Conditionally active anti-CD46 antibodies, antibody fragments, immunoconjugates thereof and uses thereof

Abstract

The present invention provides an isolated polypeptide having a heavy chain variable region and / or a light chain variable region that specifically binds to the CD46 protein, as well as antibodies and antibody fragments comprising the heavy chain variable region and / or the light chain variable region that binds to the CD46 protein. Also provided are immunoconjugates, pharmaceutical compositions, and kits comprising the polypeptides and antibodies and antibody fragments comprising the polypeptides.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 040913, filed June 18, 2020, the entire disclosure of which is specifically incorporated herein by reference. References to ASCII text sequence tables are included The Sequence Listing (13,000 bytes in size) submitted herewith as a text file named "BIAT-1032WO_ST25", created on May 25, 2021, is hereby incorporated by reference in its entirety. (Technical field) The present disclosure relates to anti-CD46 antibodies, antibody fragments and immunoconjugates of such antibodies and antibody fragments, and the use of the antibodies, antibody fragments and immunoconjugates in diagnostic and therapeutic methods. [Background technology]

[0002] CD46 is a membrane cofactor protein (MCP). It is a widely expressed type I transmembrane protein, but many isoforms exist as a result of alternative exon splicing and glycosylation. Recently, Karosi et al., Laryngoscope 118:1669-1676 (September 2008), reported the detection of 14 isoforms. The mRNA is transcribed from a single gene located on chromosome 1q32, and extensive alternative splicing generates multiple transcripts encoding various protein isoforms. Of the 14 exons, exons 1-6 are conserved in all CD46 protein isoforms, whereas exons 7-9 encode a variably utilized serine-threonine-proline ("STP")-rich region, likely contributing to the hypervariability of protein isoforms. Exons 11 and 12 encode the transmembrane domain of CD46, and exons 13 and 14 encode the cytoplasmic tail of the protein.

[0003] The longest mRNA transcript, variant A (NM_002389), contains sequences from all 14 exons of the gene. Alternative splicing of exons 7, 8, 9, and 13 is thought to generate most of the 14 isoforms of CD46. Alternative insertions or exclusions of exon 8 result in the predominantly observed 66- and 56-kDa protein isoforms. Alternative insertions / exclusions of exon 13 result in changes in the encoded sequence of the cytoplasmic tail of the molecule, and these changes may affect the intracellular trafficking, stability, and 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 and encoding protein NP_722548), isoform F contains exons 1-6, 9-12, and 14 (corresponding to sequence NM_172353 and encoding protein NP_758863), and isoform J contains exons 1-6, 8, 10-12, and 14 (corresponding to sequence NM_172356 and encoding protein NP_758866). Specifically, the CD46 molecule consists of four N-terminal short consensus repeat (SCR) modules ("Sushi" domains: four cysteines with a 1-3, 2-4 topology). These SCR domains are encoded by the first six exons of the gene. The SCR2, 3, and 4 modules are responsible for C3b / C4b binding and regulatory activity (see below), while the SCR1 module and distal sequence of SCR4 are dispensable for complement regulation. The membrane-proximal extracellular sequence, alternately utilized by exons 7-9 and 10, is heavily glycosylated, primarily via O-linked glycans.

[0005] For the purposes of this disclosure, the term "CD46," unless the context dictates otherwise, shall be deemed to mean any of the above proteins, including splice variants or immunoreactive fragments thereof, and any nucleic acid sequence encoding such a protein, splice variant, or fragment.

[0006] CD46 has been implicated in many biological functions, many of which are involved in regulating the immune system. One of the major immunoregulatory functions of CD46 is the regulation of complement proteins, which are part of the innate immune response in higher eukaryotes, to protect host cells from damage by these proteins. Specifically, CD46 is a cofactor for factor I-mediated proteolytic cleavage of the complement proteins C3b and C4b. CD46 has been shown to activate C3 convertase, a molecule that cleaves C3b into inactive fragments, 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 through CD46 leads to T cell proliferation and differentiation into a specific class of regulatory T cells, called Tr1, which are characterized by high production of the anti-inflammatory cytokine IL-10. High levels of CD46 are also expressed on sperm, suggesting that CD46 is involved in reproduction, possibly in sperm-egg fusion. High levels of CD46 also appear in the placenta, where it may help protect the fetus from maternal immune rejection.

[0008] CD46 has also been shown to be ubiquitously expressed on most normal human cells, with the exception of erythrocytes. For example, CD46 is reported to be highly expressed on epithelial cells, moderately expressed on lymphocytes and endothelium, and weakly expressed on other cell types, such as osteoclasts, osteocytes, stromal cells, and muscle cells. Because of its widespread expression, many human pathogens have developed strategies to utilize CD46 as a receptor or coreceptor for cell binding as a precursor to infection. These pathogens include human herpesvirus 6, measles virus, several adenovirus serotypes, and pathogenic species of the commensal Neisseria family. Certain retroviruses are thought to evade complement-mediated immunity by carrying CD46 mimics on their surface (Stoiber et al., Molecular Immunology 2005; Saifuddin et al., J Gen Virol 1997).

[0009] In addition to its presence on normal cells, CD46 expression levels can be increased in certain cancers. For example, elevated CD46 expression has been shown to be associated with breast cancer (Thorsteinsson et al., APMIS 106:869-78 (1998); Hofman et al., Breast Cancer Res. Treat. 32:213-9 (1994)); colon / 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. Respir. Cell. Mol. Bioi. 19:522-9 (1998)); ovarian cancer (Bjorge et al., Int. J. Cancer 70: 14-25 (1997)); kidney 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 that are suitable for therapeutic and diagnostic uses, particularly for the diagnosis and treatment of cancer. Some of these anti-CD46 antibodies or antibody fragments may have higher binding activity or affinity for CD46 in a tumor microenvironment compared to the binding activity or affinity for CD46 present in a non-tumor microenvironment. These anti-CD46 antibodies or antibody fragments typically have efficacy at least equivalent to that of known anti-CD46 antibodies or antibody fragments. Furthermore, the anti-CD46 antibodies or antibody fragments of the present invention may have relatively lower binding activity or affinity for CD46 in the non-tumor microenvironment present in normal tissues, and as a result, may exhibit reduced side effects compared to monoclonal anti-CD46 antibodies known in the art. These advantages provide more selective targeting of CD46 expressed in tumors, and the selectivity of the antibodies for CD46 present in the tumor microenvironment may allow the use of higher doses of these anti-CD46 antibodies or antibody fragments, thereby achieving more effective therapeutic treatment without a corresponding increase in undesirable side effects.

[0011] (Summary of the Invention) In one aspect, the present invention provides an isolated polypeptide that specifically binds to human CD46. The isolated polypeptide comprises a light chain variable region having three complementarity determining regions (CDRs) having sequences L1, L2, and L3, The L1 sequence is RAX1QX2IX3NYLN (SEQ ID NO: 1), The L2 sequence is YTSSLX4X5 (SEQ ID NO: 2), a light chain variable region, the L3 sequence of which is 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 (SEQ ID NO: 8), The H2 sequence is VIWTDGGTNYNSAFMS (SEQ ID NO: 9), Heavy chain variable region where the H3 sequence is VYDGYPWFAY (SEQ ID NO: 10) Including, 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 simultaneously be S, G, S, H, and S, respectively.

[0012] The polypeptide 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 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 foregoing embodiments, 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 sequences: SEQ ID NO:12, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO: 15, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or SEQ ID NO:22, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or SEQ ID NO:22, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:22, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0014] In another embodiment, an isolated polypeptide of the invention comprises 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 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 said isolated polypeptide specifically binds to 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, an isolated polypeptide of the invention comprises 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.

[0017] In another embodiment, the invention provides a light chain variable region having three complementarity determining regions (CDRs) having sequences L1, L2, and L3, The L1 sequence is RAX1QX2IX3NYLN (SEQ ID NO: 1), The L2 sequence is YTSSLX4X5 (SEQ ID NO: 2), a light chain variable region, the L3 sequence of which is 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 (SEQ ID NO: 8), The H2 sequence is VIWTDGGTNYNSAFMS (SEQ ID NO: 9), the H3 sequence comprises a heavy chain variable region that is VYDGYPWFAY (SEQ ID NO: 10); 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, with the proviso that X1, X2, X3, X4, and X5 cannot simultaneously be S, G, S, H, and S, respectively.

[0018] In one embodiment, the antibody or antibody fragment may have an L1 sequence selected from the amino acid sequence 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 have an L2 sequence selected from the amino acid sequence YTSSLHS (SEQ ID NO:6), YTSSLFS (SEQ ID NO:17), and YTSSLHE (SEQ ID NO:19). In each of the foregoing antibody or antibody fragment embodiments, 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 comprise a set of six CDRs selected from the following sets of six CDRs: SEQ ID NO:12, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO: 15, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or SEQ ID NO:22, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or SEQ ID NO:22, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:22, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0020] In one embodiment, an antibody or antibody fragment of the invention can comprise 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 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 the antibody or antibody fragment specifically binds to human CD46 protein.

[0021] In one embodiment, the antibody or antibody fragment of the invention may comprise 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, an antibody or antibody fragment of the invention competes with any of the antibodies or antibody fragments described above for binding to human CD46.

[0023] In each of the foregoing embodiments, the antibody or antibody fragment may have higher binding activity to CD46 protein at a value of a condition in a tumor microenvironment compared to a different value of the same condition occurring in a 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 foregoing embodiments, the isolated polypeptide, antibody, or antibody fragment may have an antigen-binding activity at pH 6.0 that is at least 70% of the antigen-binding activity of the parent polypeptide, antibody, or antibody fragment at pH 6.0, and the isolated polypeptide, antibody, or antibody fragment may have an antigen-binding activity at pH 7.4 that is less than 50%, or less than 40%, or less than 30%, or less than 20%, or less than 10% of the antigen-binding activity of the parent polypeptide, antibody, or antibody fragment at pH 7.4. The antigen-binding activity may be binding to the CD46 protein.

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

[0026] In yet another aspect, the present invention provides an immunoconjugate comprising any of the above-described antibodies or antibody fragments of the invention, wherein the antibody or antibody fragment is optionally conjugated to an agent selected from a chemotherapeutic agent, a radioactive atom, a cytostatic agent, and a cytotoxic agent.

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

[0028] A single dose of the pharmaceutical composition can comprise an amount of about 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 an amount of polypeptide, antibody or 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.

[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 foregoing 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 a WNT protein, which may 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 diagnostic or therapeutic kit comprising any of the above-described polypeptides, antibodies or antibody fragments, immunoconjugates or pharmaceutical compositions of the present invention. [Brief explanation of the drawings]

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

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

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

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

[0037] [Figure 5] FIG. 5 shows the binding activity of exemplary anti-CD46 CAB ADCs and WT ADCs of the 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 invention to HEK 293 cells expressing human CD46 at pH 6.0, as measured by fluorescence-activated cell sorting (FACS).

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

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

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

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

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

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

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

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

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

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

[0049] [Figure 17] FIG. 17 shows the protein sequences of representative conditionally active antibodies of the present invention.

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

[0051] The term "about," as used herein in connection with a measured quantity, refers to normal variations in the measured quantity that would be expected by one of ordinary skill in the art making and handling the measurement commensurate with the purpose of the measurement and the precision of the measuring device used. Unless otherwise indicated, "about" refers to a + / - 10% variation of the provided value.

[0052] The term "affinity," as used herein, refers to the strength of the sum of 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 a 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 the 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 with one or more modifications in one or more heavy or light chain variable regions that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess such modifications.

[0054] As used herein, the term "amino acid" refers to any organic compound containing an amino group (--NH) and a carboxyl group (--COOH), preferably either as a free group or alternatively after condensation as part of a peptide bond. "The 20 naturally encoded polypeptide-forming α-amino acids" are art-recognized and 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, such as Fab, Fab', (Fab')2, Fv, and SCA fragments, that are capable of binding to an epitope of an antigen. These antibody fragments retain some ability to selectively bind to the antigen (e.g., polypeptide antigen) of the antibody from which they are derived and can be produced using methods well known in the art (see, e.g., Harlow and Lane, supra) and are further described below. Antibodies can be used to isolate preparative quantities of antigens by immunoaffinity chromatography. Various other uses of such antibodies are for the diagnosis and / or staging of disease (e.g., neoplasia), as well as therapeutic applications for treating disease (e.g., neoplasia, autoimmune disease, AIDS, cardiovascular disease, infectious disease, etc.). Chimeric, human-like, humanized, or fully human antibodies are particularly useful for administration to human patients.

[0056] A Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule and can be produced by digestion of whole antibody molecules with the enzyme papain to yield a fragment consisting of an intact light chain and a portion of the heavy chain.

[0057] Fab' fragments of antibody molecules can be obtained by treating whole antibody molecules with pepsin, followed by reduction, to yield molecules consisting of an intact light chain and a portion of the heavy chain, resulting in two Fab' fragments per antibody molecule so treated.

[0058] (Fab')2 fragments of antibodies can be obtained by treating whole antibody molecules 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 engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.

[0060] The term "antibody fragment" as used herein refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab'), 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 "antibody that binds to CD46" refer to an antibody that can bind to CD46 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeting CD46. In one embodiment, the extent of binding of an anti-CD46 antibody to an unrelated, non-CD46 protein is less than about 10% of the binding of the antibody to the CD46 protein, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to the CD46 protein has an affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, e.g., 10 -9 M~10 -13 In certain embodiments, the anti-CD46 antibody binds to an epitope of CD46 that is conserved among CD46 from different species, e.g., the extracellular domain of CD46.

[0062] The term "binding" as used herein refers to the interaction of an antibody variable region or Fv with an antigen, with the interaction being dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody variable region or Fv recognizes and binds to a specific protein structure rather than proteins in general. As used herein, the terms "specifically binding" or "binding specifically" mean that an antibody variable region or Fv binds or associates with a particular antigen more frequently, rapidly, for a longer duration, and / or with a higher affinity than with other proteins. For example, an antibody variable region or Fv specifically binds to the antigen with higher affinity, avidity, ease, and / or duration than it binds to other antigens. In another example, an antibody variable region or Fv binds to a cell surface protein (antigen) with substantially higher affinity than the affinity for related proteins or other cell surface proteins or antigens commonly recognized by polyreactive natural antibodies (i.e., natural antibodies known to bind to a variety of antigens naturally found in humans). However, "specifically binds" does not necessarily require exclusive or undetectable binding of another antigen, as is meant by the term "selective binding." In one example, "specific binding" of an antibody variable region or Fv (or other binding region) means that the antibody variable region or Fv binds to an 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" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancer include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. More specific examples of such cancer 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, hepatoma, 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, and various types of head and neck cancer.

[0064] As used herein, the terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0065] As used herein, the term "chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins, particularly delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (including synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; kallistatin; CC-1065 (and including synthetic analogs of adozelesin, carzelesin, and bizelesin; podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eluterobin; pancratistatin; sarcodictin; spongistatin; chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, metronidazole, methadone ... nitrogen mustards such as chlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, fenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma 11 and calicheamicin omega 11 (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33:183-186 (1994)); the oral alpha-4 integrin inhibitor CDP323;Dynemicins, including dynemicin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, 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®), peglylated liposomal doxorubicin (including CAELYX®, and deoxydoxorubicin), mitomycins such as epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, 5-fluorouracil (5-FU); denopterin, Folic acid analogues such as methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calucelone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine;Diazicon; elformitin; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, anguidine); urethane; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, such as paclitaxel (TAXOL®), albumin-modified nanoparticle formulation of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chlorambucil; 6-thioguanine; mercaptopurine; methicone platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas that prevent tubulin polymerization to form microtubules, such as vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMF®); retinoids such as retinoic acid, including bexarotene (TARGRETIN®);clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), bisphosphonates such as NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as, for example, PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as the THERATOPE® vaccine and gene therapy vaccines, for example, the ALLOVECTIN® vaccine, the LEUVECTIN® vaccine, and the VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib, Bayer); SU-11248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors such as oblimersen sodium (GENASENSE®); pixantro EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNE®); farnesyltransferase inhibitors such as lonafarnib (SCH6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; combinations of two or more of the above, such as CHOP (an abbreviation for cyclophosphamide, doxorubicin, vincristine, and prednisolone combination therapy);and FOLFOX (an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin);

[0066] Chemotherapeutic agents, as defined herein, include "antihormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth. They may themselves be hormones, including, but not limited to, antiestrogens with mixed agonist / antagonist profiles, including selective estrogen receptor modulators (SERMs) such as tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and SERM3; pure antiestrogens without the agonist properties of fulvestrant (FASLODEX®) and EM800 (agents that may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), as well as anastrazole (ARIMIDEX®), letrozole (FEMARA®). and aminoglutethimide, and other aromatase inhibitors including vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, 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 androgens / retinoids such as fluoxymesteron, all-trans retionic acid, and fenretinide; onapristone; antiprogesterones; estrogen receptor downregulators (ERDs); 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 and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived 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 conditions in a tumor microenvironment than under conditions in a non-tumor microenvironment. Conditions in a tumor microenvironment include lower pH, higher concentrations of lactate and pyruvate, low oxygen, lower concentrations of glucose, and slightly higher temperatures compared to a non-tumor microenvironment. For example, a conditionally active antibody is virtually inactive at normal body temperature but active at the higher temperatures in a tumor microenvironment. In yet another embodiment, a conditionally active antibody is less active in normal, oxygenated blood but more active in the hypoxic environment present in a tumor. In yet another embodiment, a conditionally active antibody is less active at a normal physiological pH of 7.2-7.8 but more active at an acidic pH of 5.8-7.0 or 6.0-6.8 present in a tumor microenvironment. Other conditions known to those skilled in the art exist in the tumor microenvironment and can be used as conditions in the present invention under which an anti-CD46 antibody has a different binding affinity for the CD46 protein.

[0069] The term "cytostatic agent" as used herein refers to a compound or composition that arrests cell proliferation either in vitro or in vivo. Thus, a cytostatic agent may significantly reduce the proportion of cells in S phase. Further examples of cytostatic agents 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 cytostatic agent that induces G0 / G1 arrest. Classical M-phase blockers include vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors (such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin). Certain agents that arrest G1, e.g., DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C, also spill over 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 anticancer drugs" by Murakami et al. (WB Saunders, Philadelphia, 1995), e.g., p. 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analog of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, leading to the inhibition of mitosis in cells.

[0070] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents cellular 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 radioactive isotopes of Lu), chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents), growth inhibitory agents, enzymes such as nucleases and fragments thereof, antibiotics, toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof, and various anti-tumor or anti-cancer agents disclosed below.

[0071] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L ) light chain variable domain (V L ) bound to the heavy chain variable domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create 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. Representative examples of useful detectable labels include, but are not limited to, the following: molecules or ions that are directly or indirectly detectable based on light absorption, fluorescence, reflectance, light scattering, phosphorescence, or luminescence properties; molecules or ions that are radioactively detectable; and molecules or ions that are nuclear magnetic resonance or paramagnetically detectable. For example, among the group of molecules that are indirectly detectable based on light absorption or fluorescence, are various enzymes that convert an appropriate substrate, for example, from a non-light-absorbing molecule to a light-absorbing molecule, or from a non-fluorescent molecule to a fluorescent molecule.

[0073] As used herein, the term "diagnosis" refers to determining the subject's susceptibility to disease or disorder, determining whether the subject currently suffers from disease or disorder, prognosing the subject suffering from disease or disorder (for example, identifying the pre-metastatic or metastatic cancerous state, the stage of cancer, or the response of cancer to treatment), and treatment strategy (for example, monitoring the subject's condition to provide information on the effectiveness or efficacy of treatment).In some embodiments, the diagnostic method of the present invention is particularly useful for detecting early cancer.

[0074] The term "diagnostic agent" as used herein refers to a molecule that can be detected directly or indirectly and is used for diagnostic purposes. The diagnostic agent may be administered to a subject or a sample. The diagnostic agent may be provided by itself or may be conjugated to a vehicle such as a conditionally active antibody.

[0075] As used herein, the term "effector function" refers to the biological activities attributable to the Fc region of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0076] As used herein, the term "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0077] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. This term includes native-sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy terminus of the heavy chain. However, the C-terminal lysine (Lys447) 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 is according to 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 of a variable domain other than residues in the complementarity determining regions (CDRs or H1-3 in the heavy chain, L1-3 in the light chain). The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the CDR and FR sequences are generally expressed as a V H (or V L ), they generally 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 L), as well as a light chain constant domain (CL) and a heavy chain constant domain (CH1, CH2, and CH3). The constant domains may be native-sequence constant domains (e.g., human native-sequence constant domains) or amino acid sequence variants thereof. Depending on the amino acid sequence of the constant domain of their heavy chain, full-length antibodies can be assigned to different "classes." There are five major 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 the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0080] As used herein, the term "function-conservative variant" refers to a protein or enzyme in which a given amino acid residue has been altered without altering the overall conformation and function of the polypeptide, including, but 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 designated as conserved amino acids may vary in a protein, resulting in a change in the percent sequence similarity of proteins or amino acids between any two functionally similar proteins, e.g., 70%-99% when similarity is determined according to an alignment scheme such as a clustering method based on the MEGALIGN algorithm. "Function-conservative variants" also include polypeptides 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%, as determined by the BLAST or FASTA algorithm, with the native or parent protein to which they are compared, and possessing the same or substantially similar properties or functions.

[0081] As used herein, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," and include the primary transformed cell and its progeny, regardless of the number of transfers. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0082] As used herein, the term "human antibody" refers to an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an amino acid sequence derived from a non-human source that utilizes the coding sequence of a human antibody repertoire or other human antibody. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0083] As used herein, the term "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDRs and human FRs. In certain embodiments, a humanized antibody comprises 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. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "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" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0085] As used herein, the term "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, 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 "inhibiting 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 that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or greater than 99% purity, as determined, for example, 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 a review of methods for assessing antibody purity, 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 the antibody, including such nucleic acid molecules in a single vector or in separate vectors, and including such nucleic acid molecules present in one or more locations within a host cell.

[0089] As used herein, the term "metastasis" refers to all CD46-mediated processes that support cancer cell dispersal from a primary tumor, penetration into lymphatics and / or blood vessels, circulation through the bloodstream, and proliferation at distant foci (metastases) in normal tissues elsewhere in the body. In particular, it refers to tumor cell cellular events, such as proliferation, migration, anchorage independence, evasion of apoptosis, or secretion of angiogenic factors, that underlie metastasis and are stimulated or mediated by CD46.

[0090] As used herein, the term "microenvironment" refers to any part or region of a tissue or body that has permanent or temporary, physical or chemical differences from other regions of the tissue or body. In the case of tumors, the term "tumor microenvironment" refers to the environment in which the tumor resides, including the acellular regions within the tumor and the regions 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 interact. Tumors can alter their microenvironment, which can affect tumor growth and spread. Typically, tumor microenvironments have a low pH, ranging from 5.0 to 7.0, or from 5.0 to 6.8, or from 5.8 to 6.8, or from 6.2 to 6.8. Normal physiological pH, on the other hand, is in the range of 7.2 to 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 to 1°C higher than normal physiological temperature. The tumor microenvironment is discussed in Gillies et al., "MRI of the Tumor Microenvironment," Journal of Magnetic Resonance Imaging, vol. 16, pp. 430-450, 2002, which is incorporated herein by reference in its entirety. The term "non-tumor microenvironment" refers to the microenvironment at a site other than a tumor.

[0091] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, but excludes variant antibodies that contain, for example, naturally occurring mutations or that may arise during production of the monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies specific for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is specific for a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as obtained from a population of substantially homogeneous antibodies and should not be construed as requiring 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 not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; 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 to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in pharmaceutical formulations.

[0093] As used herein, the term "package insert" refers to instructions customarily included in commercial packaging for a therapeutic product, which contain information about the indications, uses, dosages, administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0094] As used herein, the term "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been submitted, along with user documentation, to 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 the source code. The ALIGN-2 program must 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 vary.

[0095] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with or to a given amino acid sequence B (alternatively, it can be expressed as a given amino acid sequence A having or containing a particular % amino acid sequence identity with or to a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, 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 will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the previous section using the ALIGN-2 computer program.

[0096] As used herein, the term "pharmaceutical formulation" refers to a formulation that is in a form that is effective for the biological activity of the active ingredient contained therein and that does not contain additional ingredients that are unacceptably toxic to the subject to whom the formulation is administered.

[0097] As used herein, the term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to a subject. 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 an antibody or nucleotide sequence according to the present invention and indicate that the designated molecule is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" preferably means that at least 75% by weight of the same biological macromolecules is present, more preferably at least 85% by weight, even more preferably 95% by weight, and most preferably at least 98% by weight. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the polypeptide, although the molecule may contain some additional bases or moieties that do not adversely affect the basic characteristics of the composition.

[0099] As used herein, the term "recombinant antibody" refers to an antibody (e.g., a chimeric, humanized, or human antibody, or an antigen-binding fragment thereof) expressed by a recombinant host cell containing nucleic acid encoding the antibody. Examples of "host cells" for producing recombinant antibodies include: (1) mammalian cells, such as Chinese hamster ovary (CHO), COS, myeloma cells (including Y0 and NS0 cells), baby hamster kidney (BHK), Hela cells, and Vero cells; (2) insect cells, such as sf9, sf21, and Tn5; (3) plant cells, such as plants belonging to the genus Nicotiana (e.g., Nicotiana tabacum); (4) yeast cells, such as those belonging to the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or Aspergillus (e.g., Aspergillus niger); and (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells.

[0100] As used herein, the term "single-chain Fv" ("scFv") refers to a covalently linked V H ::V L A heterodimer, which typically consists of two genes linked by a peptide-encoded linker, V H and V L"dsFv" is a disulfide bond stabilized V H ::V L Divalent and multivalent antibody fragments can form spontaneously by association of monovalent scFvs or can be generated by linking monovalent scFvs with peptide linkers (such as bivalent sc(Fv)2).

[0101] The term "therapeutically effective amount" of an antibody of the present invention refers to a sufficient amount of antibody to treat the cancer in question, at a reasonable benefit-to-risk ratio applicable to any medical treatment. However, it will be understood that the total daily usage of the antibodies and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the disorder being treated and the severity of the disorder, the activity of the specific antibody used, the specific composition used, the patient's age, weight, general health, sex, and diet, the time of administration, route of administration, and excretion rate of the specific antibody used, the duration of treatment, drugs used in combination with or concomitantly with the specific antibody used, and similar factors known in the medical arts. For example, it is known in the art to start administering compounds at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0102] As used herein, the terms "treatment," "treat," or "treating" refer to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, remission or palliation of the disease state, and improvement in remission or prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of the disease.

[0103] As used herein, the term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive as referred to herein.

[0104] As used herein, the term "variable region" or "variable domain" refers to the domains of an antibody heavy or light chain that are involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (V, respectively) of a native antibody are H and V L ) have a generally similar structure, with each domain containing four conserved framework regions (FR) and three complementarity-determining regions (CDR). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single V H or V L The V domain may be sufficient to confer specificity of antigen binding. H or V L Domains can be used to isolate antibodies that bind to a specific antigen and screen libraries of complementary VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol., vol. 150, pp. 880-887, 1993; 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 linked. The term includes vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors." (Mode for Carrying Out the Invention)

[0106] For illustrative purposes, the principles of the present invention are described with 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 the disclosed embodiments of the present invention in detail, it should be understood that the present invention is not limited in its application to the details of any particular embodiment shown. In addition, the terminology used herein is for purposes of description and not limitation. Furthermore, although certain methods are described with reference to steps presented herein in a particular order, in many cases these steps can be performed in any order as would be understood by one of ordinary skill in the art, and thus the novel methods are not limited to the particular arrangement of steps disclosed herein.

[0107] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Additionally, 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 expressing quantities of ingredients, properties such as molecular weight, percentages, ratios, reaction conditions, and the like used in the specification and claims, whether or not the term "about" is present, should be understood to be modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed at least in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0109] It is understood that each component, compound, substituent, or parameter disclosed herein is to be construed as disclosed for use alone or in combination with one or more of each other component, compound, substituent, or parameter disclosed herein.

[0110] Also, each amount / value or amount / value range of each component, compound, substituent, or parameter disclosed herein should be construed as being disclosed in combination with each amount / value or amount / value range disclosed for any other component, compound, substituent, or parameter disclosed herein; thus, for purposes of this description, any combination of amounts / values ​​or amount / value ranges for two or more components, compounds, substituents, or parameters disclosed herein should also be understood to be disclosed in combination with each other.

[0111] It is further understood that each lower limit of each range disclosed herein should be interpreted as a disclosure 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 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 with each upper limit of each range, etc. Furthermore, a specific amount / value of a component, compound, substituent, or parameter disclosed in the description or examples should be interpreted as a disclosure of either a lower or upper limit of a range and, therefore, can be combined with any other lower or upper limit or specific amount / value of a range for 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 and light chain variable regions of the present invention were derived from parent antibodies using the methods disclosed in U.S. Patent Nos. 8,709,755 and 8,859,467, respectively. This method of producing heavy and light chain variable regions, as well as methods of producing antibodies and antibody fragments, is disclosed in U.S. Patent Nos. 8,709,755 and 8,859,467, and is hereby incorporated by reference herein.

[0113] In one aspect, the present invention provides an isolated polypeptide that specifically binds to human CD46. The isolated polypeptide comprises a light chain variable region having three complementarity determining regions (CDRs) having sequences L1, L2, and L3, The L1 sequence is RAX1QX2IX3NYLN (SEQ ID NO: 1), The L2 sequence is YTSSLX4X5 (SEQ ID NO: 2), a light chain variable region in which the L3 sequence is 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 (SEQ ID NO: 8), The H2 sequence is VIWTDGGTNYNSAFMS (SEQ ID NO: 9), the H3 sequence comprises a heavy chain variable region that is VYDGYPWFAY (SEQ ID NO: 10); 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 simultaneously be S, G, S, H, and S, respectively.

[0114] The polypeptide 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 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 foregoing embodiments, 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 sequences: SEQ ID NO:12, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO: 15, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or SEQ ID NO:22, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or SEQ ID NO:22, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:22, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0116] In another embodiment, an isolated polypeptide of the invention comprises 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 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 said isolated polypeptide specifically binds to 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, an isolated polypeptide of the invention comprises 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.

[0119] In another embodiment, the invention provides a light chain variable region having three complementarity determining regions (CDRs) having sequences L1, L2, and L3, The L1 sequence is RAX1QX2IX3NYLN (SEQ ID NO: 1), The L2 sequence is YTSSLX4X5 (SEQ ID NO: 2), a light chain variable region in which the L3 sequence is 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 (SEQ ID NO: 8), The H2 sequence is VIWTDGGTNYNSAFMS (SEQ ID NO: 9), the H3 sequence comprises a heavy chain variable region that is VYDGYPWFAY (SEQ ID NO: 10); 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, with the proviso that X1, X2, X3, X4, and X5 cannot simultaneously be S, G, S, H, and S, respectively.

[0120] In one embodiment, the antibody or antibody fragment may have an L1 sequence selected from the amino acid sequence 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 have an L2 sequence selected from the amino acid sequence YTSSLHS (SEQ ID NO:6), YTSSLFS (SEQ ID NO:17), and YTSSLHE (SEQ ID NO:19). In each of the foregoing antibody or antibody fragment embodiments, 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 comprise a set of six CDRs selected from the following sets of six CDRs: SEQ ID NO:12, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO: 15, SEQ ID NO: 6, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or SEQ ID NO:22, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 3, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or SEQ ID NO:22, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or SEQ ID NO:22, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

[0122] In one embodiment, an antibody or antibody fragment of the invention can comprise 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 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 the antibody or antibody fragment specifically binds to human CD46 protein.

[0123] In one embodiment, the antibody or antibody fragment of the invention may comprise 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, an antibody or antibody fragment of the invention competes with any of the antibodies or antibody fragments described above for binding to human CD46.

[0125] In each of the foregoing embodiments, the antibody or antibody fragment may have higher binding activity for CD46 protein at a value of a condition in a tumor microenvironment compared to a different value of the same condition occurring in a 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 foregoing embodiments, the isolated polypeptide, antibody, or antibody fragment may have an antigen-binding activity at pH 6.0 that is at least 70% of the antigen-binding activity of the parent polypeptide, antibody, or antibody fragment at pH 6.0, and the isolated polypeptide, antibody, or antibody fragment may have an antigen-binding activity at pH 7.4 that is less than 50%, or less than 40%, or less than 30%, or less than 20%, or less than 10% of the antigen-binding activity of the parent polypeptide, antibody, or antibody fragment at pH 7.4. The antigen-binding activity may be binding to the CD46 protein.

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

[0128] Antibodies and antibody fragments containing these heavy 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 a tumor microenvironment (e.g., pH 6.0-6.8) than at the pH of a non-tumor microenvironment (e.g., pH 7.0-7.6). Consequently, the anti-CD46 antibodies or antibody fragments of the present invention have higher binding activity to CD46 in a tumor microenvironment compared to their binding activity to CD46 in a typical normal (non-tumor) tissue microenvironment.

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

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

[0131] The processes described herein serve as a guide for deriving these variants. Variants of the heavy and light chain variable regions can be prepared by introducing appropriate modifications into the nucleotide sequences encoding the heavy and light chain variable regions or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequences of the heavy and light chain variable regions. The antibodies or antibody fragments of the present invention can be achieved by any combination of deletions, insertions, and substitutions, so long as they have the desired properties, such as antigen-binding ability and / or conditional activity against human CD46.

[0132] Substitution, insertion, and deletion variants In certain embodiments, antibody or antibody fragment variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include CDRs and framework regions (FRs). Conservative substitutions are shown in Table 1 under the heading "Conservative Substitutions." More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions," and are further described below with respect to classes of amino acid side chains. Amino acid substitutions can be introduced into the antibody or antibody fragment of interest, and the products can be screened for desired activity, such as retention / improvement of antigen binding, or reduced immunogenicity. JPEG0007795206000001.jpg160170

[0133] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln (3) Acidic: Asp, Glu (4) Basic: His, Lys, Arg (5) Residues that affect chain orientation: Gly, Pro (6) Aromatic: Trp, Tyr, Phe

[0134] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0135] One type of substitutional variant involves substituting residues in one or more complementarity-determining regions of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further testing has an altered (e.g., improved) biological property relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or substantially retains a biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques, such as those described herein. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0136] For example, modifications (e.g., substitutions) may be made in the CDRs to improve antibody affinity. Such modifications can be made in CDR "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol., vol. 207, pp. 179-196, 2008), and / or in the SDRs (a-CDRs), resulting in variants V H or V LThe resulting antibody variants are tested for binding affinity. Affinity maturation by constructing and reselecting from a secondary library is described, for example, in Hoogenboom et al. in Methods in Molecular Biology, vol. 178, pp. 1-37, 2001. In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then generated. The library is then screened to identify any antibody variants 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 run) 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, as long as such changes do not substantially reduce the ability of the antibody or antibody fragment to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding activity or binding affinity may be made in the CDRs. Such modifications may be outside the "hot spots" of the CDRs or SDRs. Variant V provided above H and V L In certain embodiments of the sequences, each CDR is either unaltered or does not contain one, two, 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, a target residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody or antibody fragment with the antigen is affected. Further substitutions may be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, a crystal structure of an 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 may be eliminated. Variants may be screened to determine whether they contain desired properties.

[0139] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions, ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibodies include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0140] Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding, activity, affinity, and / or other biological properties of the antibody. V of antibodies derived from non-human animals H and V L Only the CDRs in the V region of a human antibody are H and V LIt is known that when a humanized antibody is produced by grafting FRs into the V region of a non-human antibody, the antigen-binding activity is reduced compared to that of the original antibody derived from a non-human animal. H and V L Some amino acid residues in the V region of a human antibody are thought to be directly or indirectly involved in antigen-binding activity. H and V L Substitution of different amino acid residues from the FR of the human antibody will reduce the binding activity. H and V L In the amino acid sequence of the FR, it is necessary to attempt to identify amino acid residues that are directly involved in binding to the antibody, or that interact with amino acid residues in the CDR, or that maintain the three-dimensional structure of the antibody and are directly involved in binding to the antigen. Reduced antigen-binding activity can be increased by substituting the identified amino acids with amino acid residues of the original antibody derived from a non-human animal.

[0141] Modifications and changes can be made in the structure of the antibodies of the present invention, and in the DNA sequences that encode them, while still obtaining a functional molecule that encodes an antibody with desired characteristics.

[0142] When making changes to the amino acid sequence, the hydrophilicity index of the amino acids may be taken into consideration. The importance of the hydrophilicity index in conferring interactive biological function on a protein is generally understood in the art. It is accepted that the relative hydrophilicity characteristics of amino acids contribute to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydrophilicity index based on its hydrophobicity and charge characteristics: 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-conservative variants of the antibodies of the invention.

[0144] Two amino acid sequences are "substantially homologous" or "substantially similar" if they are more than 80%, preferably more than 85%, preferably more than 90% identical in amino acids over the entire length of the shorter sequence, or are about 90%, preferably more than 95%, similar (functionally identical). Preferably, similar or homologous sequences are identified by alignment using, for example, a GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wis.) pileup program, or one of the sequence comparison algorithms such as BLAST or FASTA.

[0145] For example, certain amino acids can be substituted for other amino acids in a protein structure without appreciable loss of activity. Because the interaction capacity and properties of a protein determine its biological functional activity, certain amino acid substitutions can be made in a protein sequence, and of course, in its DNA coding sequence, while still obtaining a protein with similar properties. Thus, it is contemplated that various changes can be made in the sequences of the antibodies or antibody fragments of the present invention, or the corresponding DNA sequences encoding the antibodies or antibody fragments, without significantly impairing their biological activity.

[0146] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydrophilicity index or score and still result in a protein with similar biological activity, i.e., still resulting in a biologically functional equivalent protein.

[0147] As outlined above, amino acid substitutions are therefore generally based on the relative similarity of the amino acid side-chain substituents, e.g., their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions that take into account various of the above characteristics are well known to those of skill 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] Glycosylation variants In certain embodiments, the anti-CD46 antibodies or antibody fragments provided herein are modified to increase or decrease the extent to which the antibody or antibody fragment is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by modifying the amino acid sequence so that one or more glycosylation sites are created or removed.

[0149] If the antibody contains an Fc region, the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, generally N-linked to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al., TIBTECH, vol. 15, pp. 26-32, 1997. The oligosaccharides can contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharides in the antibodies of the present invention can be made to create antibody variants with improved properties.

[0150] In one embodiment, antibody variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can 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 glycan at Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, e.g., as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues). However, due to slight sequence variations in antibodies, Asn297 may be located approximately ±3 amino acids upstream or downstream of position 297, i.e., positions 294 to 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US2003 / 0157108 (Presta, L.) and US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108, WO2000 / 61739, WO2001 / 29246, US2003 / 0115614, US2002 / 0164328, US2004 / 0093621, and US2004 / 01321. 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, 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, which are deficient in 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)), and 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 biantennary oligosaccharides are also provided, for example, biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878, U.S. Patent No. 6,602,684, and US2005 / 0123546. Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087, WO1998 / 58964, and WO1999 / 22764.

[0152] Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an anti-CD46 antibody or antibody fragment provided herein to generate an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0153] In certain embodiments, the present invention contemplates antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications in which in vivo antibody half-life is important but certain effector functions (such as ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, whereas monocytes 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 to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see also, e.g., 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; U.S. Pat. No. 5,821,337 (see also, Bruggemann et al., J. Exp. Med., vol. 166, pp. 1351-1361, 1987). Alternatively, non-radioactive assay methods can be used (see, e.g., ACTI™ Non-Radioactive Cytotoxicity Assay for Flow Cytometry (CellTechnology, Inc. Mountain View, Calif.), and 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 assessed in vivo, for example, in an animal model such as that 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 is unable to bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, 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). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol., vol. 18, pp. 1759-1769, 2006).

[0154] Antibody or antibody fragment variants with reduced effector function include those with substitutions at one or more 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 Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0155] Certain antibody variants with improved or diminished binding to FcRs have been described (see, for example, U.S. Pat. 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 comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbering of residues).

[0157] In some embodiments, alterations that result in altered (i.e., improved or diminished) C1q binding and / or complement-dependent cytotoxicity (CDC), such as those described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol., vol. 164, pp. 4178-4184, 2000, are made in the Fc region.

[0158] Antibodies with improved binding to the neonatal Fc receptor (FcRn), which is involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., vol. 117, pp. 587-593, 1976 and Kim et al., J. Immunol., vol. 24, p. 249, 1994), and increased half-lives are described in US2005 / 0014934. These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include variants with substitutions at 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., a substitution at residue 434 in the Fc region; U.S. Pat. No. 7,371,826). For other examples of Fc region variants, see also Duncan & Winter, Nature, vol. 322, pp. 738-740, 1988; U.S. Pat. Nos. 5,648,260 and 5,624,821; and WO 94 / 29351.

[0159] Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine-engineered antibodies, e.g., "thioMAbs," 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 sites accessible to the antibody. By substituting these residues with cysteine, reactive thiol groups are placed at accessible sites on the antibody, allowing the antibody to be conjugated to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. 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-engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0160] antibody derivative In certain embodiments, the anti-CD46 antibodies or antibody fragments provided herein may be further modified to contain additional nonproteinaceous moieties known in the art and readily available. Suitable moieties for derivatizing antibodies or antibody fragments include, but are not limited to, water-soluble polymers. Non-limiting 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), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, 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 can be of any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody or antibody fragment can vary, and if two or more polymers are attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody or antibody fragment to be improved, whether the derivative will be used in therapy under certain conditions, etc.

[0161] In another embodiment, a conjugate of an antibody or antibody fragment and a nonproteinaceous moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the nonproteinaceous 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, including but not limited to, wavelengths that do not harm normal cells but heat the nonproteinaceous moiety to a temperature that kills cells in close proximity to the antibody-nonproteinaceous moiety.

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

[0163] In certain embodiments, the anti-CD46 antibodies or antibody fragments of the invention have a dissociation constant (Kd) with CD46 under conditions in a tumor microenvironment of about ≦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 -13In one embodiment, the ratio of the Kd of the antibody or antibody fragment to CD46 under conditions in a non-tumor microenvironment to the Kd under the same conditions in a 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 conditions in a non-tumor microenvironment to the binding activity under the same conditions in a 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 a Fab version of its antigen using the following assay: The solution binding affinity of a Fab for an antigen is determined by binding the Fab to a minimum concentration ( 125I) Measurements are made by equilibrating with labeled antigen and then capturing the bound antigen using a plate coated with an anti-Fab antibody (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 [I]-antigen is mixed 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)). The Fab of interest is then incubated overnight, although incubation may continue for a longer period (e.g., approximately 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added, and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). A concentration of each Fab that provides 20% or less of maximum binding is selected for use in competitive binding assays.

[0166] According to another embodiment, Kd is measured at approximately 10 response units (RU) using a surface plasmon resonance assay with a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) using an immobilized antigen CM5 chip at 25°C. Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide 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 then injected at a flow rate of 5 μl / min to achieve approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in 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 binding model (BIACORE® evaluation software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. off / k on The on-rate is calculated as a ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). When the on-rate is 10 by the surface plasmon resonance assay described above, 6 M -1 s -1Above 1000 kJ / s, the on-rate can be determined 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 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C with increasing concentrations of antigen, as measured in a spectrometer such as a stopped-flow equipped spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[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 is used, such as an Fv, Fab, Fab', Fab'-SH, scFv, diabody, triabody, tetrabody, or F(ab')2 fragment formed from an antibody fragment, and a multispecific antibody. In another embodiment, the antibody is a full-length antibody, such as an intact IgG antibody, or other antibody class or isotype as defined herein. For a review of certain antibody fragments, see Hudson et al. Nat. Med., vol. 9, pp. 129-134, 2003. For a review of scFv fragments, see, e.g., Pluckthuen, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor-binding epitope residues and have increased in vivo half-lives, see U.S. Pat. No. 5,869,046.

[0168] The diabodies of the present invention can be bivalent or bispecific. For examples of diabodies, see, for example, EP 404,097, WO 1993 / 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 invention includes single-domain antibody fragments, comprising all or a portion of a heavy chain variable domain, or all or a portion of a light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Patent No. 6,248,516 B1).

[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., E. 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 Morrison et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp. 6851-6855, (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody in which the class or subclass of the antibody is changed compared to the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0172] In certain embodiments, the chimeric antibody of the present invention is a humanized antibody. Typically, such non-human antibodies are humanized to reduce 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, in which the CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. Optionally, the humanized antibody may also comprise at least a portion of a human constant region. In some embodiments, some FR residues of the humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0173] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008, and further described, for example, in 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. al., Methods, vol. 36, pp. 25-34, 2005 (describing SDR (a-CDR) grafting), Padlan, Mol. Immunol., vol. 28, pp. 489-498, 1991 (describing "resurfacing"), Dall'Acqua et al., Methods, vol. 36, pp. 43-60, 2005 (describing "FR shuffling"), and Osbourn et al., Methods, vol. 36, pp. 61-68, 2005 and Klimka et al., Br. J. Cancer, vol. 83, pp. 252-260, 2000 (describing a "guided selection" approach to FR shuffling).

[0174] Human framework regions that can 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 a particular subgroup of light 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 mature (somatically mutated) 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 (see, e.g., Baca et al. 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 antibodies of the present invention are multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In some embodiments, one binding specificity is for CD46 and the other is for another antigen. In some embodiments, bispecific antibodies can bind to two different epitopes of CD46. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing CD46. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0176] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature, vol. 305, pp. 537-540, 1983; WO 93 / 08829; and Traunecker et al., EMBO J., vol. 10, pp. 3655-3659, 1991), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by manipulating electrostatic steering effects to create antibody Fc-heterodimeric molecules (WO2009 / 089004A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, vol. 229, pp. 81-83, 1985), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., vol. 148, pp. 1547-1553, 1992), using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., J. Immunol., vol. 148, pp. 1547-1553, 1992), and using "diabody" technology to generate bispecific antibody fragments (see, e.g., Hollinger et al., J. Immunol., vol. 148, pp. 1547-1553, 1992). al., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993), and by using single-chain Fv (scFv) dimers (see, for example, Gruber et al., J. Immunol., vol. 152, pp. 5368-5374, 1994), as well as by preparing triabodies (see, for example, Tutt et al. J. Immunol., vol. 147, pp. 60-69, 1991).

[0177] Engineered antibodies with three or more functional antigen binding sites are also included herein, including "Octopus antibodies" (see, eg, US2006 / 0025576A1).

[0178] The anti-CD46 antibodies or antibody fragments of the invention can be produced using recombinant methods and compositions described in detail in US2016 / 0017040.

[0179] The physical / chemical properties and / or biological activity of the anti-CD46 antibodies or antibody fragments of the present invention can be tested and measured by various assays known in the art, some of which are described in U.S. Patent No. 8,853,369.

[0180] B. Immunoconjugates In another aspect, the present invention also provides immunoconjugates, comprising an anti-CD46 antibody or antibody fragment conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), and radioactive isotopes.

[0181] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC), in which an antibody or antibody fragment is conjugated to one or more drugs, including, but not limited to, maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. EP 0425235 B1), auristatins such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (U.S. Pat. No. 5,635,483 Nos. 5,780,588 and 7,498,298), dolastatins, calicheamicin or derivatives thereof (see U.S. Pat. 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 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 (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., al.,Bioorg.& Med.Chem.Letters,vol.12,vol.1529-1532,2002, King et al.,J.Med.Chem.,vol.45,pp.4336-4343, 2002, and U.S. Patent No. 6,630,579), methotrexate, vindesine, taxanes (such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel), trichothecenes, and CC1065.

[0182] In another embodiment, the immunoconjugate comprises an antibody or antibody fragment described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0183] In another embodiment, the immunoconjugate comprises an antibody or antibody fragment described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212, and radioactive isotopes of Lu. When a radioconjugate is used for detection, it may contain a radioactive atom for scintigraphy studies, such as tc99m or I123, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, and iron.

[0184] In some embodiments, the immunoconjugate comprises a radioactive agent that may be selected from an alpha emitter, a beta emitter, and a gamma emitter. Examples of alpha emitters include: 211 At, 210 Bi, 212 Bi, 211 Bi, 223 Ra, 224 Ra, 225 Ac, and 227 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 It's Ba.

[0185] Conjugates of antibodies / antibody fragments and cytotoxic agents can be made 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), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science, vol. 238, pp. 1098-, 1987. Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic agent inside the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res., vol. 52, pp. 127-131, 1992; U.S. Patent No. 5,208,020) can be used.

[0186] Immunoconjugates encompassed herein are expressly contemplated, but include, but are not limited to, commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SLAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as immunoconjugates prepared with cross-linking reagents including, but not limited to, SVSB (succinimidyl-(4-vinylsulfone)benzoate).

[0187] Exemplary embodiments of ADCs include a tumor cell-targeting antibody or antibody fragment (Ab), a drug moiety (D), and a linker moiety (L) that connects the Ab to D. In some embodiments, the antibody is attached to the linker moiety (L) via one or more amino acid residues, such as lysine and / or cysteine.

[0188] An exemplary ADC is Ab-(LD) p wherein p is 1 to about 20. In some embodiments, the number of drug moieties that can be conjugated to an 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 methods described herein. Exemplary ADCs of Formula I include, but are not limited to, antibodies with one, two, three, or four engineered cysteine ​​amino acids (Lyon et al., Methods in Enzym., vol. 502, pp. 123-138, 2012). In some embodiments, one or more free cysteine ​​residues are already present in the antibody without modification, in which case the existing free cysteine ​​residues can be used to conjugate the antibody to a drug. In some embodiments, the antibody is exposed to reducing conditions to generate one or more free cysteine ​​residues prior to conjugation of the antibody.

[0189] Linkers are used to conjugate moieties to antibodies to form immunoconjugates, such as ADCs. Suitable linkers are described in WO2017 / 180842.

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

[0191] Drug moieties also include compounds with nucleolytic activity (eg, ribonucleases or DNA endonucleases).

[0192] In certain embodiments, the immunoconjugate may include a highly radioactive atom. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 In some embodiments, when the immunoconjugate is used for detection, it can be used with a radioactive atom for scintigraphy studies, e.g., Tc 99 Or I 123 or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging (MRI)), such as zirconium-89, iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron. Zirconium-89 can be complexed to various metal chelators and, for example, conjugated to antibodies for PET imaging (WO2011 / 056983).

[0193] Radiolabels or other labels may be incorporated into the immunoconjugate by known methods. For example, peptides may be biosynthesized or chemically synthesized using suitable amino acid precursors, e.g., containing one or more fluorine-19 atoms in place of one or more hydrogen atoms. In some embodiments, Tc 99 , I 123 ,Re 186 ,Re 188 , and In 111 Labels such as iodine-123 can be attached via cysteine ​​residues in the antibody. In some embodiments, yttrium-90 can be attached via lysine residues of 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, an immunoconjugate may comprise 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 WO 81 / 01145) into an active drug, such as an anti-cancer drug. Such immunoconjugates are, in some embodiments, useful in antibody-dependent enzyme-mediated prodrug therapy ("Adept"). Enzymes that can be conjugated to the antibody include, but are not limited to, alkaline phosphatase, useful for converting phosphate-containing prodrugs to free drugs; arylsulfatases, useful for converting sulfate-containing prodrugs to free drugs; cytosine deaminase, useful for converting non-toxic 5-fluorocytosine to the anti-cancer drug 5-fluorouracil; proteases useful for converting peptide-containing prodrugs to free drugs, such as Serratia protease, pyrolysis, subtilisin, carboxypeptidase, and cathepsins (such as cathepsins B and L); D-amino acid Examples of suitable enzymes include D-alanylcarboxypeptidase, useful for converting prodrugs containing substituent groups; carbohydrate-cleaving enzymes, such as β-galactosidase and neuraminidase, useful for converting glycosylated prodrugs into free drugs; β-lactamase, useful for converting β-lactam-derivatized drugs into free drugs; and penicillin amidases, such as penicillin V amidase and penicillin G amidase, useful for converting amine nitrogen-derivatized drugs bearing phenoxyacetyl or phenylacetyl groups into free drugs. In some embodiments, enzymes can be covalently bound to antibodies 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 loading in the conjugate is represented by p, the average number of drug moieties per antibody. Drug loading can range from 1 to 20 drug moieties per antibody. The conjugates of the present invention can have a range of 1 to 20 drug moieties. The average number of drug moieties per antibody used to prepare 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), drug loading may be limited by the number of conjugation sites on the antibody. For example, when the linkage is a cysteine ​​thiol, as in certain exemplary embodiments described above, the antibody may have only one or a few cysteine ​​thiol groups, or only one or a few sufficiently reactive thiol groups to which a linker may be attached. In certain embodiments, higher drug loading, e.g., p>5, may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading of an ADC ranges from 1 to about 8, from about 2 to about 6, or from about 3 to about 5. Indeed, it has been shown that for certain ADCs, the optimal ratio of drug moieties per antibody may be less than 8, from about 2 to about 5 (U.S. Patent No. 7,498,298).

[0197] In certain embodiments, fewer than the theoretical maximum number of drug moieties are conjugated to the antibody during the conjugation reaction.Antibodies may contain lysine residues that do not react with drug-linker intermediates or linker reagents, for example, as discussed below.In general, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be linked to drug moieties.In fact, most cysteine ​​thiol residues in antibodies exist as disulfide bridges.In certain embodiments, antibodies can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) to generate reactive cysteine ​​thiol groups under partial or total reduction conditions.In certain embodiments, antibodies are subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.

[0198] The loading (drug / antibody ratio) of ADCs can be controlled in different ways, for example, by (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reduction conditions of cysteine ​​thiol modifications.

[0199] C. Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the anti-CD46 antibodies or antibody fragments provided herein can be used to detect the presence of CD46 in a biological sample, either quantitatively or qualitatively, hi certain embodiments, the biological sample comprises cells or tissues, such as breast, pancreatic, esophageal, lung, and / or brain cells or tissues.

[0200] A further aspect of the present invention relates to an anti-CD46 antibody or antibody fragment of the 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 can be labeled with a detectable molecule or substance, such as the above-mentioned fluorescent molecule, radioactive molecule, or any other label known in the art. For example, the antibody or antibody fragment of the present invention can 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 Radioactive atoms used in scintigraphy studies, such as Re, may also be labeled with spin labels 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 imaging.

[0202] The antibodies or antibody fragments of the present invention may be useful for diagnosing and staging cancers and diseases associated with CD46 overexpression, including 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, blood cancer (leukemia), astrocytoma, and various head and neck cancers, or hyperproliferative diseases with CD46 expression or overexpression.

[0203] The antibodies or antibody fragments of the present invention may be useful for diagnosing diseases other than cancer in which CD46 expression is increased or decreased. (Both soluble and cellular forms of CD46 can be used for such diagnosis. Typically, such diagnostic methods involve the use of a biological sample obtained from a patient. Biological samples encompass a variety of sample types obtained from a subject that can be used in diagnostic or monitoring assays. Biological samples include, but are not limited to, blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom, and the progeny thereof. For example, biological samples include cells obtained from tissue samples recovered from individuals suspected of having a cancer 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 include clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0204] In a specific embodiment, the invention is a method for diagnosing cancer associated with CD46 overexpression in a subject by detecting CD46 on cells from the subject using an antibody of the invention. In particular, the method comprises: 1) contacting a biological sample of a subject with an antibody or antibody fragment according to the present invention under conditions suitable for the antibody or antibody fragment to form a complex with cells of the biological sample that express CD46; and (b) detecting and / or quantifying said complex, wherein detection of said complex is indicative of a cancer associated with CD46 overexpression.

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

[0206] In certain embodiments, the present invention provides methods for diagnosing diseases associated with CD46 expression or overexpression, such as cancer, human immune disorders, thrombotic diseases (thrombosis and atherothrombosis), and cardiovascular diseases.

[0207] In one embodiment, an anti-CD46 antibody or antibody fragment is provided for use in a method of diagnosis or detection. In a further aspect, a method for detecting the presence of CD46 in a biological sample is provided. In a further aspect, a method for quantifying the amount of CD46 in a biological sample is provided. In certain embodiments, the method comprises contacting a biological sample with an 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 methods may be performed in vitro or in vivo. In one embodiment, the anti-CD46 antibody or antibody fragment is used to select subjects eligible for therapy. In some embodiments, the therapy comprises administering an 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, labels or moieties that are directly detected (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties such as enzymes or ligands that are indirectly detected (e.g., via enzymatic reaction or molecular interaction). Exemplary labels include, but are not limited to, radioisotopes ( 32 P, 14 C. 125 I, 3 H, and 131I), or fluorophores (rare earth chelates, or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.), luciferases (e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456)), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (uricase, xanthine oxidase, etc.), coupled enzymes that oxidize dye precursors using hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), biotin / avidin, bacteriphage labels, stable free radicals, etc.

[0209] D. Pharmaceutical Preparations Anti-CD46 antibodies or antibody fragments have cell-killing activity. This cell-killing activity extends to multiple different cell line types. Furthermore, when these antigens or antibody fragments are conjugated to cytotoxic agents, they can reduce tumor size and exhibit reduced toxicity. Therefore, anti-CD46 antibodies, their fragments, or immunoconjugates can be useful for treating 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] Anti-CD46 antibodies or antibody fragments can be used to treat diseases associated with CD46 expression, overexpression, or activation. There is no particular limit to the types of cancers or tissues that can be treated, even if they do not require CD46 expression. Examples 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, blood cancer (leukemia), astrocytoma, and various head and neck cancers. 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 to treat 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, e.g., vaccines, and as anti-infective agents against, for example, 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 atherothrombosis. Anti-CD46 antibodies or antibody fragments can also be used to protect against, prevent, or treat cardiovascular diseases, as well as to prevent or inhibit the invasion of viruses, such as Lassa and Ebola viruses, and to treat viral infections.

[0213] In each of the embodiments of the methods of treatment described herein, the anti-CD46 antibody, antibody fragment, or immunoconjugate of an anti-CD46 antibody or antibody fragment may be delivered in a manner consistent with conventional methods associated with the management of the disease or disorder for which treatment is sought. In accordance with the disclosure herein, an effective amount of the antibody, antibody fragment, or immunoconjugate is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent or treat the disease or disorder. Accordingly, one aspect of the present invention relates to a method of treating a disease associated with CD46 expression, comprising administering a therapeutically effective amount of an antibody, antibody fragment, or immunoconjugate of the present invention to a subject in need of treatment for the disease associated with CD46 expression.

[0214] For administration, anti-CD46 antibodies, antibody fragments, or immunoconjugates can 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, therapeutic molecules are typically combined with a mixture, solution, or composition containing a pharmaceutically acceptable carrier.

[0215] Pharmaceutically acceptable carriers are substances that can be tolerated by recipient patients. 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 comprise one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, etc.

[0216] The form, administration route, dosage, and regimen of the pharmaceutical composition will naturally depend on the condition to be treated, the severity of the disease, the age, weight, and sex of the patient, etc. These considerations can be taken into account by those skilled in the art to formulate a suitable pharmaceutical composition. The pharmaceutical composition of the present invention can be formulated for topical administration, oral administration, parenteral administration, intranasal administration, intravenous administration, intramuscular administration, subcutaneous administration, or intraocular administration, etc.

[0217] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for injectable preparations. These may be present in a particular isotonic sterile saline solution (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. or a mixture of such salts), or in a dried, especially lyophilized, composition that allows the constitution of an injectable solution upon addition of, for example, sterile water or saline.

[0218] In some embodiments, stabilizers, sometimes known as etc. Tonicity agents are present to adjust or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with the charged groups of amino acid side chains, reducing the likelihood of inter- and intra-molecular interactions. etc. The tonicity agent may be present in any amount from 0.1 to 25% by weight of the pharmaceutical composition, preferably from 1 to 5% by weight. etc. The tonicity agent includes polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0219] Additional excipients include agents that can function as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers, and (4) agents that prevent denaturation or adhesion to container walls. 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, myo-inisitose, myonisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol), These may 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] Non-ionic surfactants or detergents (also known as "wetting agents") can be used to aid in solubilizing the therapeutic agent and to protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stresses without causing denaturation of the active therapeutic protein or antibody. The non-ionic 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 (e.g., 20, 40, 60, 65, 80), poloxamers (e.g., 184, 188), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (e.g., TWEEN®-20, TWEEN®-80), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Usable anionic detergents include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0222] The dose used for administration can be adapted depending on various parameters, in particular depending on the mode of administration used, the pathology involved, or alternatively the desired duration of treatment. To prepare a pharmaceutical composition, an effective amount of the 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 including sesame oil, peanut oil, or aqueous propylene glycol, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0224] The solution of active compound as free base or pharmacologically acceptable salt can be prepared in water with suitable surfactant mixture.Dispersion can also be prepared in glycerol, liquid polyethylene glycol and their mixture, and in oil.Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0225] Anti-CD46 antibodies or antibody fragments can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) and are formed with inorganic acids (e.g., hydrochloric acid or phosphoric acid), or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with the free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxide), and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0226] The carrier can also be a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption, for example, aluminum monostearate and gelatin, in the composition.

[0227] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent, which contains one or more other components than those listed above as needed, and then filtration sterilization.Generally, dispersion is prepared by incorporating various sterilized active components into sterile vehicle, and contains basic dispersion medium and other components that are required from those listed above.For the sterile powder that is used to prepare sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, and obtain the powder of active component and any additional desired component from the solution that has been previously sterilized by filtration.

[0228] Preparation of larger or more concentrated solutions for direct injection is also contemplated, and it is envisioned that the use of dimethyl sulfoxide (DMSO) as a solvent will result in very rapid penetration, delivering high concentrations of active agent to small tumor areas.

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

[0230] For parenteral administration in aqueous solution, the solution should be suitably buffered, if necessary, and the liquid diluent should first be rendered 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 will be known to those skilled in the art in light of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion therapy fluid or injected at the proposed infusion site (see, e.g., "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 dose for the individual subject.

[0231] The antibody or antibody fragment can be formulated to deliver 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 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose in a 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, as well as any other dosage form currently in use.

[0233] In certain embodiments, the use of liposomes and / or nanoparticles for the introduction of antibodies or antibody fragments into host cells is contemplated. The formation and use of liposomes and / or nanoparticles is known to those of skill in the art.

[0234] Nanocapsules can generally entrap compounds in a stable and reproducible manner.To avoid the side effects of intracellular polymer overload, these ultrafine particles (sizes of around 0.1 μm) are generally designed using polymers that can be degraded in vivo.The biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are intended for use in the present invention, and these particles can be easily produced.

[0235] Liposomes are formed from phospholipids dispersed in an aqueous medium that spontaneously form multilamellar concentric bilayer vesicles (also called multilamellar vesicles, or MLVs). MLVs generally have diameters between 25 nm and 4 μm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters ranging from 200 to 500 Å, which contain aqueous solution within their cores. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0236] Pharmaceutical formulations containing the anti-CD46 antibodies or antibody fragments described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies or antibody fragments having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to, buffers (such as phosphate, citric acid, 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), low molecular weight polypeptides (about 1 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 dextrins), 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 non-ionic surfactants (such as polyethylene glycol (PEG)).

[0237] Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0238] Exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and WO 2006 / 044908, the latter formulations comprising a histidine acetate buffer.

[0239] The formulations herein may also contain two or more active ingredients, as needed for the particular indication being treated. Preferably, ingredients with complementary activities that do 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 suitably present in combination in amounts effective for the intended purpose.

[0240] In one embodiment, an anti-CD46 antibody, antibody fragment, or immunoconjugate of the 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, and WNT16). The combination may be in the form of two separate molecules, i.e., an anti-CD46 antibody, antibody fragment, or immunoconjugate of the invention and another antibody or antibody fragment. Alternatively, the combination may also be in the form of a single molecule, which has binding activity or affinity for both CD46 and another antigen, thus forming a multispecific (eg, bispecific) antibody.

[0241] Active ingredients can be encapsulated in microcapsules, for example, by coacervation technique or prepared by interfacial polymerization.For example, hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules can be used in colloid drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions, respectively.This technique is disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0242] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody or antibody fragment, which matrices can be in the form of shaped articles, e.g., films, or microcapsules.

[0243] Formulations to be used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0244] E. Therapeutic Methods and Compositions Any of the anti-CD46 antibodies or antibody fragments provided herein can be used in therapeutic methods. In one aspect, an anti-CD46 antibody or antibody fragment is provided for use as a pharmaceutical. In a further aspect, an anti-CD46 antibody or antibody fragment is provided for use in treating 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 certain embodiments, an anti-CD46 antibody or antibody fragment is provided for use in a therapeutic method. In one embodiment, the present invention provides an anti-CD46 antibody or antibody fragment for use in a method of treating an individual with cancer, comprising administering to the individual an effective amount of an anti-CD46 antibody or antibody fragment. In one embodiment, the present invention provides an anti-CD46 antibody or antibody fragment for use in a method for treating an individual having an immune disorder (e.g., an autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus), or diabetes, comprising administering to the individual an effective amount of an anti-CD46 antibody or antibody fragment. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as one described below. In a further embodiment, the present invention provides an anti-CD46 antibody or antibody fragment for use in inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral vasculature or tumor-associated vasculature), and / or inhibiting tumor stromal function.

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

[0246] In a further aspect, the present invention provides use of an anti-CD46 antibody or antibody fragment in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of cancer (in some embodiments, 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 further embodiment, the medicament is used in a method of treating cancer, comprising administering an effective amount of the medicament to an individual having cancer. In a further embodiment, the medicament is used in a method of treating an immune disorder (e.g., an 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 an anti-CD46 antibody or antibody fragment to an individual. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent, such as: In a further embodiment, the medicament is an agent for inhibiting angiogenesis, cell proliferation, immune function, secretion of inflammatory cytokines (e.g., from tumor-associated macrophages), tumor vasculature (e.g., intratumor vasculature or tumor-associated vasculature), and / or tumor stromal function. In a further embodiment, the medicament is used in a method for inhibiting angiogenesis, cell proliferation, immune function, inflammatory cytokine secretion (e.g., from tumor-associated macrophages), tumor vasculature (e.g., intratumor vasculature or tumor-associated vasculature), and / or tumor stromal function in an individual, the method comprising administering an effective amount of the medicament to the individual to inhibit angiogenesis, inhibit cell proliferation, promote immune function, induce inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibit tumor vasculature development (e.g., intratumor vasculature or tumor-associated vasculature), and / or inhibit tumor stromal function. An "individual" according to any of the above embodiments may be a human.

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

[0248] In a further aspect, the present invention provides a method of treating an immune disorder (e.g., an autoimmune disorder), a cardiovascular disorder (e.g., atherosclerosis, hypertension, thrombosis), an infectious disease (e.g., Ebola virus, Marburg virus), or diabetes. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent described below. An "individual" according to any of the above embodiments may be a human.

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

[0250] In a further aspect, the present invention provides pharmaceutical formulations comprising any of the anti-CD46 antibodies or antibody fragments provided herein, e.g., for use in any of the above-described methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-CD46 antibodies or antibody fragments provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-CD46 antibodies or antibody fragments provided herein and at least one additional therapeutic agent, e.g., as described below.

[0251] In each and every treatment described above, the antibody or antibody fragment of the present invention can be used alone, as an immunoconjugate, or in combination with other agents in the treatment. For example, the antibody of the present invention can be co-administered 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 cytostatic agent. 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 immunity or immune system.

[0252] Such combination therapy as described above encompasses combined administration (wherein two or more therapeutic agents are contained in the same or separate formulations) as well as separate administration, where administration of the antibody or antibody fragment may occur prior to, concurrently with, and / or after administration of the additional therapeutic agent and / or adjuvant. The antibody or antibody fragment may also be combined with radiation therapy.

[0253] Anti-CD46 antibodies or antibody fragments can be formulated, dosed, and administered in a manner consistent with good medical practice. Relevant factors to consider include the particular disorder being treated, the particular 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 physicians. The antibody or antibody fragment is optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody or antibody fragment present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and by any route of administration described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route determined empirically / clinically appropriate.

[0254] The appropriate dosage of an antibody or antibody fragment for disease prevention or treatment (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 being administered for prophylactic 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 suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, an initial candidate dosage for administration to a patient may be about 1 μg of antibody or antibody fragment / kg of patient body weight to 40 mg of antibody or antibody fragment / kg of patient body weight, for example, whether by one or more separate administrations or by continuous infusion. A typical daily dosage may range from about 1 μg of antibody or antibody fragment / kg of patient body weight to 100 mg of antibody or antibody fragment / kg of patient body weight or more, depending on the factors mentioned above. For repeated administration over several days or longer, depending on the condition, treatment will generally be sustained until a desired suppression of disease symptoms occurs. Such doses can be administered intermittently, for example, every week or every three weeks (e.g., the patient receives from about 2 to about 20, or, for example, about 6, doses of the antibody or antibody fragment). A higher initial loading dose, followed by one or more lower doses, can be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0255] Specific dosages of the anti-CD46 antibodies or antibody fragments of the present invention that can be administered for the prevention or treatment of a subject's disease can 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 of antibody or antibody fragment per kg of patient body weight. In specific embodiments, dosages can 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 of antibody or antibody fragment per kg of patient body weight. The dosage of the antibody or antibody fragment is the same 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 immunoconjugate. Additionally, the polypeptide having anti-CD46 activity is administered in the same amount as the antibody or antibody fragment.

[0256] A single dose of the pharmaceutical formulation of the invention can contain about 45 μg to about 13,600 mg of an antibody or antibody fragment of the invention, or about 45 μg to about 5,440 mg of an anti-CD46 antibody or antibody fragment. In some embodiments, a single dose of the pharmaceutical formulation of the invention can contain 135 mg to 1,387 mg of an anti-CD46 antibody or antibody fragment of the invention, or amounts such as 135, 235, 335, 435, 535, 635, 735, 835, 935, 1035, 1135, 1235, or 1,387 mg. In certain embodiments, the amount of the anti-CD46 antibody or antibody fragment of the present invention in a single dose of a pharmaceutical formulation ranges from 135 to 235 mg, 235 to 335 mg, 335 to 435 mg, 435 to 535 mg, 535 to 635 mg, 635 to 735 mg, 735 to 835 mg, 835 to 935 mg, 935 to 1035 mg, 1035 to 1135 mg, 1135 to 1235 mg, or 1235 to 1387 mg. The amount of antibody or antibody fragment in a single dose of a pharmaceutical formulation remains the same when administered in the form of a bispecific antibody, in combination with another immune checkpoint inhibitor, as an immunoconjugate, 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 a pharmaceutical formulation.

[0257] In one example, 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 can be an anti-CD46 antibody or antibody fragment disclosed in the present application and an immune checkpoint inhibitor molecule administered as separate molecules or as a bispecific antibody, which has a binding activity to CD46 and a second binding activity to an immune checkpoint.

[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). Additional 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 will be understood that any of the above formulations or methods of treatment may be practiced using an antibody fragment or immunoconjugate of the invention in place of, or in addition to, an anti-CD46 antibody.

[0262] Improving host immune function to eradicate tumors is a topic of increasing interest. Conventional methods include (i) enhancing APCs, such as (a) injecting DNA encoding foreign MHC alloantigens into tumors, or (b) transfecting biopsied tumor cells with genes that increase the probability of tumor immune antigen recognition (e.g., immunostimulatory cytokines, GM-CSF, costimulatory molecules B7.1, B7.2), and (iii) adoptive cellular immunotherapy, or treatment with activated tumor-specific T cells. Adoptive cellular immunotherapy involves isolating tumor-infiltrating host T lymphocytes and expanding the population in vitro, for example, through stimulation with IL-2, tumor, or both. Furthermore, isolated dysfunctional T cells can also be activated by in vitro application of the anti-PD-L1 antibodies of the invention. The activated T cells can then be readministered to the host. One or more of these methods can be used in combination with the administration of the antibodies, antibody fragments, or immunoconjugates of the invention.

[0263] Traditional therapies for cancer include: (i) radiation therapy (e.g., radiotherapy, X-ray therapy, irradiation), or the use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy can be administered via external beam radiation therapy (EBRT) or internally via brachytherapy; (ii) chemotherapy, or the application of cytotoxic drugs that generally affect rapidly dividing cells; (iii) targeted therapy, or agents that specifically affect deregulated proteins in cancer cells (e.g., tyrosine kinase inhibitors imatinib, gefitinib; monoclonal antibodies, photodynamic therapy); (iv) immunotherapy, or enhancement of the host's immune response (e.g., vaccines); (v) hormone therapy, or hormone blockade (e.g., when the tumor is hormone-sensitive); (vi) angiogenesis inhibitors, or blocking blood vessel formation and growth; and (vii) palliative care, or treatments aimed at improving 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 allow for more aggressive treatment regimens.

[0264] In the treatment of cancer, any of the aforementioned conventional treatments for cancer immunity can be administered before, after, or simultaneously with the administration of an anti-CD46 antibody or antibody fragment. In addition, the anti-CD46 antibody or antibody fragment can be administered before, after, or simultaneously with conventional cancer treatments, such as the administration of a tumor-binding antibody (e.g., a monoclonal antibody, a toxin-conjugated monoclonal antibody) and / or the administration of a chemotherapeutic agent.

[0265] F. Products and Kits In another aspect of the present invention, an article of manufacture is provided that includes an anti-CD46 antibody or antibody fragment and other materials useful for the treatment, prevention, and / or diagnosis of the disorders described above. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous infusion bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds the composition, either by itself or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and can have a sterile access port (e.g., the container can be an intravenous infusion bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is the antibody or antibody fragment of the present invention. The label or package insert indicates that the composition is used for treating the selected condition. Additionally, the article of manufacture may include (a) a first container (containing a composition, the composition including an antibody or antibody fragment), and (b) a second container (containing a composition, the composition including an additional cytotoxic or other therapeutic agent). The article of manufacture in this embodiment of the invention may further include a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or in addition, the article of manufacture 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 standpoint, including other buffers, diluents, filters, needles, and syringes.

[0266] It is to be understood that any of the above products may contain an immunoconjugate of the invention instead of, or in addition to, the 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. Kits containing the polypeptides, antibodies, antibody fragments, or antibody-drug conjugates of the present invention are used in detecting CD46 expression (increase or decrease) or in therapeutic or diagnostic assays. Kits of the present invention may include antibodies coupled to a solid support, such as a tissue culture plate or beads (e.g., Sepharose beads). Kits containing antibodies for in vitro detection and quantification of CD46, for example, in ELISA or Western blot, can be provided. Such antibodies useful for detection can be provided with a label, such as a fluorescent label or a radiolabel.

[0268] The kits further include instructions for their use. In some embodiments, the instructions include those required by the U.S. Food and Drug Administration for in vitro diagnostic kits. In some embodiments, the kits further include 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 kits include one or more antibodies or antibody fragments. In other embodiments, the kits further include one or more enzymes, enzyme inhibitors, or enzyme activators. In still other embodiments, the kits further include one or more chromatography compounds. In still other embodiments, the kits further include one or more compounds used to prepare samples for spectroscopic assays. In further embodiments, the kits further include a reference material for interpreting the presence or absence of CD46 according to the intensity, color spectrum, or other physical attribute of the indicator.

[0269] The following examples are illustrative, but not limiting, of the anti-CD46 antibodies of the present disclosure. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in the art and obvious to those skilled in the art are within the scope of the present disclosure. [Example]

[0270] Antibodies or antibody fragments of the invention having the following six CDR sets were tested in the Examples: SEQ ID NO:12, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; SEQ ID NO:15, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; and SEQ ID NO:22, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. JPEG0007795206000002.jpg70170

[0271] Example 1: Binding activity of conditionally active anti-CD46 antibodies to huCD46 The binding activity of the conditionally active anti-CD46 antibodies to human CD46 was measured by ELISA using the BM (benchmark) antibody as a control. The EC50 values ​​for the binding of the conditionally active anti-CD46 antibodies to human CD46 at pH 6.0 and pH 7.4 are summarized in Table 1, and the binding activity is shown in Figures 1 and 2. JPEG0007795206000003.jpg255167

[0272] Example 2: Binding activity of conditionally active anti-CD46 antibodies to cyno CD46 The binding activity of the conditionally active anti-CD46 antibodies to cyno CD46 was measured by ELISA and is shown in Figures 3 and 4. The EC50 values ​​of the conditionally active anti-CD46 antibodies for 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 antibodies to human CD46 The binding activity of conditionally active anti-CD46 antibodies to human CD46 by pH titration was also measured by ELISA (see Figure 5). The pH inflection points of conditionally active anti-CD46 antibodies to human CD46 are summarized in Table 3.

[0274] Example 4: Binding activity of conditionally active anti-CD46 antibodies measured by FACS FACS analysis was performed using 293 cells expressing human CD46. The 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). The EC50 values ​​for binding of the conditionally active humanized anti-CD46 antibodies to 293 cells expressing human CD46 are summarized in Table 4.

[0275] Example 5: Binding activity of conditionally active anti-CD46 antibodies measured by FACS The binding activity of conditionally active anti-CD46 antibodies to CD46-expressing Colo205 cells was measured by FACS at pH 6.0 and pH 7.4. Conditionally active anti-CD46 antibodies consistently showed higher binding activity to Colo205 cells at pH 6.0 than at pH 7.4 (see Figures 8 and 9). The EC50 values ​​for binding of conditionally active anti-CD46 antibodies to CD46-expressing Colo205 cells are summarized in Table 5. JPEG0007795206000004.jpg255166

[0276] Similar FACS analysis was performed using 293 cells expressing cyno CD46. Furthermore, the conditionally active anti-CD46 antibodies consistently exhibited higher binding to 293 cells expressing cyno CD46 at pH 6.0 than at pH 7.4 (see Figures 10 and 11). The EC50 values ​​for binding of the conditionally active anti-CD46 antibodies to 293 cells expressing cyno CD46 are summarized in Table 6.

[0277] Example 6: In vitro cell killing of 293 cells expressing human CD46 Using 293 cells expressing human CD46, in vitro cell killing of 293 cells expressing human CD46 was analyzed at pH values ​​of 6.0 and 7.4. In vitro killing of 293 cells by conditionally active anti-CD46 antibodies is shown in Figures 12 and 13. IC50 values ​​for cell killing of 293 cells by conditionally active anti-CD46 antibodies are shown in Table 7.

[0278] Example 7: Cytotoxicity of conditionally active anti-CD46 antibodies in suppressing CD46-expressing Colo205 cells The conditionally active antibodies were used to treat CD46-expressing Colo205 cells at the tumor microenvironment pH of 6.0 and normal physiological pH of 7.4. The conditionally active antibodies induced a greater inhibition rate (IR%) at the 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 testing of conditionally active antibodies in the Colo205CDX subcutaneous administration model The objective of this study was to evaluate the in vivo antitumor efficacy of test articles in a subcutaneous Colo 205 human colon cancer xenograft model in female BALB / c nude mice. Abbreviation JPEG0007795206000005.jpg91170

[0280] Experimental design Table 1-1. Description of experimental design JPEG0007795206000006.jpg155170

[0281] Experimental methods and procedures cell culture Colo205 tumor cells (ATCC, Manassas, VA, cat # ATCC® CCL-222™) were maintained as monolayer cultures in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO in air. Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells growing in exponential growth phase were harvested and counted for tumor inoculation.

[0282] Tumor inoculation and animal grouping Each mouse received Colo205 tumor cells (5x10 6 ) was inoculated subcutaneously in 0.2 mL of PBS to allow tumor development. Treatment resulted in a mean tumor size of approximately 204 mm 3 The treatment started on day 11 after tumor inoculation, when tumor volume reached 1. Animals were assigned to groups according to tumor volume using an Excel-based stratified randomization program. Each group consisted of eight tumor-bearing mice. Test articles were administered according to the experimental design shown in Table 1-1.

[0283] Test sample preparation Table 2-1. Test Article Preparation Description JPEG0007795206000007.jpg139170

[0284] observation All procedures related to the handling, care, and treatment of animals in this study were conducted in accordance with the guidelines of the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) and approved by WuXi AppTec's Institutional Animal Care and Use Committee (IACUC). Routine monitoring included daily checks for the effects of treatment on tumor growth, as well as normal behaviors such as motility, food and water consumption (visual appearance only), weight gain / loss (measured twice weekly), eye / hair matting, and any other abnormal effects described in the protocol. Mortality and observed clinical signs were recorded based on the number of animals in each subset.

[0285] Tumor measurements and endpoints The primary endpoint was the ability to slow tumor growth. Tumor size was measured in two dimensions twice weekly using calipers and calculated using the formula: V = 0.5ax b2, where a and b are the long and short diameters of the tumor, respectively. Tumor size was used to calculate the values ​​of T / C, TGI, and RTV. The T / C value (percent) is an index of antitumor efficacy, where T and C are the mean volumes of the treatment and control groups, respectively, on a given day. The TGI for each treatment group was calculated using the following formula: TGI (%) = [1 - (Ti - T0) / (Vi - V0)] x 100; Ti is the mean tumor volume of the treatment or isotype group on a given day, T0 is the mean tumor volume of the treatment 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 particular day by its volume on day 0. The RTV value for each mouse was calculated individually and used to calculate the mean RTV for each group.

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

[0287] statistical analysis The mean tumor volume and standard error for each group at different time points were calculated (Table 3-1). For data obtained on day 25 after the start of dosing (the last day that mice remained in all groups), statistical analysis was performed to determine the difference in tumor volume between the vehicle group and the test article groups. Meanwhile, for data obtained on day 32 after the start of dosing (the last day that mice remained in all test article groups), statistical analysis was performed to determine the difference in tumor volume between the isotype group and the other test article groups. One-way analysis of variance was performed to compare the mean tumor volume and RTV between groups. A significant F-statistic was obtained, and comparisons between groups were performed using the Games-Howell test. All data were analyzed using IBM® SPSS Statistics® software (version 17.0). A p-value of <0.05 was considered statistically significant.

[0288] Tumor volume The mean tumor volume for each group is shown in Table 3-1. Table 3-1 Tumor volume JPEG0007795206000008.jpg154170

[0289] Analysis of tumor growth suppression effect Table 3-2 Tumor growth inhibitory effect compared to the vehicle group (based on data on Day 25). JPEG0007795206000009.jpg138170

[0290] Table 3-3 Tumor growth inhibitory effect compared with the isotype group (based on Day 32 data) JPEG0007795206000010.jpg130170

[0291] Tumor growth curve Tumor growth curves are shown in Figure 16. In Figure 16, data represent mean values ​​± SEM.

[0292] Summary and Discussion In this study, the therapeutic effect of conditionally active antibodies was evaluated using the Colo205 human colon xenograft model. The tumor size of each group at each time point after treatment is shown in Table 3-1, Table 3-2, and Figure 16. The mean tumor size in the vehicle group was 1,288 mm on day 25 after the start of treatment. 3 The RTV reached 6.43 ± 0.69. All mice in the vehicle and isotype groups were euthanized with PG-D32. For the other groups, observation was extended until 4 weeks after the fourth administration. All test products demonstrated significant antitumor activity (TGI > 93%, p < 0.002, PG-D25). Administration of the test products BAP133-LP1 or BA-133-00-01 (BM) and BA-133-04-04 LP1 at a dose of 3 mg / kg demonstrated dramatic antitumor activity, leading to complete remission in most mice. While tumor volume was significantly reduced with BA-133-04-01 LP1, BA-133-04-02 LP1, BA-133-04-03 LP1, and BAP133-2-02-12 LP-1, tumor regrowth was observed in several mice in these groups 10 days after the final treatment. Treatment with BAP133-2-02-12 LP-1 delayed tumor growth at the start of the study, but reversed the tumor growth rate several days after treatment was stopped (Figure 16). Isotype treatment (B12-LP1) had little effect compared to vehicle treatment (T / C=84.86%, TGI=17.99%, p-value=0.954, PG-D25). No severe weight loss or death / morbidity events were observed during the entire administration and observation period. Thus, no obvious toxicity was observed in relation to the administration of the test product at the designated doses.

[0293] Example 9: Binding activity of conditionally active anti-CD46 antibodies measured by SPR analysis The binding kinetics of anti-CD46 antibodies were measured by surface plasmon resonance (SPR) on a planar amine sensor chip using an SPR2 / 4 instrument (Sierra Sensors, Hamburg, Germany). 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 served as control surfaces for FC2 and FC4, respectively, with no protein immobilized on them.

[0294] All injections were performed at a flow rate of 25 μL / min and 25°C. The sensor surface was activated with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (200 mM / 50 mM) for 480 seconds. Human CD46-His (2 μg / mL in 10 mM NaAc, pH 5.0) was injected for 480 seconds, and the surface was deactivated by injecting 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. A control surface was activated and deactivated using the same conditions, except no protein was injected. PBST buffer (0.05% TWEEN 20 TM PBS (pH 7.4) containing 30 mM sodium bicarbonate was used as the running buffer for surface preparation. The running solution was switched to PBST with 30 mM sodium bicarbonate, and the pH was adjusted as indicated before injecting the analytes. The instrument was equilibrated with the running solution for 1 h before injecting the first analyte.

[0295] 100 μL of analyte diluted in the corresponding running solution (34.25 nM, 13.70 nM, 6.85 nM, 3.42 nM, 1.37 nM, and 0.0 nM) was injected onto flow cells 1, 2, or 3, 4. The off-rate was measured for 360 seconds. After each cycle of interaction analysis, the chip surface was regenerated by injecting 6 μL of 10 mM glycine (pH 2.0). Flow cells 1 and 3, which did not contain immobilized protein, were used as control surfaces for reference subtraction.

[0296] Additionally, data with buffer only as analyte (0 nM analyte) was subtracted from each run. The provided analysis software, Analyzer R2 (Sierra Sensors), was used to fit the doubly subtracted data using a 1:1 binding model. A molecular weight of 200 kDa was used to calculate the molar concentration of the analyte.

[0297] The binding activity of the conditionally active anti-CD46 antibodies to 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. JPEG0007795206000011.jpg255169

[0298] Experimental Protocol of the Example Examples 1 and 2 JPEG0007795206000012.jpg225170

[0299] formulation Test articles were initially diluted to 300 ng / mL in either pH 6.0 or pH 7.4 ELISA incubation buffer. The 3000 ng / mL test article was then serially diluted 3-fold in either pH 6.0 or pH 7.4 ELISA incubation buffer.

[0300] PH affinity ELISA assay 1) Coat an 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 at 4°C overnight. 3) Decant the plate and tap it gently 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 the wells and aspirate the contents completely. 5) Add 200 μL of pH 6.0 or pH 7.4 ELISA incubation buffer to the wells. Cover the plate with sealing film and place on a plate shaker set at 50 rpm for 60 minutes at room temperature. 6) Decant the plate and tap it gently against a stack of paper towels to remove any remaining liquid. 7) The test article is serially diluted in 3-fold dilutions starting at 3000 ng / mL in ELISA incubation buffer at pH 6.0 or pH 7.4. 8) Add 100 μL / well of diluted test article to the plate. 9) Cover the plate with sealing film and place on a plate shaker set at 50 rpm for 60 minutes at room temperature. 10) Decant the plate and tap it gently 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 wash buffer into the wells and aspirate the contents completely. 12) Dilute the HRP secondary antibody 1:2500 in ELISA incubation buffer pH 6.0 or pH 7.4 13) Add 100 μL of HRP secondary antibody diluted in ELISA incubation buffer, pH 6.0 or pH 7.4, to each well. 14) Cover the plate with sealing film and place on a plate shaker set at 50 rpm for 60 minutes at room temperature. 15) Decant the plate and tap it gently 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 wash buffer into the wells and aspirate the contents completely. 17) Dispense 50 μL of TMB substrate solution into all wells of the plate. Incubate at room temperature for approximately 2 minutes 15 seconds or 2 minutes. 18) Add 50 μL per well of 1N HCl to all wells of the plate. Read the plate at 450 nm using a PerkinElmer EnSpire 2300 Multilabe 1 Reader.

[0301] Example 3 JPEG0007795206000013.jpg214170

[0302] formulation Test articles were diluted to 10 ng / mL in ELISA incubation buffers of various pHs ranging from pH 5.5 to pH 7.4.

[0303] pH Range ELISA Assay 1) Coat an 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 at 4°C overnight. 3) Decant the plate and tap it gently 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 into the wells and aspirating the contents completely. 5) Add 200 μL of incubation buffer of various pH (pH 5.5, 6.0, 6.2, 6.5, 6.7, 7.0, and 7.4) to the wells. Cover the plate with sealing film and place on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. 6) Decant the plate and tap it gently against a stack of paper towels to remove any remaining liquid. 7) Serially dilute test substances to 30 ng / mL in incubation buffers of various pHs (pH 5.5, 6.0, 6.2, 6.5, 6.7, 7.0, and 7.4). 8) Add 100 μL / well of diluted test substance to the plate. 9) Cover the plate with sealing film and place on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. 10) Decant the plate and tap it gently against a stack of paper towels to remove any remaining liquid. 11) Wash the wells three times by dispensing 200 μL of wash buffer of various pH (pH 5.5, 6.0, 6.2, 6.5, 6.7, 7.0, and 7.4) into the wells and aspirating the contents completely. 12) Dilute the HRP secondary antibody 1:2500 in incubation buffers of various pHs (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 pHs (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 on a plate shaker (set at 200 rpm) at room temperature for 60 minutes. 15) Decant the plate and tap it gently against a stack of paper towels to remove any remaining liquid. 16) Wash the wells three times by dispensing 200 μL of wash buffer of various pH (pH 5.5, 6.0, 6.2, 6.5, 6.7, 7.0, and 7.4) into the wells and aspirating the contents completely. 17) Dispense 50 μL of TMB substrate solution into all wells of the plate. Incubate at room temperature for 3 minutes. 18) Add 50 μL per well of 1N HCl to all wells of the plate. Read the plate at 450 nm using a PerkinElmer EnSpire 2300 Multilabe 1 Reader.

[0304] Examples 4 and 5 JPEG0007795206000014.jpg222170

[0305] formulation Test articles were initially diluted to 30 μg / mL in either pH 6.0 or pH 7.4 FACS buffer and then serially diluted 3-fold in either pH 6.0 or pH 7.4 FACS buffer. cell culture 293-huCD46 and 293-cynoCD46 cells were maintained in stable cell line medium (MEM + 10% FBS + 1 mg / mL G418). CD46-expressing colo205 cells (ATCC, Cat# CCL222) were maintained in colo205 medium (RPMI1640 + 10% FBS). Cells were routinely subcultured twice weekly. Cells were harvested during exponential growth phase and counted for plating.

[0306] Cell staining with test antibodies 1) Inoculate 3x10 cells into a T-75 flask and culture medium according to the vendor's instructions. 6 Cells are seeded. 2) On the day of 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 gently pipetting. 6) Pool the cells and transfer the cell suspension to a 50 mL conical tube. 7) Count the cells using trypan blue staining, then centrifuge at 1500 rpm for 5 minutes at 4°C. 8) Wash the cells once with PBS. 9) 3.5 x 10 cells in FACS buffer, pH 6.0 or pH 7.4 6 Resuspend cells to 100 cells / mL. 10) In a 96-well U-bottom plate, incubate 3.5 x 10 cells in 100 µL of FACS buffer, pH 6.0 or pH 7.4. 5 Aliquot the cells. 11) Spin down the cells and discard the buffer. 12) Dilute the test article in three-fold serial dilutions starting at 30 μg / mL in FACS buffer pH 6.0 or pH 7.4. 13) Add 100 μL / well of diluted test article to the cells, mix the wells gently, and incubate on ice for 1 hour with shaking (200 rpm). 14) Centrifuge the cells at 1500 rpm for 5 minutes at 4° C. Wash the cells twice with 150 μL of pH 6.0 or pH 7.4 wash buffer. 15) Dilute goat anti-human IgG AF488 antibody 1:300 in FACS buffer pH 6.0 or pH 7.4. 16) Add 100 μL of diluted antibody from the above step to the cells and incubate with shaking (200 rpm) on ice for 45 minutes, protected from light. 17) Pellet the cells and wash three times with 150 μL of pH 6.0 or pH 7.4 wash buffer. 18) Fix the cells with 4% PFA diluted in 1x PBS for 10 minutes at room temperature, then wash the cells with 1x PBS. 19) Resuspend the cells in 100 μL of 1x PBS. 20) Analyze cells by NovoCyte flow cytometer using Ex 488nm / Em 530nm. Collect at least 5,000 singlet cells for 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 JPEG0007795206000015.jpg212170

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

[0310] formulation 1) 10x test article ADC or B12 isotype ADC stock is serially diluted 5-fold starting from 50 μg / mL in assay medium at pH 6.0 or pH 7.4. 2) Centrifuge the plate, gently remove the culture medium, and then add 90 μL of pH assay medium before adding the ADC. 3) Add 10 μL of serially diluted 10x ADC or B12 sample stock to wells containing 3000 cells (final starting concentration 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 CellTiter-Glo buffer and allow to equilibrate to room temperature before use. 2) Allow lyophilized CellTiter-Glo substrate to equilibrate to room temperature before use. 3) Reconstitute the lyophilized enzyme / substrate mixture by transferring the entire liquid volume of CellTiter-Glo Buffer into the amber bottle containing CellTiter-Glo Substrate. This forms the CellTiter-Glo Reagent. 4) Mix by gently vortexing to obtain a homogeneous solution. 5) Allow the plate and its contents to equilibrate to room temperature. 6) Add 70 μL of CellTiter-Glo reagent to each well. Mix on an orbital shaker at 100 rpm for 2 minutes to induce cell lysis. 5) Incubate the plate at room temperature for 10 minutes to stabilize the luminescent signal. 6) Luminescence is recorded on a SpectraMax i3X plate reader.

[0312] Data analysis The inhibition rates of different doses of test antibody were plotted against the concentration-response luminescence signal to calculate IC50. Data were interpreted using GraphPad Prism software.

[0313] Although many of the features and advantages of the present invention have been set forth in the foregoing description, together with details of its structure and function, it is to be understood, however, that the disclosure is illustrative only and that changes in detail may be made, particularly in matters of shape, size and arrangement of parts within the principles of the invention, within the full scope indicated by the broad general meaning of the terms in which the appended claims are expressed.

[0314] All documents mentioned in this specification are incorporated herein by reference in their entirety or alternatively provide the disclosure on which they are specifically relied upon. The applicant does not intend to dedicate the disclosed embodiments to the public, and to the extent that the disclosed modifications or variations may not literally fall within the scope of the claims, they are considered part of the present invention under the doctrine of equivalents.

[0315] Polypeptide (including antibody) sequences of the present invention JPEG0007795206000016.jpg214170JPEG0007795206000017.jpg234170

Claims

1. An isolated antibody or antigen-binding antibody fragment thereof, 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.

2. The antibody or antigen-binding antibody fragment thereof of claim 1, wherein the L1 sequence is selected from the amino acid sequences RASQWISNYLN (SEQ ID NO: 12), RASQGIANYLN (SEQ ID NO: 15), and RALQGISNYLN (SEQ ID NO: 22).

3. The antibody or antigen-binding antibody fragment thereof of claim 1, wherein the L2 sequence is selected from the amino acid sequences YTSSLFS (SEQ ID NO: 17) and YTSSLHE (SEQ ID NO: 19).

4. The antibody or antigen-binding antibody fragment thereof of claim 1, comprising a set of six CDRs selected from the following six CDR sets: SEQ ID NO:12, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; SEQ ID NO:15, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; SEQ ID NO:5, SEQ ID NO:17, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; and SEQ ID NO:22, SEQ ID NO:6, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:

10.

5. The antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding antibody fragment thereof has higher binding activity to CD46 protein in a pH range of 5.0 to 6.8 in a tumor microenvironment compared to a pH range of 7.0 to 7.6 in a non-tumor microenvironment.

6. The antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 5, having a ratio of its binding activity to human CD46 protein in a tumor microenvironment at a pH range of 5.0 to 6.8 to its binding activity to human CD46 protein in a non-tumor microenvironment at a pH range of 7.0 to 7.6 of at least 1.5:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, or at least 7:

1.

7. An immunoconjugate comprising the antibody or antigen-binding antibody fragment thereof according to any one of claims 1 to 6.

8. The immunoconjugate of claim 7 , wherein the immunoconjugate comprises at least one agent selected from a chemotherapeutic agent, a radioactive agent, a cytostatic agent, and a cytotoxic agent.

9. The immunoconjugate of claim 8 comprising at least two of said agents.

10. The immunoconjugate of claim 8 , wherein the at least one agent is a radioactive agent.

11. 11. The immunoconjugate of claim 10, wherein the radioactive agent is selected from an alpha emitter, a beta emitter, and a gamma emitter.

12. The immunoconjugate of claim 8 , wherein the antibody or antigen-binding antibody fragment thereof and the at least one agent are covalently attached to a linker molecule.

13. 13. The immunoconjugate of any one of claims 7 to 12, wherein one of the at least one agent is selected from maytansinoids, auristatins, dolastatins, calicheamicins, pyrrolobenzodiazepines, and anthracyclines.

14. A pharmaceutical composition comprising the antibody or antigen-binding antibody fragment thereof of any one of claims 1 to 6, or the immunoconjugate of any one of claims 7 to 13, and a pharmaceutically acceptable carrier.

15. The pharmaceutical composition of claim 14, further comprising an isotonicity agent.

16. The pharmaceutical composition of claim 15 in single-dose form, comprising 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 the antibody or antigen-binding antibody fragment thereof of any one of claims 1 to 6, or the immunoconjugate of any one of claims 7 to 13.

17. The pharmaceutical composition of claim 15 in single-dose form, comprising an amount in the range of 135 to 235 mg, 235 to 335 mg, 335 to 435 mg, 435 to 535 mg, 535 to 635 mg, 635 to 735 mg, 735 to 835 mg, 835 to 935 mg, 935 to 1035 mg, 1035 to 1135 mg, 1135 to 1235 mg, or 1235 to 1387 mg of the antibody or antigen-binding antibody fragment thereof of any one of claims 1 to 6, or the immunoconjugate of any one of claims 7 to 13.

18. 16. The pharmaceutical composition of claim 15, further comprising an immune checkpoint inhibitor molecule.

19. 19. The pharmaceutical composition of claim 18, wherein the immune checkpoint inhibitor molecule is an antibody or antibody fragment against an immune checkpoint.

20. 20. The pharmaceutical composition of claim 19, 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.

21. The pharmaceutical composition of claim 20, wherein the immune checkpoint is CTLA4, PD-1, or PD-L1.

22. The pharmaceutical composition of any one of claims 14 to 20, further comprising an antibody or antigen-binding antibody fragment thereof against an antigen selected from CTLA4, PD1, PD-L1, AXL, ROR2, CD3, HER2, B7-H3, ROR1, SFRP4, and a WNT protein.

23. 23. A pharmaceutical composition for treating cancer, comprising the antibody or antigen-binding antibody fragment thereof of any one of claims 1 to 6, the immunoconjugate of any one of claims 7 to 13, or the pharmaceutical composition of any one of claims 14 to 22.

24. 26. Use of the antibody or antigen-binding antibody fragment thereof of any one of claims 1 to 6, the immunoconjugate of any one of claims 7 to 13, or the pharmaceutical composition of any one of claims 14 to 22 in the manufacture of a medicament for the treatment of cancer.

25. 23. A kit for diagnosis or treatment, comprising the antibody or antigen-binding antibody fragment thereof of any one of claims 1 to 6, the immunoconjugate of any one of claims 7 to 13, or the pharmaceutical composition of any one of claims 14 to 22, and instructions for using the antibody or antigen-binding antibody fragment thereof, the immunoconjugate, or the pharmaceutical composition for diagnosis or treatment.

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