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 212, and radioisotopes of Lu. When a radiolabeled conjugate is used for detection, it may include a radioactive atom for scintigraphy studies, such as tc99m or I123, or iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, and iron, etc., a spin label for nuclear magnetic resonance (NMR) imaging methods (also known as magnetic resonance imaging, MRI).

[0184] In some embodiments, the immunoconjugate includes a radiopharmaceutical that can be selected from alpha emitters, beta emitters, and gamma emitters. Examples of alpha emitters are 211 At, 210 Bi, 212 Bi, 211 Bi, 223 Ra, 224 Ra, 225 Ac, and 227 Th. Examples of beta emitters are 67 Cu, 90 Y, 131 I, 153 Sm, 166 Ho, and 186 Re. Examples of gamma emitters are 60 Co, 137 Ce, 55 Fe, 54 Mg, 203 Hg, and 133 Ba.

[0185] 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 radioactive nucleotides to antibodies. See WO94 / 11026. The linker may be a “cleavable linker” that facilitates the release of cytotoxic agents 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).

[0186] The immunoconjugates included herein are, but are not limited to, those expressly intended to be, 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 immunoconjugates prepared with crosslinking reagents including, but not limited to, SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0187] 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.

[0188] An example ADC is Ab-(LD) p The ADC has formula I as follows, 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 the use of modifications, in which case the antibody can be conjugated to the drug using the existing free cysteine ​​residues. In some embodiments, the antibody is exposed to reducing conditions before antibody conjugation to generate one or more free cysteine ​​residues.

[0189] A linker is used to conjugate the moiety to an antibody to form an immunoconjugate such as an ADC. Suitable linkers are described in WO2017 / 180842.

[0190] Some drug moieties that can be conjugated to an antibody are described in WO2017 / 180842.

[0191] The drug moiety also includes compounds having nuclease activity (e.g., ribonuclease or DNA endonuclease).

[0192] In certain embodiments, the immunoconjugate may contain a radioisotope. Various radioisotopes are available for the production of radiolabeled conjugated antibodies. Examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and radioisotopes of Lu. In some embodiments, when the immunoconjugate is used for detection, it may contain a radioactive atom for a scintigraphy test, e.g., Tc 99 or I 123 , or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging (MRI)), e.g., zirconium-89, iodine-character="123", iodine-character="131", indium-character="111", fluorine-character="19", carbon-character="13", nitrogen-character="15", oxygen-character="17", gadolinium, manganese or iron. Zirconium-89 can be complexed to various metal chelating agents and conjugated to an antibody for, e.g., PET imaging (WO2011 / 056983).

[0193] 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.

[0194] 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.

[0195] 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.

[0196] 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).

[0197] In some embodiments, fewer drug moieties than the theoretical maximum are conjugated to the antibody during the conjugation reaction. 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.

[0198] 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.

[0199] C. Methods and compositions for diagnosis and detection In certain embodiments, any of the anti-CD46 antibodies or antibody fragments provided herein may be used to detect the presence of CD46 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.

[0200] A further aspect of the present invention relates to an anti-CD46 antibody or antibody fragment of the present invention for diagnosing and / or monitoring cancer or another disease in which CD46 expression levels are increased or decreased from normal physiological levels at at least one location in the body.

[0201] In a preferred embodiment, the antibody or antibody fragment of the present invention may be labeled with a detectable molecule or substance, such as the fluorescent molecule, radioactive molecule, or any other label known in the art. 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.

[0202] The antibodies or antibody fragments of the present invention may be useful in the diagnosis and staging of cancers and diseases associated with CD46 overexpression. Cancers associated with CD46 overexpression may include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma (hepatocellular carcinoma), breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, sarcoma, hematological cancer (leukemia), astrocytoma, and various head and neck cancers, or hyperproliferative diseases of CD46 expression or overexpression.

[0203] The antibodies or antibody fragments of the present invention may be useful in the diagnosis of non-cancer diseases characterized by increased or decreased CD46 expression. (Both soluble and cellular CD46 forms can be used in such diagnoses. 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 recovered from individuals suspected of having cancers associated with CD46 overexpression, and, in preferred embodiments, glioma, gastric cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, liver cancer, and endometrial cancer. Biological samples encompass clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0204] In certain embodiments, the present invention is a method for diagnosing cancer associated with CD46 overexpression in a subject by detecting CD46 on cells from the subject using the antibody of the present invention. In particular, the method is 1) The step of bringing a target biological sample into contact with the antibody or antibody fragment according to the present invention under conditions suitable for forming a complex between the antibody or antibody fragment and cells of the biological sample expressing CD46; and (b) The step may include detecting and / or quantifying the complex (detection of the complex indicates cancer associated with CD46 overexpression).

[0205] To monitor cancer progression, the method can be repeated at different time points to determine whether antibody binding to the sample increases or decreases, and from there, it can be determined whether the cancer is progressing, regressing, or stable.

[0206] In certain embodiments, the present invention provides a method for diagnosing diseases associated with the expression or overexpression of CD46. Examples of such diseases include cancer, human immunodeficiency, thrombotic disorders (thrombosis and atherothrombosis), and cardiovascular diseases.

[0207] In one embodiment, an anti-CD46 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 CD46 in a biological sample is provided. In a further embodiment, a method for quantifying the amount of CD46 in a biological sample is provided. In one embodiment, the method includes contacting a biological sample with the anti-CD46 antibody or antibody fragment described herein under conditions that allow binding of the anti-CD46 antibody or antibody fragment to CD46, and detecting whether a complex is formed between the anti-CD46 antibody or antibody fragment and CD46. Such a method may be carried out in vitro or in vivo. In one embodiment, the anti-CD46 antibody or antibody fragment is used to select a subject eligible for a treatment. In some embodiments, the treatment includes administering the anti-CD46 antibody or antibody fragment to the subject.

[0208] In one embodiment, a labeled anti-CD46 antibody or antibody fragment is provided. 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) or a fluorophore (such as a rare earth chelate, or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.), luciferase (such as 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 oxidase (such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (such as uricase, xanthine oxidase, etc.), a coupled enzyme that oxidizes a dye precursor using hydrogen peroxide (such as HRP, lactoperoxidase, or microperoxidase), biotin / avidin, bacteriophage labeling, stable free radicals, etc.

[0209] D. Pharmaceutical Formulations Anti-CD46 antibodies or antibody fragments have cytotoxic activity. This cytotoxic activity extends to multiple different cell line types. Furthermore, these antigens or antibody fragments, when conjugated to a cytotoxic agent, can reduce tumor size and exhibit reduced toxicity. Therefore, anti-CD46 antibodies, their fragments or immunoconjugates may be useful for the treatment of proliferative diseases associated with CD46 expression. The antibodies, fragments, or immunoconjugates can be used alone or in combination with any suitable drug or other conventional treatment.

[0210] Diseases associated with the expression, overexpression, or activation of CD46 can be treated using anti-CD46 antibodies or antibody fragments. There is no particular limitation on the type of cancer or tissue that can be treated other than the requirement for CD46 expression. For example, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glioma tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma (hepatocellular carcinoma), breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, sarcoma, blood cancer (leukemia), astrocytoma, and various head and neck cancers, etc. are included. More preferred cancers are glioblastoma, gastric cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, liver cancer, and endometrial cancer.

[0211] Anti-CD46 antibodies or antibody fragments are potent activators of the innate immune response and can therefore be used in the treatment of human immune disorders, such as sepsis. The anti-CD46 antibodies or antibody fragments of the present invention can also be used as adjuvants for immunization, such as for vaccines, and as anti-infective agents, for example, against bacteria, viruses, and parasites.

[0212] Anti-CD46 antibodies or antibody fragments can be used to protect against, prevent, or treat thrombotic diseases, such as venous and arterial thrombosis and atherosclerotic thrombosis. Anti-CD46 antibodies or antibody fragments can be used to protect against, prevent, or treat cardiovascular diseases, and to prevent or inhibit the entry of viruses, such as Lassa and Ebola viruses, and to treat viral infections.

[0213] In each embodiment of the therapeutic methods described herein, an anti-CD46 antibody, antibody fragment, or an immunoconjugate of an anti-CD46 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 CD46 expression-related disease, comprising administering a therapeutically effective amount of the antibody, antibody fragment, or immunoconjugate of the present invention to a subject requiring treatment of a CD46 expression-related disease.

[0214] For administration, anti-CD46 antibodies, antibody fragments, or immunoconjugates may be formulated as pharmaceutical compositions. Pharmaceutical compositions containing anti-CD46 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.

[0215] 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.

[0216] 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 taken into account 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.

[0217] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These may be present in a specific isotonic sterile saline solution (such as monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, or a mixture of such salts), or in a dry, particularly lyophilized, composition that allows for the formation of an injectable solution upon addition of sterile water or saline.

[0218] 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.

[0219] 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).

[0220] 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.

[0221] 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.

[0222] The dose used for administration can be adapted according to various parameters, particularly the method of administration used, the associated lesion, or alternatively, the desired duration of treatment. To prepare the pharmaceutical composition, an effective amount of antibody or antibody fragment can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0223] 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.

[0224] 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.

[0225] Anti-CD46 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.

[0226] 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.

[0227] 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.

[0228] Also, for direct injection, the preparation of more solution or a higher concentration of solution is also contemplated, and by using dimethyl sulfoxide (DMSO) as a solvent, very rapid penetration is brought about, and it is assumed that a high concentration of the active agent is delivered to a small tumor area.

[0229] Upon formulation, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are readily administered in a variety of dosage forms such as those of the injectable solutions described above, but drug release capsules and the like can also be used.

[0230] In the case of parenteral administration in an aqueous solution, if necessary, the solution is preferably buffered and the liquid diluent is first made isotonic with sufficient saline or glucose. These particular 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 in light of the present disclosure. For example, one dosage can be dissolved in 1 ml of isotonic NaCl solution, added to 1000 ml of liquid for subcutaneous drip therapy, or injected into the proposed injection site (see, for example, “Remington’s Pharmaceutical Sciences” 15th Edition, pages 1035 - 1038 and 1570 - 1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will in any event determine the appropriate dosage for an individual subject.

[0231] The antibody or antibody fragment can be formulated to deliver from 0.0001 to 10.0 milligrams, or about 0.001 to 5 milligrams, or about 0.001 to 1 milligram, or about 0.001 to 0.1 milligram, or about 0.1 to 1.0 or even about 10 milligrams per dose within the therapeutic mixture. Multiple doses can also be administered at selected time intervals.

[0232] 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.

[0233] 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.

[0234] 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 the present invention, and such particles can be readily fabricated.

[0235] Liposomes are dispersed in an aqueous medium and are formed from phospholipids that spontaneously form multilayer concentric bilayer vesicles (also called multilayer vesicles (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.

[0236] Pharmaceutical formulations containing anti-CD46 antibodies or antibody fragments as described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antibody fragments having a desired degree of purity with one or more optionally selected 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 dosage and concentration 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)).

[0237] Examples of pharmaceutically acceptable carriers as described herein include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, 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.

[0238] Examples of lyophilized antibody preparations include aqueous antibody preparations described in U.S. Patent No. 6,267,958, as well as those described in U.S. Patent No. 6,171,586 and International Publication No. 2006 / 044908, the latter of which contains histidine acetate buffer.

[0239] The formulations described herein may also contain two or more active ingredients as needed for the specific indication being treated. Preferably, ingredients having complementary activity that does not adversely affect each other may be combined in a single formulation. 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.

[0240] In one embodiment, the anti-CD46 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, HER2, 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-CD46 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, possessing binding activity or affinity to both CD46 and other antigens, and thus forming a multispecific (e.g., bispecific) antibody.

[0241] 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).

[0242] 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.

[0243] Preparations used for in vivo administration are generally sterile. Sterilization can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0244] E. Treatment methods and compositions Any anti-CD46 antibody or antibody fragment provided herein can be used in a therapeutic method. In one embodiment, an anti-CD46 antibody or antibody fragment is provided for use as a pharmaceutical. In a further embodiment, an anti-CD46 antibody or antibody fragment is provided for use in the treatment of cancer (e.g., breast cancer, non-small cell lung cancer, pancreatic cancer, brain tumor, pancreatic, brain, kidney, ovarian, stomach, leukemia, endometrial, colon, prostate, thyroid, liver cancer, osteosarcoma, and / or melanoma). In a particular embodiment, an anti-CD46 antibody or antibody fragment is provided for use in a therapeutic method. In a particular embodiment, the present invention provides an anti-CD46 antibody or antibody fragment for use in a method of treating an individual having cancer, comprising administering an effective amount of the anti-CD46 antibody or antibody fragment to the individual. In one embodiment, the present invention provides an anti-CD46 antibody or antibody fragment used in a method of treating an individual having an immune disorder (e.g., autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus), or diabetes, comprising administering the individual an effective amount of an anti-CD46 antibody or antibody fragment. In one such embodiment, the method further comprises administering the individual an effective amount of at least one additional therapeutic agent, e.g., the following. In a further embodiment, the present invention provides an anti-CD46 antibody or antibody fragment used in inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (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.

[0245] In certain embodiments, the present invention provides an anti-CD46 antibody or antibody fragment to be used in a method for inhibiting angiogenesis, cell proliferation, immune function, inflammatory cytokine secretion (e.g., from tumor-associated macrophages), tumor vascularization (e.g., intratumor vascularization or tumor-associated vascularization), and / or tumor stromal function in an individual, the method comprising administering an effective amount of the anti-CD46 antibody or antibody fragment to an individual to inhibit angiogenesis, cell proliferation, immune function, inflammatory cytokine secretion (e.g., from tumor-associated macrophages), tumor vascularization (e.g., intratumor vascularization or tumor-associated vascularization), and / or tumor stromal function. The "individual" in any of the above embodiments is preferably a human.

[0246] In further embodiments, the present invention provides the use of anti-CD46 antibodies or antibody fragments in the manufacture or preparation of pharmaceuticals. In one embodiment, the pharmaceutical is for the treatment of cancer (in some embodiments, breast cancer, non-small cell lung cancer, pancreatic cancer, brain tumors, cancers of the pancreas, brain, kidney, ovary, stomach, leukemia, endometrium, colon, prostate, thyroid, liver, osteosarcoma, and / or melanoma). In further embodiments, the pharmaceutical is used in a method of treating cancer, comprising administering an effective amount of the pharmaceutical to an individual having cancer. In further embodiments, the pharmaceutical is used in a method of treating an immune disorder (e.g., autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus), or diabetes, comprising administering an effective amount of anti-CD46 antibodies or antibody fragments to an individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to an individual, e.g., the following: In further embodiments, the pharmaceutical is an agent for inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), tumor vascular structure (e.g., intratumor vascular structure or tumor-associated vascular structure), and / or tumor stromal function. In further embodiments, the pharmaceutical is used in a method for inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), inhibition of tumor vascular systems (e.g., intratumor vascular systems or tumor-associated vascular systems), and / or tumor stromal function in an individual, the method comprising administering an effective amount of the pharmaceutical to an individual to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), inhibit tumor vascular development (e.g., intratumor vascular systems or tumor-associated vascular systems), and / or tumor stromal function. The “individual” in any of the above embodiments may be a human.

[0247] In further embodiments, the present invention provides a method for treating cancer. In one embodiment, the method comprises administering an effective amount of an anti-CD46 antibody or antibody fragment to an individual having such cancer. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent described below to the individual. The “individual” in any of the above embodiments may be a human.

[0248] In further embodiments, the present invention provides methods for treating immune disorders (e.g., autoimmune disorders), cardiovascular disorders (e.g., atherosclerosis, hypertension, thrombosis), infectious diseases (e.g., Ebola virus, Marburg virus), or diabetes. In one such embodiment, the method further comprises administering an effective amount of at least one of the following additional therapeutic agents to an individual. The “individual” in any of the above embodiments may be a human.

[0249] In further embodiments, the present invention provides methods for inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), tumor vascular structure (e.g., intratumor vascular structure or tumor-associated vascular structure), and / or tumor stromal function in an organism. In one embodiment, the method comprises administering an effective amount of anti-CD46 antibody or antibody fragment to an organism to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), inhibit tumor angiogenesis (e.g., intratumor vascular system or tumor-associated vascular system), and / or inhibit tumor stromal function. In one embodiment, “organism” is a human.

[0250] In further embodiments, the present invention provides a pharmaceutical formulation comprising, for example, one of the anti-CD46 antibodies or antibody fragments provided herein, used in any of the therapeutic methods described above. In one embodiment, the pharmaceutical formulation comprises one of the anti-CD46 antibodies or antibody fragments provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises one of the anti-CD46 antibodies or antibody fragments provided herein and at least one additional therapeutic agent, for example, the following:

[0251] 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.

[0252] 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.

[0253] Anti-CD46 antibodies or antibody fragments may be formulated, administered, and given in a manner consistent with good medical practice. Factors relevant to consideration include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the scheduling of administration, and other factors known to the physician. Antibodies or antibody fragments may be formulated, optional but not always necessary, 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.

[0254] 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). A higher initial loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may be useful. The progress of this treatment is readily monitored by conventional techniques and assays.

[0255] Specific doses of the anti-CD46 antibody or antibody fragment of the present invention, which may be administered for the prevention or treatment of the target disease, may be approximately 0.3, 0.6, 1.2, 18, 2.4, 3.0, 3.6, 4.2, 4.8, 5.4, 6.0, 6.6, 7.2, 7.8, 8.4, 9.0, 9.6, or 10.2 mg / kg of patient body weight of the antibody or antibody fragment. In certain embodiments, the dose may range from 0.3 to 2.4, 2.4 to 4.2, 4.2 to 6.0, 6.0 to 7.8, 7.8 to 10.2, 10.2 to 12, 12 to 14, 14 to 16, 16 to 18, or 18 to 20 mg / kg of patient body weight of the antibody or antibody fragment. 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 dose of the antibody or antibody fragment is the same. Furthermore, polypeptides possessing anti-CD46 activity are administered in the same amount as the antibody or antibody fragment.

[0256] A single dose of the pharmaceutical formulation of the present invention may include an amount of approximately 45 μg to approximately 13,600 mg of the antibody or antibody fragment of the present invention, or an amount of approximately 45 μg to approximately 5,440 mg of anti-CD46 antibody or antibody fragment. In some embodiments, a single dose of the pharmaceutical formulation of the present invention may include 135 mg to 1,387 mg of the anti-CD46 antibody or antibody fragment of the present invention, or an amount such as 135, 235, 335, 435, 535, 635, 735, 835, 935, 1035, 1135, 1235, 1387 mg. In certain embodiments, the amount of the anti-CD46 antibody or antibody fragment of the present invention in a single dose of the pharmaceutical formulation is in the range of 135-235, 235-335, 335-435, 435-535, 535-635, 635-735, 735-835, 835-935, 935-1035, 1035-1135, 1135-1235, and 1235-1387 mg. The amount of the antibody or antibody fragment in a single dose of the pharmaceutical formulation remains the same when administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor, or as an immune conjugate, or in combination with another antibody or antibody fragment against another antigen disclosed herein. Furthermore, the polypeptide having anti-CD46 activity will be included in the same amount as the antibody or antibody fragment in a single dose of the pharmaceutical formulation.

[0257] In one embodiment, an anti-CD46 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.

[0258] The combination may be an anti-CD46 antibody or antibody fragment disclosed in this application and an immune checkpoint inhibitor molecule administered as a separate molecule or as a bispecific antibody. Such a bispecific antibody has binding activity to CD46 and a second binding activity to immune checkpoints.

[0259] 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).

[0260] The immune checkpoint is preferably CTLA4, PD-1, or PD-L1.

[0261] 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 instead of, or in addition to, the anti-CD46 antibody.

[0262] Enhancing the host's immune function to eradicate tumors is a subject of increasing interest. Conventional methods include (i) enhancement of APCs, e.g., (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, co-stimulatory molecules B7.1, B7.2), and (iii) adoptive cell immunotherapy or treatment with activated tumor-specific T cells. Adoptive cell immunotherapy involves isolating tumor-infiltrating host T lymphocytes and expanding the population in vitro, e.g., through stimulation by IL-2 or the tumor or both. Furthermore, dysfunctional isolated T cells can also be activated by in vitro application of the anti-PD-L1 antibody of the present invention. 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 antibody, antibody fragment, or immune conjugate of the present invention.

[0263] 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.

[0264] In cancer treatment, any of the aforementioned conventional therapies for cancer immunotherapy may be administered before, after, or concurrently with the administration of anti-CD46 antibodies or antibody fragments. In addition, anti-CD46 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.

[0265] F. Products and Kits In another aspect of the present invention, a product is provided comprising an anti-CD46 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 include (a) a first container (containing the composition, which contains an antibody or antibody fragment), and (b) a second container (containing the composition, which contains a further cytotoxic agent or other therapeutic agent). The product in this embodiment of the present invention may further include a document indicating that the composition can be used to treat a particular pathological condition. Alternatively, the product may further include 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 include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0266] 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-CD46 antibody or antibody fragment.

[0267] Finally, the present invention also provides a kit comprising at least one antibody or antibody fragment of the present invention. A kit containing the polypeptide, antibody or antibody fragment, or antibody-drug conjugate of the present invention is used in the detection of CD46 expression (increase or decrease) or in therapeutic or diagnostic assays. The kit of the present invention may comprise an antibody coupled to a solid support, such as a tissue culture plate or beads (e.g., Sepharose beads). For example, a kit containing an antibody for in vitro detection and quantification of CD46 in ELISA or Western blotting can be provided. Such an antibody useful for detection may be provided with labeling, such as fluorescent or radiolabeling.

[0268] 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 cerebrospinal fluid in a sample based on the presence or absence of CD46 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 CD46 according to the intensity, color spectrum, or other physical attributes of an indicator.

[0269] The following examples illustrate, but are not limited to, the anti-CD46 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]

[0270] The antibody or antibody fragment of the present invention having the following set of six CDRs was tested in the examples. Sequence IDs 12, 6, 3, 8, 9, and 10; Sequence IDs 15, 6, 3, 8, 9, and 10; Sequence IDs 5, 17, 3, 8, 9, and 10; Sequence ID 5, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10; and Sequence IDs 22, 6, 3, 8, 9, and 10. JPEG2026053430000002.jpg70170

[0271] Example 1: Binding activity of conditionally active anti-CD46 antibody to huCD46 The binding activity of conditionally active anti-CD46 antibodies to human CD46 was measured by ELISA using a BM (benchmark) antibody as a control. Table 1 shows the EC50 values ​​of conditionally active anti-CD46 antibodies binding to human CD46 at pH 6.0 and pH 7.4, and Figures 1 and 2 summarize the binding activity. JPEG2026053430000003.jpg255167

[0272] Example 2: Binding activity of conditionally active anti-CD46 antibody to cyno-CD46 The binding activity of conditionally active anti-CD46 antibodies to cyno-CD46 was measured by ELISA and is shown in Figures 3 and 4. The EC50 values ​​of conditionally active anti-CD46 antibodies binding to cyno-CD46 at pH 6.0 and pH 7.4 are summarized in Table 2.

[0273] Example 3: Binding activity of conditionally active anti-CD46 antibody to human CD46 The binding activity of conditionally active anti-CD46 antibodies to human CD46 was similarly measured by ELISA using pH titration. See Figure 5. The pH inflection points of conditionally active anti-CD46 antibodies against human CD46 are summarized in Table 3.

[0274] Example 4: Binding activity of conditionally active anti-CD46 antibody as measured by FACS. FACS analysis was performed using 293 cells expressing human CD46. Conditionally active anti-CD46 antibodies consistently showed higher binding activity to 293 cells expressing human CD46 at pH 6.0 than at pH 7.4. See Figures 6 and 7. Table 4 summarizes the EC50 values ​​of binding of conditionally active humanized anti-CD46 antibodies to 293 cells expressing human CD46.

[0275] Example 5: Binding activity of conditionally active anti-CD46 antibody as measured by FACS The binding activity of a conditionally active anti-CD46 antibody to CD46-expressing Colo205 cells was measured by FACS at pH 6.0 and pH 7.4. The conditionally active anti-CD46 antibody consistently showed higher binding activity to Colo205 cells at pH 6.0 than at pH 7.4. See Figures 8 and 9. Table 5 summarizes the EC50 values ​​of binding of the conditionally active anti-CD46 antibody to CD46-expressing Colo205 cells. JPEG2026053430000004.jpg255166

[0276] Furthermore, a similar FACS analysis was performed using 293 cells expressing cyno CD46. The conditionally active anti-CD46 antibody consistently showed higher binding affinity to cyno CD46-expressing 293 cells at pH 6.0 than at pH 7.4. See Figures 10 and 11. Table 6 summarizes the EC50 values ​​of binding of the conditionally active anti-CD46 antibody to cyno CD46-expressing 293 cells.

[0277] Example 6: In vitro cell sterilization of 293 cells expressing human CD46 We analyzed in vitro cell toxicity of human CD46-expressing 293 cells at pH values ​​of 6.0 and 7.4 using 293 cells. Figures 12 and 13 show the in vitro toxicity of 293 cells by a conditionally active anti-CD46 antibody. Table 7 shows the IC50 values ​​for toxicity of 293 cells by a conditionally active anti-CD46 antibody.

[0278] Example 7: Cytotoxicity of a conditionally active anti-CD46 antibody in the suppression of CD46-expressing Colo205 cells. Conditionally active antibodies were used to treat CD46-expressing Colo205 cells at tumor microenvironment pH 6.0 and normal physiological pH 7.4. The conditionally active antibodies induced a greater inhibitory rate (IR%) at tumor microenvironment pH than at normal physiological pH. Figures 14 and 15. IC50 values ​​are shown in Table 8 below.

[0279] Example 8: In vivo efficacy study of a conditionally active antibody in a Colo205CDX subcutaneous administration model. The purpose of this study was to evaluate the in vivo antitumor effect of the test product in a subcutaneous Colo 205 human colorectal cancer xenograft model of female BALB / c nude mice. Abbreviation JPEG2026053430000005.jpg91170

[0280] Experimental Design Table 1-1. Description of the experimental design JPEG2026053430000006.jpg155170

[0281] Experimental methods and procedures cell culture Colo205 tumor cells (ATCC, Manassas, VA, cat # ATCC® CCL-222TM) were maintained in monolayer culture at 37°C in RPMI-1640 medium supplemented with 10% thermoinactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, under 5% CO2 in air. 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.

[0282] Tumor inoculation and animal grouping Colo205 tumor cells (5x10) were placed in the right flank of each mouse. 6 Tumors were induced by subcutaneous inoculation of 0.2 mL of PBS. The treatment was performed when the average tumor size was approximately 204 mm. 3 When the tumor volume reached a certain level, the study was initiated on day 11 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 1-1.

[0283] Test sample preparation Table 2-1. Explanation of test sample preparation JPEG2026053430000007.jpg139170

[0284] observation 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). Routine monitoring included daily checks for the effects of treatment on tumor growth, motility, food and water consumption (visual only), weight gain / loss (measured twice weekly), normal behaviors such as eye / hair matting, and other abnormal effects described in the protocol. Deaths and observed clinical symptoms were recorded based on the number of animals in each subset.

[0285] Tumor measurement and endpoints The primary endpoint was whether tumor growth could be slowed. Tumor size was measured two-dimensionally twice a week using calipers and calculated using the following formula: V = 0.5ax b², where a and b are the major and minor diameters of the tumor, respectively. Tumor size was used to calculate T / C, TGI, and RTV values. The T / C ratio (percentage) is an indicator of antitumor efficacy, where T and C represent the mean volumes of the treated group and the control group, respectively, on a given day. The TGI for each treatment group was calculated using the following formula: TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100; where Ti is the mean tumor volume of the treatment group or isotype group on a given day, T0 is the mean tumor volume of the treatment group or isotype group on day 0, Vi is the mean tumor volume of the vehicle control group on the same day as Ti, and V0 is the mean tumor volume of the vehicle group on day 0. Individual RTV (relative tumor volume) was calculated by dividing the tumor volume on a given day by its volume on day 0. The RTV value for each mouse was calculated individually and used to calculate the average RTV for each group.

[0286] sampling 50–60 μL of serum was collected from three mice in each group 24 hours and 96 hours after the first dose (immediately before the second dose).

[0287] statistical analysis The mean tumor volume and standard error for each group at different time points were calculated (Table 3-1). Statistical analysis of the difference in tumor volume between the vehicle group and the test product group was performed on data obtained on day 25 after the start of administration (the last day when mice remained in all test product groups). On the other hand, statistical analysis of the difference in tumor volume between the isotype group and the other test product groups was performed on data obtained on day 32 after the start of administration (the last day when mice remained in all test product groups). 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-value < 0.05 was considered statistically significant.

[0288] Tumor volume The average tumor volume for each group is shown in Table 3-1. Table 3-1 Tumor Volume JPEG2026053430000008.jpg154170

[0289] Analysis of tumor growth inhibitory effects Table 3-2. Tumor growth inhibitory effect compared to the vehicle group (based on data from Day 25). JPEG2026053430000009.jpg138170

[0290] Table 3-3 Tumor growth inhibitory effect compared to isotype group (based on data from Day 32) JPEG2026053430000010.jpg130170

[0291] Tumor growth curve The tumor growth curve is shown in Figure 16. In Figure 16, the data represents the mean ± SEM.

[0292] Summary and Discussion In this study, the therapeutic effect of conditionally active antibodies was evaluated using the Colo205 human colorectal xenograft model. The tumor size of each group at each time point after treatment is shown in Tables 3-1, 3-2, and Figure 16. The mean tumor size of the vehicle group was 1,288 mm² at 25 days after the start of administration. 3 (RTV = 6.43 ± 0.69) was reached. All mice in the vehicle group and isotype group were euthanized with PG-D32. For the other groups, observation was extended to 4 weeks after the 4th dose. All test products showed significant antitumor activity (TGI > 93%, p < 0.002, PG-D25). Administration of test products BAP133-LP1 or BA-133-00-01(BM) and BA-133-04-04 LP1 at a dose of 3 mg / kg resulted in dramatic antitumor activity leading to complete remission in most mice. A clear reduction in tumor volume was observed with BA-133-04-01 LP1, BA-133-04-02 LP1, BA-133-04-03 LP1, and BAP133-2-02-12 LP-1, although several mice in these groups showed tumor regrowth 10 days after the final dose. Treatment with BAP133-2-02-12 LP-1 delayed tumor growth at the start of the study, but the tumor growth rate reversed a few days after discontinuation of administration (Figure 16). Isotyped treatment (B12-LP1) was largely ineffective compared to vehicle-based treatment (T / C=84.86%, TGI=17.99%, p-value=0.954, PG-D25). No severe weight loss or death / morbidity events were observed throughout the entire administration and observation period. Thus, no apparent toxicity was observed in relation to the administration of the test product at the specified dose.

[0293] Example 9: Binding activity of conditionally active anti-CD46 antibody measured by SPR analysis The binding kinetics of anti-CD46 antibodies were measured using an SPR2 / 4 instrument (Sierra Sensors, Hamburg, Germany) and surface plasmon resonance on a flat amine sensor tip. The SPR sensor contains four flow cells (FC1-FC4), each of which can be addressed individually or in groups. huCD46-His was immobilized on FC2, and cynoCD46-His was immobilized on FC4. FC1 and FC3, used as control surfaces for FC2 and FC4, were unimmobilized with proteins.

[0294] All injections were performed at a flow rate of 25 μL / min and at 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 CD46-His (2 μg / mL in 10 mM NaAc, pH 5.0) for 480 seconds and 1 M ethanolamine-HCl for 480 seconds. cynoCD46-His was immobilized under the same conditions as described for huCD46-His, except that it was diluted in 10 mM NaAc buffer pH 4.5. The control surface was activated and deactivated using the same conditions, except that no protein was injected. PBST buffer (0.05% TWEEN20) TM PBS (pH 7.4) containing [specific component] 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 instrument was equilibrated in the electrophoresis solution for 1 hour before injecting the first analyte.

[0295] 100 μL of analytes (34.25 nM, 13.70 nM, 6.85 nM, 3.42 nM, 1.37 nM, and 0.0 nM), diluted with the corresponding electrophoretic solution, were injected onto flow cells 1, 2, 3, or 4. Off-rates were 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 cells 1 and 3, which did not contain immobilized proteins, were used as control surfaces for reference subtraction.

[0296] Furthermore, data containing only the buffer solution 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.

[0297] The binding activity of conditionally active anti-CD46 antibodies against human CD46 and cyno-CD46 at pH 6.0, pH 6.5, and pH 7.4 was measured by SPR analysis and is shown in Tables 9 and 10 below, respectively. JPEG2026053430000011.jpg255169

[0298] Experimental protocol for the example Examples 1 and 2 JPEG2026053430000012.jpg225170

[0299] formulation The test sample was first diluted to 300 ng / mL in ELISA incubation buffer at pH 6.0 or pH 7.4. Then, the 3000 ng / mL test sample was serially diluted three-fold in ELISA incubation buffer at pH 6.0 or pH 7.4.

[0300] PH affinity ELISA assay 1) Coat the ELISA plate with 1 μg / mL recombinant human CD46 antigen or CynoCD46 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) The test sample is serially diluted in ELISA incubation buffer at pH 6.0 or pH 7.4, starting with a 3-fold dilution at 3000 ng / mL. 8) Add 100 μL / well of the diluted test sample to the plate. 9) Cover the plate with sealing film and place it in a plate shaker set to 50 rpm for 60 minutes at room temperature. 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 pH 6.0 or pH 7.4 ELISA washing buffer 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 pH 6.0 or pH 7.4 ELISA washing buffer 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.

[0301] Example 3 JPEG2026053430000013.jpg214170

[0302] formulation The test samples were diluted to 10 ng / mL in ELISA incubation buffers of various pH levels ranging from pH 5.5 to pH 7.4.

[0303] pH range ELISA assay 1) Coat the ELISA plate with 1 μg / mL recombinant human CD46 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 then aspirate the contents completely. 5) Add 200 μL of incubation buffers of various pH levels (pH 5.5, 6.0, 6.2, 6.5, 6.7, 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 30 ng / mL in incubation buffers of various pH levels (pH 5.5, 6.0, 6.2, 6.5, 6.7, 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 at various pH levels (pH 5.5, 6.0, 6.2, 6.5, 6.7, 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 values ​​(pH 5.5, 6.0, 6.2, 6.5, 6.7, 7.0, and 7.4). 13) Add 100 μL of HRP secondary antibody diluted in incubation buffers of various pH values ​​(pH 5.5, 6.0, 6.2, 6.5, 6.7, 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 at various pH levels (pH 5.5, 6.0, 6.2, 6.5, 6.7, 7.0, and 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 3 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.

[0304] Examples 4 and 5 JPEG2026053430000014.jpg222170

[0305] formulation The test samples were first diluted to 30 μg / mL in FACS buffer at pH 6.0 or pH 7.4, and then sequentially diluted threefold in FACS buffer at pH 6.0 or pH 7.4. cell culture 293-huCD46 and 293-cynoCD46 cells were maintained using a stable cell line culture medium (MEM + 10% FBS + 1 mg / mL G418). CD46-expressing colo205 cells (ATCC, Cat#CCL222) were maintained in colo205 culture medium (RPMI1640 + 10% FBS). Cells were routinely subcultured twice a week. Cells were harvested during the exponential growth phase and counted for plating.

[0306] 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 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 FAC S buffer in a 96-well U-bottom plate. 5 Aliquot the cells. 11) Centrifuge the cells and discard the buffer solution. 12) The test sample is serially diluted in FACS buffer at pH 6.0 or pH 7.4, starting with a 3-fold dilution at 30 μg / mL. 13) Add 100 μL / well of the diluted test sample 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 wash buffer at pH 6.0 or pH 7.4. 15) Dilute the goat anti-human IgG AF488 antibody 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% PFA 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 1×PBS. 20) Analyze cells using a NovoCyte flow cytometer with Ex488nm / Em530nm. Collect at least 5,000 singlet cells at each data point.

[0307] FACS Data Analysis The MFI of AF488 in cell singlets was plotted using GraphPad Prism software version 7.03.

[0308] Examples 6 and 7 JPEG2026053430000015.jpg212170

[0309] cell culture 293-huCD46 cells were maintained in a stable cell line culture medium (MEM + 10% FBS + 1 mg / mL G418). CD46-expressing colo205 cells (ATCC, Cat#CCL222) were maintained in colo205 culture medium (RPMI1640 + 10% FBS). Cells were routinely subcultured twice a week. Cells were harvested during the exponential growth phase and counted for plating.

[0310] formulation 1) Dilute a stock of 10× test ADC or B12 isotype ADC fivefold in assay medium at pH 6.0 or pH 7.4, starting from 50 μg / mL. 2) Centrifuge the plate and gently remove the culture medium. Then, add 90 μL of pH assay medium before adding ADC. 3) Add 10 μL of serially diluted 10×ADC or B12 sample stock to a well containing 3000 cells (final starting concentration is 5 μg / mL). 4) Incubate the treated cells in a 37°C, 5% CO2 incubator for 72 hours.

[0311] CELLTITER-GLO Luminescent Cell Viability Assay 1) Thaw the CellTiter-Glo buffer and allow it to equilibrate at room temperature before use. 2) Allow the lyophilized CellTiter-Glo substrate to equilibrate at room temperature before use. 3) Transfer the entire volume of the CellTiter-Glo buffer liquid to the amber bottle containing the CellTiter-Glo substrate to reconstitute the lyophilized enzyme / substrate mixture. This forms the CellTiter-Glo reagent. 4) Mix gently while vortexing to obtain a homogeneous solution. 5) Allow the plate and its contents to equilibrium at room temperature. 6) Add 70 μL of CellTiter-Glo reagent to each well. Mix for 2 minutes using a 100 rpm orbital shaker to induce cell lysis. 5) To stabilize the luminescence signal, incubate the plate at room temperature for 10 minutes. 6) Record the flash emission using a SpectraMax i3X plate reader.

[0312] Data Analysis The inhibition rates of different doses of the test antibody were plotted using concentration-response luminescence signals, and the IC50 was calculated. The data were interpreted using GraphPad Prism software.

[0313] 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.

[0314] 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.

[0315] The polypeptide sequence (containing an antibody) of the present invention JPEG2026053430000016.jpg214170JPEG2026053430000017.jpg234170

Claims

1. An isolated polypeptide comprising a light chain variable region and a heavy chain variable region, The light chain variable region has three complementarity-determining regions (CDRs) having sequences L1, L2, and L3. The L1 array is RAX 1 QX 2 IX 3 NYLN (Sequence ID 1) The L2 array is YTSSSLX 4 X 5 (Sequence code 2) The L3 sequence is QQYIKLPWT (Sequence ID 3), and The heavy chain variable region has three complementarity-determining regions (CDRs) having sequences H1, H2, and H3. The H1 sequence is GGSVSYDIS (sequence number 8), The H2 sequence is VIWTDGGTNYNSAFMS (sequence number 9), The H3 sequence is VYDGYPWFAY (sequence number 10), In the formula, X 1 is S or L, X 2 is G or W, X 3 is S or A, X 4 is H or F, X 5 is S or E, provided that X 1 , X 2 , X 3 , X 4 and X 5 cannot simultaneously be S, G, S, H, and S respectively, a polypeptide.

2. The polypeptide according to claim 1, wherein the L1 sequence is selected from the amino acid sequences of RASQWISNYLN (SEQ ID NO: 12), RASQGIANYLN (SEQ ID NO: 15), and RALQGISNYLN (SEQ ID NO: 22).

3. The polypeptide according to claim 1, wherein the L2 sequence is selected from the amino acid sequences of YTSSLFS (SEQ ID NO: 17) and YTSSLHE (SEQ ID NO: 19).

4. The polypeptide according to claim 1, comprising six sets of CD-Rs selected from the following six sets of CD-Rs. Sequence ID 12, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10; Sequence ID 15, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10; Sequence ID 5, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10; Sequence ID 5, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10; and Sequence ID 22, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10.

5. An isolated peptide comprising a light chain variable region and a heavy chain variable region, wherein each light chain variable region and heavy chain variable region independently has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with a pair of amino acid sequences selected from SEQ ID NOs: 11 and 13, SEQ ID NOs: 14 and 13, SEQ ID NOs: 16 and 13, SEQ ID NOs: 18 and 13, SEQ ID NOs: 20 and 13, or SEQ ID NOs: 21 and 13, and specifically binds to the human CD46 protein.

6. The isolated peptide according to claim 5, comprising a light chain variable region and a heavy chain variable region having a pair of sequences selected from SEQ ID NOs: 11 and 13, SEQ ID NOs: 14 and 13, SEQ ID NOs: 16 and 13, SEQ ID NOs: 18 and 13, SEQ ID NOs: 20 and 13, or SEQ ID NOs: 21 and 13.

7. An isolated antibody or antibody fragment comprising a light chain variable region and a heavy chain variable region, The light chain variable region has three complementarity-determining regions (CDRs) having sequences L1, L2, and L3. The L1 array is RAX 1 QX 2 IX 3 NYLN (Sequence ID 1) The L2 array is YTSSSLX 4 X 5 (Sequence code 2) The L3 sequence is QQYIKLPWT (Sequence ID 3), and The heavy chain variable region has three complementarity-determining regions (CDRs) having sequences H1, H2, and H3. The H1 sequence is GGSVSYDIS (sequence number 8), The H2 sequence is VIWTDGGTNYNSAFMS (sequence number 9), The H3 sequence is VYDGYPWFAY (sequence number 10), In the formula, X 1 is S or L, X 2 is G or W, X 3 is S or A, X 4 is H or F, X 5 is S or E, however X 1 , X 2 , X 3 , X 4 and X 5 These are isolated antibodies or antibody fragments that cannot simultaneously be S, G, S, H, and S, respectively.

8. The antibody or antibody fragment according to claim 7, wherein the L1 sequence is selected from the amino acid sequences of RASQWISNYLN (SEQ ID NO: 12), RASQGIANYLN (SEQ ID NO: 15), and RALQGISNYLN (SEQ ID NO: 22).

9. The antibody or antibody fragment according to claim 7, wherein the L2 sequence is selected from the amino acid sequences of YTSSLFS (SEQ ID NO: 17) and YTSSLHE (SEQ ID NO: 19).

10. The antibody or antibody fragment according to claim 7, comprising a set of six CDRs selected from the following six sets of CDRs. Sequence ID 12, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10; Sequence ID 15, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10; Sequence ID 5, Sequence ID 17, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10; Sequence ID 5, Sequence ID 19, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10; and Sequence ID 22, Sequence ID 6, Sequence ID 3, Sequence ID 8, Sequence ID 9, and Sequence ID 10.

11. An antibody or antibody fragment comprising a light chain variable region and a heavy chain variable region, wherein each light chain variable region and heavy chain variable region independently has at least 80%, 85%, 90%, 95%, 98%, or 99% identity with respect to a pair of amino acid sequences selected from SEQ ID NOs: 11 and 13, SEQ ID NOs: 14 and 13, SEQ ID NOs: 16 and 13, SEQ ID NOs: 18 and 13, SEQ ID NOs: 20 and 13, or SEQ ID NOs: 21 and 13, and specifically binds to the human CD46 protein.

12. The antibody or antibody fragment according to claim 7, comprising a light chain variable region and a heavy chain variable region having a pair of sequences selected from SEQ ID NOs: 11 and 13, SEQ ID NOs: 14 and 13, SEQ ID NOs: 16 and 13, SEQ ID NOs: 18 and 13, SEQ ID NOs: 20 and 13, and SEQ ID NOs: 21 and 13.

13. An isolated antibody or antibody fragment that competes with any one of the antibodies or antibody fragments described in any one of claims 7 to 12 for binding to human CD46.

14. The antibody or antibody fragment according to any one of claims 7 to 13, wherein the antibody or antibody fragment has higher binding activity to the CD46 protein at the value of the conditions in the tumor microenvironment compared to different values ​​of the same conditions occurring in a non-tumor microenvironment.

15. The antibody or antibody fragment according to claim 14, wherein the condition is pH.

16. The antibody or antibody fragment according to claim 15, wherein the pH in the tumor microenvironment is in the range of 5.0 to 6.8, and the pH in the non-tumor microenvironment is in the range of 7.0 to 7.

6.

17. An antibody or antibody fragment according to any one of claims 7 to 16, having a ratio of binding activity to the human CD46 protein at a pH in the tumor microenvironment to binding activity to the CD46 protein at a 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.

18. An immunoconjugate comprising an antibody or antibody fragment according to any one of claims 7 to 17.

19. The immune conjugate according to claim 18, wherein the immune conjugate comprises at least one agent selected from chemotherapeutic agents, radioactive atoms, cell division inhibitors, and cytotoxic agents.

20. The immunoconjugate according to claim 19, comprising at least two of the aforementioned agents.

21. The immunoconjugate according to any one of claims 18 to 20, wherein the at least one of the drugs is a radioactive agent.

22. The immunoconjugate according to claim 21, wherein the radioactive agent is selected from an α-emitter, a β-emitter, and a γ-emitter.

23. The immunoconjugate according to any one of claims 18 to 22, wherein the antibody or antibody fragment and the at least one drug are covalently bonded to a linker molecule.

24. An immunoconjugate according to any one of claims 18 to 23, wherein one of the at least one agents is selected from mytansinoids, auristatin, drastatin, calicheamicin, pyrrolobenzodiazepines, and anthracyclines.

25. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 6, an antibody or antibody fragment according to any one of claims 7 to 17, or an immunoconjugate according to any one of claims 18 to 24, and a pharmaceutically acceptable carrier.

26. The pharmaceutical composition according to claim 25, further comprising an isotonic agent.

27. A single dose of the pharmaceutical composition according to any one of claims 25 to 26, comprising approximately 135 mg, 235 mg, 335 mg, 435 mg, 535 mg, 635 mg, 735 mg, 835 mg, 935 mg, 1035 mg, 1135 mg, 1235 mg, or 1387 mg of polypeptide, antibody or antibody fragment, or immunoconjugate.

28. A single dose of the pharmaceutical composition according to claim 25 or 26, comprising an amount of polypeptide, antibody, antibody fragment, or immunoconjugate in the range of 135-235 mg, 235-335 mg, 335-435 mg, 435-535 mg, 535-635 mg, 635-735 mg, 735-835 mg, 835-935 mg, 935-1035 mg, 1035-1135 mg, 1135-1235 mg, or 1235-1387 mg.

29. The pharmaceutical composition according to claim 25 or 26, further comprising an immune checkpoint inhibitor molecule.

30. The pharmaceutical composition according to claim 29, wherein the immune checkpoint inhibitor molecule is an antibody or antibody fragment against an immune checkpoint.

31. The pharmaceutical composition according to claim 30, 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.

32. The pharmaceutical composition according to claim 30, wherein the immune checkpoint is CTLA4, PD-1, or PD-L1.

33. A pharmaceutical composition according to any one of claims 29 to 32, further comprising an antibody or antibody fragment against an antigen selected from CTLA4, PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4, and WNT proteins.

34. A method for treating cancer, comprising the step of administering to a patient having cancer a polypeptide according to any one of claims 1 to 6, an antibody or antibody fragment according to any one of claims 7 to 17, an immune conjugate according to any one of claims 18 to 24, or a pharmaceutical composition according to any one of claims 25 to 33.

35. A kit for diagnosis or treatment, the kit comprising: a polypeptide according to any one of claims 1 to 6; an antibody or antibody fragment according to any one of claims 7 to 17; an immunoconjugate according to any one of claims 18 to 24; or a pharmaceutical composition according to any one of claims 25 to 33; and instructions for using the antibody or antibody fragment, the immunoconjugate, and / or the pharmaceutical composition for diagnosis or treatment.

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