Anti-ROR2 antibodies, antibody fragments, immunoconjugates thereof and uses thereof

Abstract

Polypeptides having a heavy and / or light chain variable region that specifically binds to ROR2 protein, and antibodies and antibody fragments comprising a heavy and / or light chain variable region that binds to ROR2 protein, pharmaceutical compositions and kits comprising the polypeptides or antibodies and antibody fragments comprising the polypeptides, and the use of such polypeptides or antibodies or antibody fragments in methods of treating ROR2-expressing cancers are also provided.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a sequence listing that has been submitted as a 174,000 byte XML file "BIAT-1035WOSequence Listing", created on February 10, 2023. The material contained in this text file is incorporated herein by reference.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 313943, filed February 25, 2022, the entire disclosure of which is incorporated by reference herein as if fully set forth herein.

[0003] The present disclosure relates to anti-ROR2 antibodies, antibody fragments and immunoconjugates of such antibodies and antibody fragments, and to the use of the antibodies, antibody fragments and immunoconjugates in diagnostic and therapeutic methods. [Background technology]

[0004] Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that regulate a range of normal cellular processes through ligand-regulated tyrosine kinase activity. Over the past two decades, RTK dysfunction has been shown to play an important role in cancer development and progression. RTKs are now recognized as prognostic molecular biomarkers and as targets for cancer therapeutics.

[0005] ROR2, also called receptor tyrosine kinase-like orphan receptor 2, is a membrane-bound RTK that is activated by non-canonical Wnt signaling through its association with Wnt5A glycoprotein during normal bone and cartilage development. ROR2 has only one transmembrane domain that separates its extracellular and intracellular domains (Figure 1). ROR2 is known to play an important role in the normal development of various organs and tissues. In mammals, ROR2- and Wnt5A-deficient mice show similar abnormalities during developmental morphology, reflecting their defects in convergent extension movements and planar cell polarity. Furthermore, mutations in the human ROR2 gene are responsible for the genetic skeletal disorders dominant brachydactyly type B and recessive Robinow syndrome. ROR2 mediates polarized cell migration, and ROR2 dysfunction has been found to lead to inherited skeletal disorders and tumor invasion (Minami et al., “Ror-family receptor tyrosine kinases in noncanonical Wnt signaling: their implications in developmental morphogenesis and human diseases,” Dev Dyn., vol. 239, pp. 1-15, 2010).

[0006] ROR2 has also been reported to have tumor-promoting effects. US Patent Publication No. 2014 / 0322234 discloses that the expression and activity of ROR2 in various cancers differs from normal tissues. Thus, it is suggested that ROR2 dysfunction plays a role in the pathogenesis of various human cancers. US Patent Publication No. 2014 / 0322234 also contemplates that antibodies against ROR2 can be used in cancer diagnosis and inhibition of cancer cell growth. For example, such antibodies can be conjugated to cytotoxic agents that have a high degree of cytotoxicity for cancer cells that express ROR2, so that the cytotoxic agents can effectively kill the cancer cells. ROR2 gene can also be used in the classification of cancer according to the ROR2 expression pattern in cancer.

[0007] Ford et al. ("The dual role of the novel Wnt receptor tyrosine kinase, ROR2, in human carcinogenesis," International Journal of Cancer, vol. 133, pp. 779-787, 2013) further explores the mechanism of ROR2 in cancer development. This reference discloses that ROR2 is involved in cancer development and progression. Specifically, ROR2 has been found to play a vital role in the oncogenesis of numerous cancers, such as colon cancer, hepatocellular carcinoma, metastatic melanoma and renal cell carcinoma. For example, ROR2 is overexpressed in osteosarcoma, melanoma, renal cell carcinoma, prostate cancer, squamous cell carcinoma of the head and neck and stromal tumors. Thus, ROR2 has the potential to be a drug target for cancer treatment by inhibition of the Wnt signaling pathway.

[0008] Furthermore, Debebe et al., ("ROR2 as a therapeutic target in cancer," Pharmacol. Ther., vol. 50, pp. 143-148, 2015) disclose that ROR2 mediates both canonical and non-canonical signaling pathways. ROR2 is highly expressed in osteosarcoma and renal cell carcinoma, as well as in melanoma, colon cancer, squamous cell carcinoma of the head and neck, and breast cancer. In the majority of these cancer types, ROR2 expression is associated with more aggressive cancer conditions. Therefore, this reference also suggests that ROR2 is a potential target for cancer therapy.

[0009] WO2013 / 103637 discloses the use of ROR2 as a therapeutic target and prognostic marker for cancer, and conjugates comprising an antibody which recognises and binds to ROR2 and a cytotoxic agent (see Abstract).

[0010] WO2017 / 197234 discloses polypeptides having a heavy chain variable region and / or a light chain variable region that specifically bind to ROR2 protein, as well as antibodies and antibody fragments comprising a heavy chain variable region and / or a light chain variable region that binds to ROR2 protein. Immunoconjugates, pharmaceutical compositions and kits comprising the polypeptides, or antibodies and antibody fragments comprising the polypeptides, are also provided.

[0011] Chang C. et al., "Novel conditionally active biologic anti-Axl antibody-drug conjugate demonstrates anti-tumor efficacy and improved safety profile" (AACR Annual Meeting, April 2016) describes a conditionally active biologic (CAB)-Axl antibody conjugated to a model toxin payload to generate a CAB-Axl-ADC (antibody-drug conjugate) (abstract).

[0012] WO2016 / 138071 describes a method for producing conditionally active biological proteins that are more active under abnormal physiological conditions than under normal physiological conditions (see abstract).

[0013] Although monoclonal antibodies against ROR2 are commercially available, anti-ROR2 antibodies suitable for cancer therapy have not been reported. The present invention provides anti-ROR2 antibodies or antibody fragments suitable for therapeutic and diagnostic use, particularly for cancer diagnosis and treatment. Some of the anti-ROR2 antibodies or antibody fragments have a higher binding affinity for ROR2 in tumors compared to ROR2 present in normal tissues. These anti-ROR2 antibodies or antibody fragments of the present invention have at least equivalent potency and longer half-life compared to monoclonal anti-ROR2 antibodies known in the art, but with reduced side effects. This may allow the use of higher dosages of these anti-ROR2 antibodies or antibody fragments, thus providing a more effective treatment option without a corresponding increase in significant side effects. Summary of the Invention [Means for solving the problem]

[0014] In one aspect, the present invention provides an isolated polypeptide that specifically binds to ROR2 protein, and use of the polypeptide in a method for treating ROR2-expressing tumors comprising administering the polypeptide to a human in need of such treatment.

[0015] The polypeptide comprises a heavy chain variable region having three complementarity determining region (CDR) H1, H2, and H3 sequences; The H1 sequence is GYTX 1 TEX 2 X 3 X 4 H (SEQ ID NO: 1) or GYSITTGX 29 YWN (SEQ ID NO:4); The H2 sequence is X 5 X 6 X 7 X 8 NNGGTGYNQKFKG (SEQ ID NO: 2) or YITYDGSX 30 NYNPSLKN (SEQ ID NO:5); The H3 sequence is X 9 X 10 X 11 SX 12 YX 13 YX 14 X 15 SYFX 16 X 17 X 18 (SEQ ID NO:3) or CSX 31 X 32 X 33 X 34 VX 35 X 36 X 37 LDX 38 (SEQ ID NO:6); X 1 is F or E, X 2 is Y or D, X 3 is T or C, X 4is M or D or E or Y, X 5 is G or S, X 6 is I or E, X 7 is N or C or L or V, X 8 is T, D or E, X 9 is A, M or T, X 10 is R or H, X 11 is G or E, X 12 is L or F, X 13 is S or G, X 14 is G or D, X 15 is N or E, X 16 is D or L, X 17 is Y or C or T, X 18 is W or L, X 29 is Y or E or R or T, X 30 is K or N, X 31 is R or G or H or W or Y, X 32 is F or C or N or Q, X 33 is E or S, X 34 is G or E or F or H or M or Q or S, X 35 is W or A or I or P or Q or T or V, X 36 is Y or G or N or Q, X 37 is G or S or T, X 38 is Y or I, and a light chain variable region having three complementarity determining region (CDR) sequences L1, L2, and L3; The L1 sequence is SATSSX 19 X 20 X 21 MX 22 (SEQ ID NO: 7) or RASESVDRYGNSX 39 IH (SEQ ID NO: 10); The L2 sequence is X 23 TSNLAS (SEQ ID NO: 8) or X 40 TYX 41 LES (SEQ ID NO:11); L3 sequence is QX 24 X 25 SX 26 YPFX 27 X 28 (SEQ ID NO: 9) or QQX 42 NX 43 DPX 44 TX 45 (SEQ ID NO: 12); X 19 is V or E, X 20 is S or D, X 21 is Y or C or D, X 22 is H, G or L, X 23 is G or C or H or P, X 24 is Q or E, X 25 is R or H, X 26 is S or D or G or I or Q or V, X 27 is T or D, X 28 is F, D, or E, X 39 is F, S or T, X 40is R or C or D or E or W, X 41 is N or D, X 42 is T, I or P, X 43 is E or V, X 44 is W or T, X 45 is either F or T.

[0016] In one embodiment of this aspect, the heavy chain variable region polypeptide comprises three complementarity determining regions, H1, H2, and H3, having amino acid sequences selected from SEQ ID NOs:18-26.

[0017] In one embodiment of this aspect, the light chain variable region polypeptide comprises three complementarity determining regions L1, L2 and L3 having an amino acid sequence selected from SEQ ID NOs: 13-17 and 27.

[0018] In another embodiment of this aspect, any one of the heavy chain variable region polypeptides as described in the above embodiments may be combined with any one of the light chain variable region polypeptides as described in the above embodiments.

[0019] In another embodiment, the present invention provides isolated polypeptides as described above, which have up to one substitution in CDRs H1, H2 and H3 relative to the parent polypeptide, and / or up to one substitution in CDRs L1, L2 and L3 relative to the parent polypeptide, including (1) isolated polypeptides having one substitution in CDRs H1, H2 and H3 relative to the parent polypeptide, (2) isolated polypeptides having one substitution in CDRs L1, L2 and L3 relative to the parent polypeptide, and (3) isolated polypeptides having one substitution in CDRs H1, H2 and H3 and one substitution in CDRs L1, L2 and L3 relative to the parent polypeptide.

[0020] In one embodiment of this aspect, possible single point mutations in CDRs H1, H2 and H3 are shown in Figures 2A-1 and 2A-2, and possible single point mutations in CDRs L1, L2 and L3 are shown in Figures 3A-1 and 3A-2.

[0021] In another embodiment of this aspect, possible single point mutations in CDRs H1, H2 and H3 are shown in Figures 2B-1 and 2B-2, and possible single point mutations in CDRs L1, L2 and L3 are shown in Figures 3B-1 and 3B-2.

[0022] In another embodiment, each of the heavy chain variable region polypeptides and each of the light chain variable region polypeptides may have one, two, three, four or five independently selected substitutions in the CDRs selected from the point mutations shown in Figures 2B-1-2 and 2A-1-2, respectively.

[0023] In yet another embodiment, the present invention provides an anti-ROR2 antibody or antibody fragment comprising any one of the polypeptides of the present invention as described above.

[0024] In one embodiment of this aspect, the invention provides an anti-ROR2 antibody or antibody fragment comprising any one of the heavy chain variable region polypeptides as described above in combination with any one of the light chain variable region polypeptides as described above.

[0025] In one embodiment of this aspect, the present invention provides an anti-ROR2 antibody or antibody fragment comprising a heavy chain variable region having an amino acid sequence selected from SEQ ID NOs: 18 to 26 in combination with a light chain variable region having an amino acid sequence selected from SEQ ID NOs: 13 to 17 and 27.

[0026] In a further embodiment of this aspect, the present invention provides an anti-ROR2 antibody or antibody fragment that combines a heavy chain variable region having an amino acid sequence selected from SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 22 with a light chain variable region having an amino acid sequence selected from SEQ ID NOs: 13 to 16.

[0027] In a further embodiment of this aspect, the invention provides an anti-ROR2 antibody or antibody fragment that specifically binds with higher binding affinity to ROR2 protein at a value of a condition in a tumor microenvironment compared to the binding affinity to ROR2 protein at a different value of the same condition occurring in a non-tumor microenvironment.

[0028] In yet another embodiment of this aspect, the tumor microenvironment condition and the non-tumor microenvironment condition is pH.

[0029] In yet another embodiment of this aspect, the pH of the tumor microenvironment is in the range of 5.8 to 6.8 and the pH of the non-tumor microenvironment is in the range of 7.0 to 7.6.

[0030] In further embodiments of this aspect, the antibody or antibody fragment has a ratio of binding affinity to ROR2 protein at a value of a condition in a tumor microenvironment to binding affinity to ROR2 protein at a different value of the same condition in a non-tumor microenvironment that 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.

[0031] In yet another embodiment of this aspect, the antibody or antibody fragment of any one of the above embodiments is a chimeric antibody, a multispecific antibody, or a humanized antibody.

[0032] In yet another aspect, the invention described above provides an immunoconjugate comprising an antibody or antibody fragment of the invention conjugated to at least one agent selected from a chemotherapeutic agent, a radioactive atom, a cytostatic agent and a cytotoxic agent.

[0033] In one embodiment of this aspect, the immunoconjugate is an antibody drug conjugate (ADC) in which a conditionally active biological (CAB) anti-ROR2 antibody or antibody fragment is linked to one or more heterologous molecules via a cleavable linker (CAB-ROR2-ADC). The CAB anti-ROR2 antibody or antibody fragment may be BA3021. BA3021 is an antibody or antibody fragment having a heavy chain variable region having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region having the amino acid sequence of SEQ ID NO: 21. The CAB-ROR2-ADC is a BA3021-cleavable linker-MMAE-ADC. (n) wherein the heterologous molecule is monomethylauristatin E (MMAE) and (n) is an integer between 1 and 4, inclusive.

[0034] In yet another aspect, the invention provides a pharmaceutical composition comprising a polypeptide, antibody or antibody fragment, or immunoconjugate of the invention together with a pharma- ceutically acceptable carrier.

[0035] In yet another aspect, the invention described above provides a diagnostic or therapeutic kit comprising a polypeptide, an antibody or antibody fragment, or an immunoconjugate of the invention.

[0036] In another aspect, the present invention provides a method of treating ROR2-expressing tumors using a polypeptide, antibody or antibody fragment, or immunoconjugate of the present invention as described above.

[0037] In one embodiment of this aspect, the method of treating a ROR2-expressing tumor of the invention comprises administering to a human subject in need of such treatment a conditionally active ROR2 polypeptide, antibody or antibody fragment, or immunoconjugate as described above.

[0038] In a further embodiment of this aspect, a method of treating a ROR2-expressing tumor comprises administering to a human subject in need of such treatment a CAB-ROR2-ADC as described above.

[0039] In a further embodiment of this aspect, the human subject in need of such treatment is a human subject with cancer or a ROR2-expressing tumor.

[0040] In certain embodiments of this aspect, the cancer may be selected from sarcoma, ovarian cancer, melanoma, non-small cell lung cancer (NSCLC), breast cancer, and head and neck cancer.

[0041] In another embodiment of this aspect, the method of treating a ROR2-expressing tumor comprises administering to a human subject in need of treatment as described in any of the above embodiments, BA3021-cleavable linker-MMAE, as described above. (n) The method includes administering a pharmaceutical composition comprising a CAB-ROR2-ADC comprising:

[0042] In yet another embodiment of this aspect, the pharmaceutical composition according to any of the above embodiments is administered every 21 days or every 3 weeks at a dosage of up to 3.3 mg / kg of body weight of a human subject.

[0043] In yet another embodiment of this aspect, the pharmaceutical composition according to any of the above embodiments is administered at a dosage of 1.8 mg / kg of body weight of a human subject every 14 days or every two weeks. [Brief description of the drawings]

[0044] [Figure 1] Schematic diagram of the structure of human ROR2 protein. The protein contains an Ig-like domain (Ig), a frizzled or cysteine-rich (CRD) domain, and a kringle (Kr) domain in the extracellular domain. The extracellular and intracellular domains are separated by a transmembrane (TM) domain. The intracellular domain contains a tyrosine kinase (TK) domain and a proline-rich domain (PR) flanked by serine / threonine (ST)-rich domains.

[0045] [Figure 2A-B]Figure 2A shows the indexes for Figures 2A-1 and 2A-2. Figure 2B shows the indexes for Figures 2B-1 and 2B-2. Figures 2A-1, 2A-2, 2B-1, and 2B-2 show sequence alignments of exemplary heavy chain variable regions of anti-ROR2 antibodies of the invention.

[0046] [Figure 3A-B] Figure 3A shows the index of Figures 3A-1 and 3A-2. Figure 3B shows the index of Figures 3B-1 and 3B-2. Figures 3A-1, 3A-2, 3B-1, and 3B-2 show sequence alignments of exemplary light chain variable regions of anti-ROR2 antibodies of the invention.

[0047] [Figure 4] 1 shows size exclusion chromatograms demonstrating that anti-ROR2 antibodies of the present invention do not aggregate, as described in Example 1.

[0048] [Diagram 5] 1 shows the pH-dependent binding profile of the anti-ROR2 antibodies of the present invention for binding to ROR2 as described in Example 1.

[0049] [Figure 6A-B] 1 shows the on- and off-rates of conditionally active antibodies of the invention as measured by surface plasmon resonance (SPR) assay as described in Example 1.

[0050] [Figure 7A-C] 1 shows the effect of treatment of xenograft mice with a paclitaxel-conjugated anti-ROR2 antibody of the present invention on tumor volume, as described in Example 2.

[0051] [Figure 8] 1 shows the pH-dependent binding affinity of an exemplary conditionally active antibody BAP048 as measured by pH titration.

[0052] [Figure 9]1 shows the binding affinity of conditionally active antibody BAP048 to human, cynomolgus monkey, and mouse ROR2 proteins.

[0053] [Figure 10] 1 shows cell killing of conditionally active antibody BAP048 conjugated to monomethyl auristatin E (MMAE) against HEK293 cells expressing human ROR2.

[0054] [Figure 11A-C] 1 shows cell killing of conditionally active antibody BAP048 conjugated to MMAE on LCLC103H cells.

[0055] [Figure 12A-C] 1 shows cell killing of conditionally active antibody BAP048 conjugated to MMAE against HT1080 cells.

[0056] [Figure 13] 1 shows treatment of LCLC103H-induced mouse tumors using the conditionally active antibody BAP048 conjugated to MMAE.

[0057] [Figure 14] 1 shows treatment of LCLC103H-induced mouse tumors using the conditionally active antibody BAP048 conjugated to MMAE via different linkers.

[0058] [Figure 15A-B] 1 shows treatment of mouse tumors induced by HT1080 or MDA-MB-436, respectively, using the conditionally active antibody BAP048 conjugated to MMAE.

[0059] [Figure 16]FIG. 16 shows the various dose-response changes by ROR tumor membrane percent score (TmPS) in NSCLC patients enrolled in a BA3021 Phase 1 study, with tumor membrane ROR2 expression associated with antitumor response in two of five NSCLC patients with evaluable ROR2 TmPS.

[0060] [Figure 17] FIG. 17 shows the dose response of two evaluable metastatic melanoma patients enrolled in a BA3021 Phase 1 study.

[0061] [Figure 18A-B] Figures 18A-18B show CT scans of a metastatic melanoma patient who received BA3021 (ozuriftamab vedotin) and achieved a complete response before (Figure 18A) and after (Figure 18B) treatment, in which one of two lung lesions had disappeared.

[0062] [Figure 19A-D] Figures 19A-19D show ROR2 staining in sarcomas at 40x magnification. Figure 19A (Q9403, % score 1+ or higher: 100, slide #88), Figure 19B (QMTB313-08, % score 1+ or higher: 70, slide #75), Figure 19C (Q9453, % score 1+ or higher: 50, slide #93) show ROR2 H-1 in sarcomas, and Figure 19D shows mouse IgG1 in sarcomas (negative control, slide #88).

[0063] [Fig. 20A-D] Figures 20A-20D show ROR2 staining in ovarian cancer at 40x magnification. Figure 20A (Q5488, % score 1+ or higher: 70, slide #120), Figure 20B (Q7499-02, % score 1+ or higher: 50, slide #124), Figure 20C (Q6946-01, % score 1+ or higher: 20, slide #122) show ROR2 H-1 in ovarian cancer, and Figure 20D shows mouse IgG1 in ovarian cancer (negative control, slide #175).

[0064] [Figure 21A-D] Figures 21A-21D show ROR2 staining in NSCLC at 40x magnification. Figure 21A (QMTB397-09, % score 1+ or higher: 100, slide #239), Figure 21B (Q4044-049, % score 1+ or higher: 10, slide #253), Figure 21C (QMTB249-02, % score 1+ or higher: 0, slide #238) show ROR2 H-1 in NSCLC, and Figure 21D shows mouse IgG1 in NSCLC (QMTB397-09, negative control, slide #294).

[0065] [Fig. 22A-D] Figures 22A-22D show ROR2 staining in triple-negative breast cancer (TNBC) at 40x magnification. Figure 22A (Q933, % score 1+ or higher: 90, slide #369), Figure 22B (Q9286, % score 1+ or higher: 60, slide #290), Figure 22C (Q9335, % score 1+ or higher: 15, slide #371) show ROR2 H-1 in TNBC, and Figure 22D shows mouse IgG1 in TNBC (negative control, slide #384).

[0066] [Diagram 23] Figure 23 shows the percentage of ROR2 positive and negative cases in each cancer indication, based on the ROR2 cutoff: intensity ≥ 1+ in ≥ 10% of tumor cells representing positive cases.

[0067] [Figure 24] FIG. 24 shows the mean ROR2 cell membrane H-score by cancer indication.

[0068] [Fig. 25A-F]Figures 25A-25F show ROR2 in normal human TMA tissues at 20x magnification. Figure 25A shows ROR2 in normal kidney, % score 1+ or more: 0 (slide #375), Figure 25B shows ROR2 in normal liver, % score 1+ or more: 0 (slide #375), Figure 25C shows ROR2 in normal intestine, % score 1+ or more: 0 (slide #375), Figure 25D shows ROR2 in normal brain, % score 1+ or more: 0 (slide #375), Figure 25E shows ROR2 in normal thyroid, % score 1+ or more: 10 (slide #375), Figure 25F shows ROR2 in normal tonsil, % score 1+ or more: 10 (slide #375).

[0069] [Figure 26] FIG. 26 shows the precision and reproducibility of ROR2 in sarcoma Q9403.

[0070] [Figure 27] FIG. 27 shows the precision and reproducibility of ROR2 in the sarcoma QMTB313-07.

[0071] [Figure 28] FIG. 28 shows the precision and reproducibility of ROR2 in ovarian cancer Q7499-02.

[0072] [Figure 29] FIG. 29 shows the precision and reproducibility of ROR2 in ovarian cancer QMTB400-05.

[0073] [Diagram 30] FIG. 30 shows the precision and reproducibility of ROR2 in NSCLC Q6949-01.

[0074] [Diagram 31] FIG. 31 shows the precision and reproducibility of ROR2 in NSCLC Q4044.

[0075] [Diagram 32] FIG. 32 shows the precision and reproducibility of ROR2 in TNBC Q9333.

[0076] [Diagram 33] FIG. 33 shows the precision and reproducibility of ROR2 in TNBC Q9250.

[0077] [Diagram 34] FIG. 34 shows an embodiment of a dosing schedule for the use of a polypeptide of the present invention for the treatment of non-small cell lung cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0078] definition To facilitate understanding of the examples provided herein, certain frequently used terms are defined herein.

[0079] The term "about" as used herein in connection with a measured quantity refers to the normal variation of that measured quantity that would be expected by one of ordinary skill in the art making the measurement and exercising a level of care commensurate with the purpose of the measurement and the precision of the measuring device being used. Unless otherwise indicated, "about" refers to a + / - 10% variation of the value provided.

[0080] The term "affinity" as used herein refers to the strength of the total 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, "binding affinity" as used herein refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by general methods known in the art, for example, those described herein. Specific explanations and exemplary embodiments for measuring binding affinity are described below.

[0081] As used herein, the term "affinity maturation" refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not possess the alterations, which alterations result in an improvement in the affinity of the antibody for an antigen. As used herein, the term "amino acid" refers to an amino group (--NH 2 ) and a carboxyl group (--COOH), preferably as a free group or after condensation as part of a peptide bond. "The 20 naturally encoded polypeptide-forming alpha-amino acids" are understood in the art 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).

[0082] The term "antibody" as used herein refers to intact immunoglobulin molecules as well as fragments of immunoglobulin molecules capable of binding to an epitope of an antigen, such as Fab, Fab', (Fab')2, Fv, and SCA fragments. 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 generated using methods well known in the art (see, e.g., Harlow and Lane, supra), and further described below. Antibodies can be used to isolate preparative amounts of antigen by immunoaffinity chromatography. Various other uses of such antibodies or antibody fragments are for diagnosing and / or staging disease (e.g., neoplasia), as well as for therapeutic applications to treat disease, such as neoplasia, autoimmune disease, AIDS, cardiovascular disease, infectious disease, and the like. Chimeric human-like humanized or fully human antibodies are particularly useful for administration to human patients.

[0083] 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.

[0084] A Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained for each antibody molecule treated in this manner.

[0085] The (Fab')2 fragment of an antibody can be obtained by treating an intact antibody molecule with the enzyme pepsin without subsequent reduction. The (Fab')2 fragment is a dimer of two Fab' fragments held together by two disulfide bonds.

[0086] An Fv fragment is defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains.

[0087] As used herein, the term "antibody fragment" 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') 2 linear antibodies; single-chain antibody molecules (eg, scFv) and multispecific antibodies formed from antibody fragments.

[0088] As used herein, the terms "anti-ROR2 antibody," "ROR2 antibody," and "antibody that binds ROR2" refer to an antibody that can bind to ROR2 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting ROr2. In one embodiment, the extent of binding of an anti-ROR2 antibody to an unrelated, non-ROR2 protein is less than about 10% of the binding of the antibody to ROR2 as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to ROR2 has an affinity of ≦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, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M) In certain embodiments, the anti-ROR2 antibody binds to an epitope of ROR2 that is conserved among ROR2 from different species.

[0089] The term "binding" as used herein refers to the interaction of an antibody variable region or Fv with an antigen, which interaction depends 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, "specifically binding" or "binding specifically" means that an antibody variable region or Fv binds or associates with a specific antigen more frequently, more rapidly, for a longer duration, and / or with greater affinity than other proteins. For example, an antibody variable region or Fv specifically binds to its antigen with greater affinity, avidity, more rapidly, and / or for a longer duration than it binds to other antigens. In another example, an antibody variable region or Fv binds to a cell surface protein (antigen) with a much greater affinity than it binds to related proteins or other cell surface proteins, or to an antigen generally recognized by a polyreactive natural antibody (i.e., a naturally occurring antibody known to bind to a variety of antigens naturally found in humans). However, "specifically binds" does not necessarily require exclusive binding or non-detectable binding of another antigen, which is what the term "selective binding" means. In one example, "specific binding" of an antibody variable region or Fv (or other binding region) that binds to an antigen means that the antibody variable region or Fv binds to the antigen with an equilibrium constant (KD) of 100 nM or less, e.g., 50 nM or less, e.g., 20 nM or less, e.g., 15 nM or less, or 10 nM or less, or 5 nM or less, 2 nM or less, or 1 nM or less.

[0090] The terms "cancer" and "cancerous" as used herein 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, melanoma, 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 (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, 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, hepatoma, leukemia and other lymphoproliferative disorders, fibrosarcoma, osteosarcoma, and various types of head and neck cancer.

[0091] 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.

[0092] The term "chemotherapeutic agent" as used herein refers to a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneimines and methylamelamines such as altretamine, triethylenemelamine, triethylenephosphoramine, methylamel ... amide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecins (e.g., synthetic analogs topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcholinesterase (HYCAMTIN ... camptothecin, scopolectin, and 9-aminocamptothecin; bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobuenbiquine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;Antibiotics, such as enediyne antibiotics (e.g., the calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II (see, e.g., Nicolaou et al., Angew. Chem. Intl. Ed. Engl., 33:183-186 (1994)); CDP323, an oral alpha-4 integrin inhibitor; dynemicins, such as dynemicin A; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, (carabicin), caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (e.g., ADRIAMYCIN®), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HC1 liposome injection (DOXIL®, liposomal doxorubicin TLC D-99 (MYOCET®), pegylated liposomal doxorubicin (CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozotocin, cin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmoful, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as florinic acid acid);aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluracil;amsacrine;bestrabucil;bisantrene;edatraxate;defofamine;demecolcine;diaziquone;elformithine;elliptinium acetate;epothilone;etoglucide;gallium nitrate;hydroxyurea;lentinan;lonidynin;maytansinoids, such as maytansine and ansamitocin;mitoguazone;mitoxantrone;mopidamol;nitraelin;pentostatin;phenamet;pirarubicin;rosoxantrone;2-ethylhydrazide;procarbazine;PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, veracrine A, roridin A and anguidine); Urethane; Vindesine (ELDISINE®, FILDESIN®); Dacarbazine; Mannomustine; Mitobronitrol; Mitolactol; Pipobroman; Gacytosine; Arabinosine thiazolidinone ("Ara-C"); thiotepa; taxoids such as paclitaxel (TAXOL®), albumin engineered nanoparticle formulations of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin;Vincas that interfere with the formation of microtubules from tubulin polymerization, such as vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS2000; difluoromethylornithine (D MF®); retinoids, such as retinoic acid, e.g., bexarotene (TARGRETIN®); bisphosphonates, such as clodronate (e.g., BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate. troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as the THERATOOPE vaccine and gene therapy vaccines, such as the ALLOVECTIN vaccine, the LEUVECTIN vaccine, and the VAXID vaccine. registered trademark) vaccines; 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®); pixantrone; 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 pharma- ceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is short for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, which is short for a treatment regimen using oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™).

[0093] Chemotherapeutic agents, as defined herein, include "anti-hormonal agents" or "endocrine therapeutic agents" that act to regulate, reduce, block, or inhibit the effects of hormones that may promote cancer growth.These may be hormones themselves, including, but not limited to, antiestrogens with mixed agonist / antagonist profiles, such as tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxyfene, ketoxifene, and selective estrogen receptor modulators (SERMs), such as SERM3; pure antiestrogens that do not 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®), and nonsteroidal aromatase inhibitors, such as anastrazole (AR IMIDEX®), letrozole (FEMARA®) and aminoglutethimide, and other aromatase inhibitors, such as vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazole; luteinizing hormone releasing hormone agonists, such as leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, such as progestins, such as For example, megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens / retinoids such as fluoxymesterone, all-trans retinoic acid and fenretinide; onapristone; antiprogesterone agents; estrogen receptor down-modulators (ERDs); anti-estrogens such as flutamide, nilutamide and bicalutamide; and pharma- ceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above.

[0094] 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, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0095] As used herein, the term "class" of an antibody refers to the type of constant domain or region carried by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and several of these have subclasses (isotypes), e.g., IgG 1 , IgG 2 , IgG 3 , IgG 4 , IgA 1 , and IgA 2 The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0096] The term "conditionally active antibody" as used herein refers to an antibody that is active under conditions in a tumor microenvironment compared to conditions in a non-tumor microenvironment. Conditions in a tumor microenvironment can include lower pH, higher concentrations of lactate and / or pyruvate, lower oxygen, lower concentrations of glucose, and slightly higher temperatures compared to conditions in a non-tumor microenvironment. For example, a conditionally active antibody is substantially inactive at normal body temperature, but active at higher temperatures in a tumor microenvironment. In yet another embodiment, a conditionally active antibody is less active in normal oxygenated blood, but active under the low oxygen environment present in a tumor. In yet another embodiment, a conditionally active antibody is less active at normal physiological pH 7.2-7.8, but more active under acidic pH 5.8-7.0, or 6.0-6.8, present in a tumor microenvironment. There are other conditions in a tumor microenvironment known to those skilled in the art, which can also be used as conditions in the present invention under which an anti-ROR2 antibody has different binding affinity for ROR2.

[0097] The term "constitutive" as used herein, for example, when applied to ROR2 activity, refers to the continuous signaling activity of the receptor kinase that is not dependent on the presence of ligand or other activating molecules. Depending on the nature of the receptor kinase, all of the activities may be constitutive, or the activity of the receptor may be further activated by the binding of other molecules (e.g., ligands). The cellular events that lead to the activation of receptor kinases are well known among those skilled in the art. For example, activation can include oligomerization into higher order receptor complexes, such as dimerization, trimerization, etc. The complexes may comprise a single type of protein, i.e., homocomplexes. Alternatively, the complexes may comprise at least two different protein species, i.e., heterocomplexes. Complex formation can be caused, for example, by overexpression of normal or mutant forms of the receptor on the surface of the cell. Complex formation can also be caused by one or more specific mutations in the receptor.

[0098] The term "cytostatic agent" as used herein refers to a compound or composition that stops cell growth either in vitro or in vivo. Thus, a cytostatic agent may significantly reduce the percentage 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. Humanized anti-Her2 antibody trastuzumab (HERCEPTIN®) 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 also spill over into S-phase arrest, such as DNA alkylating agents, e.g., tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antitineoplastic drugs", Murakami et al. (WB Saunders, Philadelphia, 1995), e.g., page 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs that are both derived from the yew tree. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, resulting in the inhibition of cell mitosis.

[0099] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , and 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 and fragments thereof, e.g., nucleases; antibiotics; toxins, e.g., 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.

[0100] As used herein, the term "diabody" refers to a diabody in which fragments are composed of a light chain variable domain (V L ) linked to a heavy chain variable domain (V H )(V H -V L This refers to a small antibody fragment that has two antigen-binding sites, each containing a single nucleotide polymorphism (nucleotide sequence) and a complementary nucleotide sequence. The use of a linker that is too short to allow pairing between the two domains on the same chain forces the domains to pair with the complementary domains of another chain, which creates two antigen-binding sites.

[0101] The term "detectably labeled" as used herein refers to any substance whose detection or measurement, either directly or indirectly, by physical or chemical means, indicates the presence of CTC in a sample.Representative examples of useful detectable labels include, but are not limited to, the following: molecules or ions that can be directly or indirectly detected based on light absorption, fluorescence, reflection, light scattering, phosphorescence, or luminescence properties; molecules or ions that can be detected by radioactivity; molecules or ions that can be detected by nuclear magnetic resonance or paramagnetics.The group of molecules that can be indirectly detected based on light absorption or fluorescence includes, for example, various enzymes that cause the conversion of suitable substrates from non-light absorbing to light absorbing molecules or from non-fluorescent to fluorescent molecules.

[0102] The term "diagnosis" as used herein refers to determining the susceptibility of a subject to disease or disorder, determining whether a subject is currently suffering from a disease or disorder, prognosing a subject suffering from a disease or disorder (e.g., identifying pre-metastatic or metastatic cancerous state, cancer stage, or cancer responsiveness to a treatment), and treatment strategy (e.g., monitoring a subject's condition to provide information on the effectiveness or efficacy of a treatment).In some embodiments, the diagnostic method of the present invention is particularly useful in detecting early cancer.

[0103] The term "diagnostic agent" as used herein refers to a molecule that can be directly or indirectly detected and used for diagnostic purposes. The diagnostic agent can be administered to a subject or a sample. The diagnostic agent can be provided by itself or can be conjugated to a vehicle, such as a conditionally active antibody.

[0104] The term "effector function" as used herein refers to the biological activity that is caused by the Fc region of antibody and varies with antibody isotype.Examples of antibody effector function include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptor (e.g., B cell receptor); and B cell activation.

[0105] 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.

[0106] As used herein, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl 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 called 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.

[0107] As used herein, the term "framework" or "FR" refers to variable domain residues other than hypervariable region (HVR or H1-3 in the heavy chain and L1-3 in the light chain) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally refer to the V H (or V L ) the following sequence: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0108] The term "full length antibody," "intact antibody," or "complete antibody" refers to an antibody that contains an antigen-binding variable region (V H or V L) and a light chain constant domain (CL) and heavy chain constant domains CH1, CH2 and CH3. The constant domains can 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 the 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 alpha, delta, epsilon, gamma, and mu, respectively. The subunits and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0109] 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", including the primary transformed cell and its progeny regardless of the number of passages. 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 in the original transformed cell are included herein.

[0110] As used herein, the term "human antibody" is an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a human antibody repertoire or other non-human source that utilizes human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies which contain non-human antigen-binding residues.

[0111] As used herein, the term "human consensus framework" refers to the human immunoglobulin V L or V HIn selecting framework sequences, the framework represents the most commonly occurring amino acid residues. L or V H The selection of sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one embodiment, the V L The subgroup for V is subgroup kappa I as in Kabat et al., supra. H The subgroup for is subgroup III as in Kabat et al., supra.

[0112] The term "humanized" antibody as used herein refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In some embodiments, a humanized antibody contains substantially all of at least one, typically two, variable domains in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally contain 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 been humanized.

[0113] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, naturally occurring four-chain antibodies contain six HVRs; H Three of them (H1, H2, H3), and V LHVRs generally include three of the following: hypervariable loops (L1, L2, L3). HVRs generally include amino acid residues from the hypervariable loops and / or from the "complementarity determining regions" (CDRs), the latter of which are the most variable in sequence and / or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol., vol. 196, pp. 901-917 1987). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. 1991). H Except for CDR1 in the middle, CDRs generally comprise amino acid residues that form hypervariable loops. CDRs also comprise "specificity determining residues", or "SDRs", which are residues that contact the antigen. SDRs are contained within regions of CDRs called truncated CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3 (see Almagro and Fransson, Front. Biosci., vol. 13, pp. 1619-1633, 2008). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0114] As used herein, the term "immunoconjugate" is an antibody conjugated to (one or more) heterologous molecules, such as, but not limited to, a cytotoxic agent.

[0115] The term "individual" or "subject" as used herein 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 some embodiments, the individual or subject is a human.

[0116] 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 the induction of cell death.

[0117] The term "isolated" antibody as used herein is an antibody that is separated from the components of its natural environment.In some embodiments, the antibody is purified to a purity of more than 95% or 99%, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase high performance liquid chromatography (HPLC)).For a general description of how to assess antibody purity, see, for example, Flatman et al., J.Chromatogr.B, vol.848, pp.79-87, 2007.

[0118] As used herein, the term "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0119] As used herein, the term "isolated nucleic acid encoding an anti-ROR2 antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), and includes such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0120] As used herein, the term "ligand-independent," for example, when applied to receptor signaling activity, refers to a signaling activity that is not dependent on the presence of a ligand. A receptor that has ligand-independent kinase activity does not necessarily preclude the binding of a ligand to the receptor to produce additional activation of the kinase activity.

[0121] The term "metastasis" as used herein refers to all ROR2-involved processes that support cancer cells to disperse from a primary tumor, infiltrate into lymphatics and / or blood vessels, circulate via the bloodstream, and grow in distant foci in normal tissues elsewhere in the body (metastasis). In particular, it refers to cellular events of tumor cells, such as proliferation, migration, anchorage independence, evasion of apoptosis, or secretion of angiogenic factors, that underpin metastasis and are stimulated or mediated by non-catalytic or catalytic activities of ROR2, preferably, for example, ROR2 phosphorylation and / or ROR2-mediated signaling.

[0122] The term "microenvironment" as used herein means any part or area of ​​a tissue or body that has a permanent or temporary physical or chemical difference from other areas of the tissue or body. For tumors, the term "tumor microenvironment" as used herein refers to the environment in which the tumor resides, which is the acellular area within the tumor and the area immediately outside the tumor tissue, but not related to the intracellular compartments of the cancer cells themselves. The tumor and the tumor microenvironment are closely related and constantly interact. A tumor can change its microenvironment, which can affect how the tumor grows and spreads. Typically, the tumor microenvironment has a low pH in the range of 5.8-7.0, more commonly in the range of 6.0-6.8, 6.2-6.8. On the other hand, normal physiological pH is in the range of 7.2-7.8. The tumor microenvironment has a low concentration of glucose and other nutrients compared to plasma, but is also known to have a high concentration of lactate. In addition, the tumor microenvironment can have a temperature 0.3-1°C higher than the normal physiological temperature. The tumor microenvironment is discussed in Gillies et al., "MRI of the Tumor Microenvironment," Journal of Magnetic Resonance Imaging, vol. 16, pp. 430-450, 2002, which is incorporated by reference in its entirety. The term "non-tumor microenvironment" refers to the microenvironment at a site other than a tumor.

[0123] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each individual antibody in the population is identical and / or binds to the same epitope, except for variant antibodies that contain, for example, naturally occurring mutations or may arise during the production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody obtained from a population of substantially homogeneous antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies that can be used according to 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 locus, and such and other exemplary methods of producing monoclonal antibodies are described herein.

[0124] 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 can be present in pharmaceutical formulations.

[0125] The term "natural antibody" as used herein refers to a naturally occurring immunoglobulin molecule with variable structure. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain contains a variable region (V), also called the variable heavy domain or heavy chain variable domain. H) followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain contains a variable region (V), also called the variable light domain or light chain variable domain. L ), followed by the constant light chain (C L The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domain.

[0126] As used herein, the term "package insert" is used to refer to instructions typically included in the commercial packaging of a therapeutic product, which contain information about the indications, usage, dosage, administration, concomitant therapy, contraindications and / or precautions regarding the use of such therapeutic product.

[0127] The term "percent (%) amino acid sequence identity" as used herein with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the 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 the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways that are 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 the appropriate parameters for aligning sequences, for example, any algorithm required to achieve maximum alignment over the entire length of the sequences being compared. However, for the purposes herein, % amino acid sequence identity values ​​are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program is authored by Genentech, Inc., and the source code has been filed with the US Copyright Office, Washington DC, 20559, and is registered under US Copyright Registration No. TXU510087, together with user documentation. 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 should be compiled for use on a UNIX operating system, e.g., Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0128] In the context of using ALIGN-2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or relative to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or containing a certain % amino acid sequence identity to, with, or relative 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 is recognized that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless otherwise specifically stated, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0129] The term "pharmaceutical formulation" as used herein refers to a preparation that is in a form that allows the effectiveness of the biological activity of the active ingredients contained therein and does not contain additional components that exhibit unacceptable toxicity to the subject to which the formulation is administered.

[0130] As used herein, the term "pharmaceutical 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.

[0131] The terms "purified" and "isolated" as used herein refer to the antibody or nucleotide sequence according to the present invention, in which the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" means that preferably at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological macromolecules are present. 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 code for a polypeptide; however, the molecule may contain some additional bases or moieties that do not adversely affect the basic characteristics of the composition.

[0132] The term "recombinant antibody" as used herein 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) cells, COS, myeloma cells (e.g., Y0 and NS0 cells), baby hamster kidney (BHK) cells, Hela 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); (5) bacterial cells, such as Escherichia coli cells or Bacillus subtilis cells.

[0133] The term "ROR2" as used herein refers to receptor tyrosine kinase-like orphan receptor 2, a predicted 943 amino acid protein with in vitro protein activity, shown in Genbank accession number AAI30523. Many lineage-restricted receptor tyrosine kinases were first identified as "orphans" homologous to known receptors, and subsequently used to identify their unknown growth factors. DeChiara et al. (2000) identified one such orphan, encoded by ROR2, shown in FIG. 1.

[0134] The term "therapeutically effective amount" of the antibody of the present invention means an amount of the antibody sufficient to treat said cancer at a reasonable benefit / risk ratio applicable to any medical treatment. However, it is understood that the total daily use of the antibody and composition of the present invention will be determined 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 age, weight, general health, sex and diet of the patient; 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 well known in the medical field. For example, it is well within the skill of the art to start the dose of a compound at a level lower than required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0135] As used herein, the term "single-chain Fv" ("scFv") refers to a VFv that is typically linked by a peptide-encoded linker. H and V L Covalently linked V expressed from a gene fusion containing the coding gene H ::V L A "dsFv" is a V heterodimer stabilized by a disulfide bond. H ::V LHeterodimers. Bivalent and multivalent antibody fragments can form spontaneously by association of monovalent scFvs or can be generated by coupling monovalent scFvs by peptide linkers (e.g., bivalent sc(Fv)2).

[0136] The terms "treatment", "treat" or "treating" as used herein refer to clinical interventions that attempt to change the natural course of the individual being treated, and can be performed for prophylaxis or in 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, improvement or mitigation of the disease state, and remission or improvement of prognosis. In some embodiments, the antibodies of the present invention are used to delay the onset of disease or slow down the progression of disease.

[0137] The term "tumor" as used herein 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 meant to be mutually exclusive herein.

[0138] As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). H or V LThe domain may be sufficient to confer antigen-binding specificity. Moreover, an antibody or antibody fragment that binds to a particular antigen may be obtained by isolating the V domain from the antibody that binds to that antigen. H or V L The complementary V domains were isolated using L or V H Libraries of domains can be screened. See, e.g., Portolano et al., J. Immunol., vol. 150, pp. 880-887, 1993; Clarkson et al., Nature, vol. 352, pp. 624-628, 1991.

[0139] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which they are introduced. Certain vectors can direct the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0140] Detailed Description For illustrative purposes, the principles of the present invention are described with reference to various exemplary embodiments. Although certain embodiments of the present invention are specifically described herein, those skilled in the art will readily recognize that the same principles are equally applicable 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. Furthermore, the terms used herein are for purposes of description and not limitation. Furthermore, although a method is described herein with reference to steps presented in a certain order, in many cases, as one skilled in the art will understand, these steps can be performed in any order; thus, the novel method is not limited to the specific order of steps disclosed herein.

[0141] 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" can be used interchangeably herein. The terms "comprising," "including," "having," and "consisting of" can also be used interchangeably.

[0142] Unless otherwise indicated, all numbers used in the specification and claims expressing amounts of ingredients, properties, such as molecular weights, percentages, ratios, reaction conditions, and the like, should be understood to be modified in all instances by the term "about", whether or not the term "about" is used. Thus, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations, which approximations may vary depending on 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 at least be construed in light of the 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.

[0143] It is to be understood that each component, compound, substituent, or parameter disclosed herein is to be construed as disclosed for use alone or in combination with each and every other component, compound, substituent, or parameter disclosed herein.

[0144] It should be understood that each amount / value or amount / value range for each component, compound, substituent, or parameter disclosed herein should also be construed as being disclosed in combination with each amount / value or amount / value range disclosed for every other component, compound, substituent, or parameter disclosed herein, and thus any combination of amounts / values ​​or amount / value ranges for two or more components, compounds, substituents, or parameters disclosed herein are also disclosed in combination with each other for the purposes of this detailed description.

[0145] It is further understood that each lower limit of each range disclosed herein should be interpreted as being disclosed in combination with each upper limit of each range disclosed herein for the same component, compound, substituent, or parameter. Thus, the disclosure of two ranges should be interpreted as a disclosure of four ranges derived by combining each lower limit of each range with each upper limit of each range. The disclosure of three ranges should be interpreted as a disclosure of nine ranges derived by combining each lower limit of each range with each upper limit of each range, etc. Furthermore, a specific amount / value of a component, compound, substituent, or parameter disclosed in this specification or examples should be interpreted as a disclosure of either the lower limit or upper limit of the range, and thus may be combined with any other lower limit or upper limit of the range or specific amount / value for the same component, compound, substituent, or parameter disclosed elsewhere in this application to form a range for that component, compound, substituent, or parameter.

[0146] A. Anti-ROR2 antibody In one aspect, the present invention provides an isolated polypeptide that specifically binds to human ROR2 protein, and use of the polypeptide in a method for treating ROR2-expressing tumors comprising administering the polypeptide to a human in need of such treatment.

[0147] The heavy chain variable region polypeptide comprises a heavy chain variable region having three complementarity determining regions H1, H2, and H3 sequences, The H1 sequence is GYTX 1 TEX 2 X 3 X 4 H (SEQ ID NO: 1) or GYSITTGX 29 YWN (SEQ ID NO:4); The H2 sequence is X 5 X 6 X 7 X 8 NNGGTGYNQKFKG (SEQ ID NO: 2) or YITYDGSX 30 NYNPSLKN (SEQ ID NO:5); The H3 sequence is X 9 X 10 X 11 SX 12 YX 13 YX 14 X 15 SYFX 16 X 17 X 18 (SEQ ID NO:3) or CSX 31 X 32 X 33 X 34 VX 35 X 36 X 37 LDX 38 (SEQ ID NO:6); X 1 is F or E, X 2 is Y or D, X 3 is T or C, X 4 is M or D or E or Y, X 5 is G or S, X 6 is I or E, X 7 is N or C or L or V, X 8 is T, D or E, X 9 is A, M or T, X10 is R or H, X 11 is G or E, X 12 is L or F, X 13 is S or G, X 14 is G or D, X 15 is N or E, X 16 is D or L, X 17 is Y or C or T, X 18 is W or L, X 29 is Y or E or R or T, X 30 is K or N, X 31 is R or G or H or W or Y, X 32 is F or C or N or Q, X 33 is E or S, X 34 is G or E or F or H or M or Q or S, X 35 is W or A or I or P or Q or T or V, X 36 is Y or G or N or Q, X 37 is G or S or T, X 38 is Y or I, and a light chain variable region having three complementarity determining regions L1, L2, and L3 sequences; The L1 sequence is SATSSX 19 X 20 X 21 MX 22 (SEQ ID NO: 7) or RASESVDRYGNSX 39 IH (SEQ ID NO: 10); The L2 sequence is X23 TSNLAS (SEQ ID NO: 8) or X 40 TYX 41 LES (SEQ ID NO:11); L3 sequence is QX 24 X 25 SX 26 YPFX 27 X 28 (SEQ ID NO: 9) or QQX 42 NX 43 DPX 44 TX 45 (SEQ ID NO: 12); X 19 is V or E, X 20 is S or D, X 21 is Y or C or D, X 22 is H, G or L, X 23 is G or C or H or P, X 24 is Q or E, X 25 is R or H, X 26 is S or D or G or I or Q or V, X 27 is T or D, X 28 is F, D, or E, X 39 is F, S or T, X 40 is R or C or D or E or W, X 41 is N or D, X 42 is T, I or P, X 43 is E or V, X 44 is W or T, X 45 is F or T, However, X 1 From X 28 cannot simultaneously be F, Y, T, M, G, I, N, T, A, R, G, L, S, G, N, D, Y, W, V, S, Y, H, G, Q, R, S, T, and F, respectively, to exclude non-conditionally active parent antibodies.

[0148] Alignments of the heavy chain variable regions are shown in Figures 2A-1, 2A-2, 2B-1 and 2B-2.

[0149] Alignments of the light chain variable regions are shown in Figures 3A-1, 3A-2, 3B-1 and 3B-2.

[0150] In another embodiment, the present invention provides isolated polypeptides as described above with up to one substitution in CDRs H1, H2 and H3 relative to the parent polypeptide and up to one substitution in CDRs L1, L2 and L3 relative to the parent polypeptide, including (1) isolated polypeptides with one substitution in CDRs H1, H2 and H3 relative to the parent polypeptide, (2) isolated polypeptides with one substitution in CDRs L1, L2 and L3 relative to the parent polypeptide, and (3) isolated polypeptides with one substitution in CDRs H1, H2 and H3 and one substitution in CDRs L1, L2 and L3 relative to the parent polypeptide.

[0151] Possible single point mutations in CDRs H1, H2, H3 are shown in Figures 2A-1 and 2A-2, possible single point mutations in CDRs L1, L2, L3 are shown in Figures 3A-1 and 3A-2, other possible single point mutations in CDRs H1, H2, and H3 are shown in Figures 2B-1 and 2B-2, other possible single point mutations in CDRs L1, L2, and L3 are shown in Figures 3B-1 and 3B-2.

[0152] In another embodiment, each of the heavy chain variable region polypeptides and each of the light chain variable region polypeptides can have 0, 1, 2, 3, 4, or 5 independently selected substitutions in the CDRs selected from the point mutations shown in Figures 2B-1-2 and 2A-1-2, respectively, so long as at least one of the heavy and light chain variable region polypeptides has at least one point mutation relative to the parent polypeptide. For example, the heavy chain variable region polypeptide can have three point mutations relative to the parent polypeptide and the light chain variable region can have five point mutations relative to the parent polypeptide.

[0153] The present invention identified these isolated heavy and light chain variable regions, respectively, from the heavy and light chain variable regions of a parent antibody via the method disclosed in U.S. Patent No. 8,709,755, which method of generating conditionally active antibodies is incorporated herein by reference.

[0154] A mutant antibody library was generated by evolving DNA encoding the parent antibody heavy and light chain variable regions using Comprehensive Positional Evolution (CPE), which randomizes each position in the parent antibody heavy and light chain variable regions one by one. Each mutant heavy / light chain in the library has only one single point mutation compared to the parent antibody heavy or light chain variable region (Figures 2A-1, 2A-2, 2B-1, 2B-2, 3A-1, 3A-2, 3B-1, and 3B-2). Mutants in the library were screened for selective binding affinity to human ROR2 at pH 6.0 compared to pH 7.4 by ELISA. Mutant heavy / light chain variable regions that are more active at pH 6.0 than at pH 7.4 were selected as the heavy / light chain variable regions of conditionally active antibodies with the indicated single point mutations in the heavy and light chain variable regions, respectively (Tables 1 and 2, Figures 2A-1, 2A-2, 2B-1, 2B-2, 3A-1, 3A-2, 3B-1, and 3B-2). [Table 1] [Table 2]

[0155] In another embodiment, the present invention includes the heavy chain variable regions depicted in Figures 2A-1, 2A-2, 2B-1, and 2B-2 and the light chain variable regions presented in 3A-1, 3A-2, 3B-1, and 3B-2. The amino acid sequences of the heavy chain variable regions are SEQ ID NOs: 18-26. The amino acid sequences of the light chain variable regions are SEQ ID NOs: 13-17 and 27. These heavy chain variable regions and light chain variable regions can specifically bind to human ROR2. Antibodies or antibody fragments comprising one of these heavy chain variable regions and light chain variable regions have been found to have a higher binding affinity for ROR2 at pH in a tumor microenvironment than at pH in a non-tumor microenvironment. For example, antibodies and antibody fragments have a higher binding affinity for ROR2 at pH 6.0 than at pH 7.4.

[0156] The anti-ROR2 antibodies or antibody fragments have a higher binding affinity to ROR2 in tumors compared to their binding affinity to ROR2 in normal tissues. These anti-ROR2 antibodies or antibody fragments have a longer half-life and reduced side effects compared to monoclonal anti-ROR2 antibodies known in the art, as well as comparable efficacy. These characteristics allow the use of higher dosages of these anti-ROR2 antibodies or antibody fragments to be delivered to patients, and are therefore a more effective therapeutic option.

[0157] The present invention includes heavy and light chain variable regions having amino acid sequences as presented in Figures 2A-1, 2A-2, 2B-1, 2B-2, 3A-1, 3A-2, 3B-1, and 3B-2 and having SEQ ID NOs: 13-24, but the present invention also provides variants thereof that can specifically bind to human ROR2. To derive these variants, the complementarity determining regions (CDRs) of the heavy chain variable region (H1-H3) and the complementarity determining regions of the light chain variable region (L1-L3) should remain intact. However, the amino acid sequences of the heavy and light chain variable regions outside the complementarity determining regions can be mutated according to the principles of substitution, insertion and deletion discussed in this application.

[0158] In the derivation of these variants, the methods described herein are guided.Variants of 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 the amino acid sequences of the heavy and light chain variable regions, and / or insertions into those sequences, and / or substitutions of residues within those sequences.Any combination of deletions, insertions, and substitutions can be made to arrive at the antibody or antibody fragment of the present invention, provided that they retain the desired characteristics, such as antigen binding to human ROR2 and / or conditional activity.

[0159] Substitution, insertion, and deletion variants In some embodiments, antibody or antibody fragment variants are provided that have one or more amino acid substitutions. Target sites for substitution mutagenesis include CDRs and framework regions (FRs). Conservative substitutions are shown in Table 3 under the heading of "conservative substitutions". More substantial changes are provided in Table 3 under the heading of "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. [Table 3]

[0160] Amino acids can be grouped according to common side chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues influencing chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0161] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0162] One type of substitution variant involves the substitution of one or more hypervariable region residues 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, reduced immunogenicity) and / or substantially retains a biological property of the parent antibody. An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated, for example, using 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).

[0163] Alterations (e.g., substitutions) can be made in the CDRs, for example, to improve antibody affinity. Such alterations can be made in the "hot spots" of the CDRs, 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), and the resulting variants VH or VL are tested for binding affinity. Affinity maturation by construction of secondary libraries and reselection from the secondary libraries is described, for example, in Hoogenboom et al. in Methods in Molecular Biology, vol. 178, pp. 1-37, 2001). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by 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 of introducing diversity involves a CDR-directed approach, in which several CDR residues (e.g., 4-6 residues at a time) are randomized. For example, alanine scanning mutagenesis or modeling can be used to specifically identify CDR residues involved in antigen binding. CDR-H3 and CDR-L3 are often targeted in particular.

[0164] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody or antibody fragment to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the CDRs. Such changes may be outside the "hot spots" of the CDRs or SDRs. Variants V provided above H and V LIn certain embodiments of the sequences, each CDR is unaltered or contains no more than one, two or three amino acid substitutions.

[0165] 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 residue or group of target residues (e.g., charged residues, e.g., 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 can be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex to identify contact points between the antibody or antibody fragment and the antigen. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. The variants can be screened to determine whether they contain the desired properties.

[0166] 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 the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0167] Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. H and V L Only the CDRs in the V H and VL It is known that when a humanized antibody is produced by simply grafting a V FR of a non-human antibody, the antibody binding activity is reduced compared to that of the original antibody derived from a non-human animal. H and V L It is considered that some amino acid residues in the V of a human antibody, not only in the CDR but also in the FR, are directly or indirectly related to the antibody binding activity. H and V L Substitution of these amino acid residues with different amino acid residues from the FR of the human antibody reduces the binding activity. H and V L Among the amino acid sequences of the FRs of the antibody, it is necessary to try to identify amino acid residues that are directly involved in binding to the antibody, or that interact with the amino acid residues of the CDRs, or that maintain the three-dimensional structure of the antibody and are directly involved in binding to the antigen. The reduced antigen binding activity can be increased by replacing the identified amino acids with amino acid residues of the original antibody derived from a non-human animal.

[0168] Modifications and changes can be made in the structure of the antibodies of the invention, and in the DNA sequences which encode them, while still obtaining a functional molecule which encodes an antibody with desired characteristics.

[0169] In making changes in amino acid sequence, the hydropathic index of amino acids can be taken into consideration. The importance of hydropathic amino acid index in conferring interactive biological function to a protein is generally understood in the art. It is accepted that the relative hydropathic characteristics of amino acids contribute to the secondary structure of the resulting protein, which in turn determines the interaction of the protein with other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydropathic 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).

[0170] A further object of the present invention also includes function-conservative variants of the antibodies of the invention.

[0171] "Function-conservative variants" are those in which a given amino acid residue in a protein or enzyme is altered without altering the overall conformation and function of the polypeptide, including, but not limited to, the replacement of an amino acid with an amino acid having similar properties (e.g., polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, etc.). Amino acids other than those shown to be conserved may differ in a protein, such that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary, e.g., according to an alignment scheme, e.g., similarity may be 70-99% as determined by the Cluster method based on the MEGALIGN algorithm. "Function-conservative variants" also include polypeptides that have at least 60%, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and even more preferably at least 95% amino acid identity as determined by the BLAST or FASTA algorithm, and have the same or substantially similar properties or functions as the native or parent protein to which they are compared.

[0172] Two amino acid sequences are "substantially homologous" or "substantially similar" if they are more than 80%, preferably more than 85%, preferably more than 90% amino acid identical, or more than about 90%, preferably more than 95%, similar (functionally identical) over the entire length of the shorter sequence. 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 any of the sequence comparison algorithms, such as BLAST, FASTA, etc.

[0173] For example, certain amino acids can be substituted by other amino acids in 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 the protein sequence, and of course in its DNA coding sequence, while still obtaining a protein with similar properties. Therefore, it is contemplated that various changes can be made in the sequence of the antibody or antibody fragment of the present invention, or the corresponding DNA sequence encoding said antibody or antibody fragment, without appreciable loss of their biological activity.

[0174] It is known in the art that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a protein having a similar biological activity, i.e., still obtain a biologically functional equivalent protein.

[0175] 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.

[0176] Glycosylation variants In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0177] If the antibody comprises an Fc region, the carbohydrate attached thereto can be altered. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides, generally attached by N-linkage to Asn297 of the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH, vol. 15, pp. 26-32, 1997. The oligosaccharides can include 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 invention can be made to generate antibody variants with certain improved properties.

[0178] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 compared to the sum of all glycan structures (e.g., complex, hybrid and high mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry as described, for example, in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 (EU numbering of Fc region residues) in the Fc region; however, Asn297 can also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in the antibody. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Application Publication No. 2003 / 0157108 (Presta, L.); US Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Patent Application Publication No. 2002 / 0164328; U.S. Patent Application Publication No. 2004 / 0093621; U.S. Patent Application Publication No. 2004 / 0132140 ... Publication No. 2004 / 0110704; U.S. Patent Application Publication No. 2004 / 0110282; U.S. Patent Application Publication No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 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 Application Publication No. 2003 / 0157108A; and WO 2004 / 056312A1, especially Example 11), as well as knockout cell lines, such as alpha-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., J. Immunol. 1999, 14:1311-1323, 2004). al., Biotechnol. Bioeng., vol. 94, pp. 680-688, 2006; and International Publication WO 2003 / 085107).

[0179] Further provided are antibody variants having bisected oligosaccharides, 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 WO 2003 / 011878; U.S. Patent No. 6,602,684; and U.S. Patent Application Publication No. 2005 / 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 WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0180] Fc region variants In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0181] In certain embodiments, the present invention contemplates antibody variants that retain some, but not all, effector functions, making them desirable candidates for applications where the half-life of the antibody in vivo is important while some effector functions (e.g., ADCC) are unnecessary or detrimental. 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 mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 5 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 (Cell Technology, 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 (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated 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. C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assay 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 MJGlennie, Blood, vol.103, pp.2738-2743, 2004). FcRn binding and in vivo clearance / half-life determination 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).

[0182] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, such as the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

[0183] Certain antibody variants with improved or diminished binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem., vol. 9, pp. 6591-6604, 2001).

[0184] In one embodiment, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, for example substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0185] In some embodiments, modifications 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.

[0186] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn), which is responsible for 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), are described in US Patent Application Publication No. 2005 / 0014934. These antibodies include an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, such as a substitution at Fc region residue 434 (U.S. Pat. No. 7,371,826). See also Duncan & Winter, Nature, vol. 322, pp. 738-740, 1988; U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0187] Cysteine ​​Engineered Antibody Variants In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced by cysteine ​​residues. In certain embodiments, the replaced residues occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby located at accessible sites of the antibody, which can be used to conjugate the antibody with other moieties, e.g., drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In some embodiments, any one or more of the following residues can be replaced by 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 can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0188] antibody derivative In certain embodiments, the antibody or antibody fragment provided herein can be further modified to contain additional non-protein moieties that are readily available and known in the art. Moieties suitable for derivatization of 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, polypropylene 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 when more than one polymer is 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, such as, but not limited to, the particular property or function of the antibody or antibody fragment to be improved, whether the derivative will be used in therapy under defined conditions, etc.

[0189] In another embodiment, a conjugate of an antibody or antibody fragment and a non-protein moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA, vol. 102, pp. 11600-11605, 2005). The radiation can be of any wavelength, including but not limited to a wavelength that does not harm normal cells but heats the non-protein moiety to a temperature that kills cells in the vicinity of the antibody-non-protein moiety.

[0190] In another aspect, the invention provides an anti-ROR2 antibody or antibody fragment comprising an isolated heavy chain variable region polypeptide or an isolated light chain variable region polypeptide. The isolated heavy chain variable region polypeptide comprises H1, H2, and H3 regions having SEQ ID NOs: 1-6. The isolated light chain variable region polypeptide comprises L1, L2, and L3 regions having SEQ ID NOs: 7-12.

[0191] The anti-ROR2 antibody or antibody fragment of the present invention has a higher binding affinity for ROR2 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 pHs. Thus, the anti-ROR2 antibody or antibody fragment of the present invention can selectively bind to ROR2 at a pH of about 5 to about 6.8, but has a lower binding affinity for ROR2 at a pH of 7.2 to 7.8 found in a normal physiological environment. As shown in Example 1, the anti-ROR2 antibody or antibody fragment has a higher binding affinity for ROR2 at pH 6.0 than at pH 7.4.

[0192] In certain embodiments, the anti-ROR2 antibodies or antibody fragments of the invention have a potency 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~10-13 M) for ROR2 under conditions in a tumor microenvironment. In one embodiment, the ratio of the Kd of the antibody or antibody fragment for ROR2 at a value for the condition in a tumor microenvironment to a different value for the same condition in a non-tumor microenvironment is at least about 1.5:1, at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 9:1, at least about 10:1, at least about 20:1, at least about 30:1, at least about 50:1, at least about 70:1, or at least about 100:1.

[0193] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab form of the antibody of interest and its antigen using the following assay: The solution binding affinity of the Fab for the antigen is determined by the minimum concentration of ( 125 I) Fab is equilibrated with labeled antigen, and then the bound antigen is captured by anti-Fab antibody-coated plates (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg / ml 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 [ 125I] The 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; however, incubation may continue for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate for incubation (e.g., 1 hour) at room temperature. The solution is then removed and the plate is washed 8 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 on a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0194] According to another embodiment, Kd is measured at about 10 response units (RU) using a surface plasmon resonance assay using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) with 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 (about 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min to achieve about 10 response units (RU) of binding 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 with 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 by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). off / k on The ratio is calculated as a ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). When the on-rate by the above surface plasmon resonance assay is 10 6 M -1 s -1If the on-rate exceeds , the on-rate can be measured by using a fluorescence quenching technique to measure 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 in the presence of increasing concentrations of antigen when measured in a spectrometer, e.g., a spectrophotometer in stopped-flow mode (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer with stirred cuvette (ThermoSpectronic).

[0195] The anti-ROR2 antibody of the present invention may be a chimeric, humanized or human antibody. In one embodiment, an anti-ROR2 antibody fragment, such as an Fv, Fab, Fab', Fab'-SH, scFv, diabody, triabody, tetrabody or F(ab') 2 Fragments and multispecific antibodies formed from antibody fragments are used. In another embodiment, the antibody is a full-length antibody as defined herein, e.g., an intact IgG antibody or other antibody class or isotype. 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. Fab and F(ab') fragments that contain salvage receptor binding epitope residues and have increased in vivo half-life 2 For a discussion of fragments, see US Pat. No. 5,869,046.

[0196] The diabody of the present invention can be bivalent or bispecific.For example, for examples of diabodies, see 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.

[0197] In some embodiments, the invention includes single domain antibody fragments that comprise all or a portion of the heavy chain variable domain or all or a portion of the 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. Pat. No. 6,248,516 B1).

[0198] Antibody fragments can be produced by a variety of 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.

[0199] In some embodiments, the anti-ROR2 antibody of the present invention can be a chimeric antibody. Some chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, vol. 81, pp. 6851-6855, 1984). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or a non-human primate, such as a monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody in which the class or subclass of the antibody has been changed relative to the class or subclass of the parent antibody. The chimeric antibody includes antigen-binding fragments thereof.

[0200] In some embodiments, the chimeric antibody of the present invention is a humanized antibody. Typically, such a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In general, a humanized antibody comprises one or more variable domains in which the CDR (or a portion thereof) is derived from a non-human antibody and the FR (or a portion thereof) is derived from a human antibody sequence. The humanized antibody may also optionally comprise at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

[0201] Humanized antibodies and methods for their production 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. Pat. 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).

[0202] 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 in specific subgroups 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).

[0203] In some embodiments, the anti-ROR2 antibody of the present invention is a multispecific antibody, e.g., a bispecific antibody. A multispecific antibody is a monoclonal antibody that has binding specificity for at least two different sites. In some embodiments, one of the binding specificities is for ROR2 and the other is for another antigen. In some embodiments, a bispecific antibody can bind to two different epitopes of ROR2. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing ROR2. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0204] 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 be produced by a number of techniques, including engineering electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1); 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., Proc. Natl. Acad. Sci. USA, vol. 90, pp. 6444-6448, 1993); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., vol. 90, pp. 6444-6448, 1993). al., J. Immunol., vol. 152, pp. 5368-5374, 1994); as well as by preparing trispecific antibodies as described, for example, in Tutt et al. J. Immunol., vol. 147, pp. 60-69, 1991.

[0205] Engineered antibodies having three or more functional antigen binding sites are also included herein, such as "Octopus antibodies" (see, eg, US Patent Application Publication No. 2006 / 0025576A1).

[0206] The antibodies or antibody fragments also include "dual action Fabs" or "DAFs" that contain antigen binding sites that bind to ROR2 and another different antigen (such as Ror1, see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0207] The anti-ROR2 antibodies or antibody fragments of the invention can be produced using recombinant methods and compositions detailed in US Patent Application Publication No. 2016 / 0017040.

[0208] The physical / chemical properties and / or biological activity of the anti-ROR2 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.

[0209] B. Immunoconjugates In another embodiment, the present invention also provides an immunoconjugate comprising an anti-ROR2 antibody or antibody fragment conjugated to one or more cytotoxic agents, e.g., a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin of bacterial, fungal, plant, or animal origin, an enzymatically active toxin, or fragments thereof), or a radioactive isotope.

[0210] 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 EP 0425235 B1); auristatins, such as the monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,208,020, 5,416,064, and EP 0425235 B1); 635,483, 5,780,588, and 7,498,298; dolastatins; calicheamicin or its derivatives (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. Pat. No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0211] 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, nonbinding 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, or curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0212] In another embodiment, the immunoconjugate comprises an antibody or antibody fragment as 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 212and radioactive isotopes of Lu. When a radioactive conjugate is used for detection, it can contain radioactive atoms for scintigraphy tests, such as tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, as well as iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0213] Conjugates of antibody / 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 (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, 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 drug in cells. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers or disulfide-containing linkers (Chari et al., Cancer Res., vol. 52, pp. 127-131, 1992; U.S. Pat. No. 5,208,020) can be used.

[0214] The immunoconjugates herein expressly contemplate conjugates prepared using cross-linking reagents, including but not limited to BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as SVSB (succinimidyl-(4-vinylsulfone)benzoate), which is commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill., USA).

[0215] Exemplary embodiments of ADCs include an antibody or antibody fragment (Ab) that targets tumor cells, a drug moiety (D), and a linker moiety (L) that attaches the Ab to D. In some embodiments, the antibody is attached to the linker moiety (L) via one or more amino acid residues, e.g., lysine and / or cysteine. In some embodiments, the drug moiety is an anti-tumor agent. For example, CAB-ROR2-ADC is composed of BA3021-cleavable linker-MMAE (n) wherein the heterologous molecule is monomethylauristatin E (MMAE) and (n) is an integer between 1 and 4, inclusive.

[0216] An exemplary ADC has the formula I: Ab-(LD) pI, where p is 1 to about 20. In some embodiments, the number of drug moieties that can be conjugated to the antibody is limited by the number of free cysteine ​​residues. In some embodiments, the 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 1, 2, 3, or 4 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 the use of engineering, 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 prior to conjugation of the antibody to generate one or more free cysteine ​​residues.

[0217] a) Exemplary Linkers A "linker" (L) is a bifunctional or multifunctional moiety that can be used to attach one or more moieties, e.g., a drug moiety (D), to an antibody or antibody fragment (Ab) to form an immunoconjugate, e.g., an ADC of formula I. In some embodiments, an ADC can be prepared using a linker that has reactive functional groups for covalently attaching to a drug and to an antibody. For example, in some embodiments, a cysteine ​​thiol of an antibody or antibody fragment (Ab) can form a bond with a reactive functional group of a linker or a drug-linker intermediate to create an ADC.

[0218] In one embodiment, linker has a functional group that can react with free cysteine ​​present on antibody to form a covalent bond.Non-limiting examples of such reactive functional groups include maleimide, haloacetamide, α-haloacetyl, activated ester such as succinimide ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, anhydride, acid chloride, sulfonyl chloride, isocyanate, and isothiocyanate.See, for example, the conjugation method in Klussman, et al, Bioconjugate Chemistry, vol.15, pp.765-773, 2004, p.766.

[0219] In some embodiments, the linker has a functional group that can react with an electrophilic group present on an antibody. Exemplary such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, the heteroatom of the reactive functional group of the linker can react with an electrophilic group on an antibody to form a covalent bond to an antibody unit. Non-limiting exemplary such reactive functional groups include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide.

[0220] The linker may include one or more linker components. Exemplary linker components include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit" or "vc"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), and 4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("MCC"). Various linker components are known in the art, some of which are described below.

[0221] The linker can be a "cleavable linker" that facilitates the release of the drug. Non-limiting examples of cleavable linkers include acid-labile linkers (e.g., containing hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992; U.S. Patent No. 5,208,020).

[0222] In one embodiment, the linker has the following formula II:-A a -W w -Y y ADCs containing a linker of formula II have the formula I(A): Ab-(A a -W w -Y y -D) p where Ab, D, and p are as defined above for Formula I. Exemplary embodiments of such linkers are described in U.S. Pat. No. 7,498,298.

[0223] In some embodiments, a linker component comprises a "stretcher unit" (A) that attaches an antibody to another linker component or to a drug moiety. Non-limiting exemplary stretcher units are shown below (where the wavy line indicates the site of covalent attachment to an antibody, a drug, or an additional linker component): [ka]

[0224] In some embodiments, the linker component comprises an "amino acid unit" (W). In some such embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate upon exposure to intracellular proteases, e.g., lysosomal enzymes (Doronina et al., Nat. Biotechnol., vol. 21, pp. 778-784, 2003). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe), phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid unit may contain naturally occurring amino acid residues and / or minor amino acids and / or non-naturally occurring amino acid analogs, such as citrulline. The amino acid unit may be designed and optimized for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0225] Typically, peptide-type linkers can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to lipid phase synthesis methods (e.g., E. Schroder and K. Luebke (1965) "The Peptides", volume 1, pp76-136, Academic Press).

[0226] In some embodiments, the linker component comprises a "spacer" unit (Y) that connects the antibody directly to the drug moiety or via a stretcher unit and / or an amino acid unit. The spacer unit may be "self-immolative" or "non-self-immolative". A "non-self-immolative" spacer unit is one in which some or all of the spacer unit remains attached to the drug moiety upon cleavage of the ADC. Examples of non-self-immolative spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. In some embodiments, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by tumor cell-associated proteases results in the release of the glycine-glycine-drug moiety from the remainder of the ADC. In some such embodiments, the glycine-glycine-drug moiety is subjected to a hydrolysis step in the tumor cell, thus cleaving the glycine-glycine spacer unit from the drug moiety.

[0227] A "self-immolative" spacer unit allows for the release of the drug moiety. In certain embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In some such embodiments, p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond, and a carbamate, methyl carbamate, or carbonate is made between the benzyl alcohol and the drug (Hamann et al. Expert Opin. Ther. Patents, vol. 15, pp. 1087-1103, 2005). In some embodiments, the spacer unit comprises p-aminobenzyloxycarbonyl (PAB). In some embodiments, the ADC comprising a self-immolative linker has the structure: [ka] (Wherein, Q is -C 1 ~C 8 Alkyl, -O-(C 1 ~C 8X is an alkyl, -alkyl, -halogen, -nitro, or -cyano; m is an integer ranging from 0 to 4; X can be one or more additional spacer units or can be absent; and p ranges from 1 to about 20. In some embodiments, p ranges from 1 to 10, 1 to 7, 1 to 5, or 1 to 4. Non-limiting exemplary X spacer units include: [ka] (In the formula, R 1 and R 2 H and C 1 ~C 6 In some embodiments, R 1 and R 2 are -CH 3 It is.

[0228] Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (U.S. Pat. No. 7,375,078; Hay et al., Bioorg. Med. Chem. Lett., vol. 9, p. 2237-, 1999) and ortho- or para-aminobenzyl acetals. In some embodiments, spacers that undergo cyclization upon amide bond hydrolysis can be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., Chemistry Biology, vol.2, pp.223-, 1995), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al., J.Amer.Chem.Soc., vol.94, p.5815-, 1972), and 2-aminophenylpropionic acid amides (Amsberry et al., J.Org.Chem., vol.55, p.5867, 1990). Attachment of a drug to the α-carbon of a glycine residue is another example of a self-immolative spacer that can be useful in ADCs (Kingsbury et al., J.Med.Chem., vol.27, p.1447, 1984).

[0229] In some embodiments, the linker L can be a dendritic-type linker for covalently linking two or more drug moieties to an antibody via a branched multifunctional linker moiety (Sun et al. Bioorganic & Medicinal Chemistry Letters, vol. 12, pp. 2213-2215, 2002; Sun et al., Bioorganic & Medicinal Chemistry, vol. 11, pp. 1761-1768, 2003). The dendritic linker can increase the drug-to-antibody molar ratio, i.e., loading, which is related to the potency of the ADC. Thus, if an antibody bears only one reactive cysteine ​​thiol group, multiple drug moieties can be attached via the dendritic linker.

[0230] Non-limiting exemplary linkers are shown below in the context of ADCs of Formula I: [ka] (In the formula, R 1 and R 2 H and C 1 ~C 6 In some embodiments, R 1 and R 2 are -CH 3 It is. [ka] (wherein n is 0 to 12). In some embodiments, n is 2 to 10. In some embodiments, n is 4 to 8.

[0231] Further non-limiting exemplary ADCs include those having the structure: [ka] Each R is independently H or C 1 ~C 6 alkyl; and n is 1 to 12.

[0232] In some embodiments, the linker is substituted with groups that modulate solubility and / or reactivity. Non-limiting examples include charged substituents, such as sulfonates (-SO 3 - ) or ammonium may increase the water solubility of the linker reagent and facilitate the coupling reaction of the linker reagent with the antibody and / or drug moiety, or may facilitate the coupling reaction of Ab-L (antibody-linker intermediate) with D, or the coupling reaction of DL (drug-linker intermediate) with Ab, depending on the synthetic route used to prepare the ADC. In some embodiments, a portion of the linker is coupled to the antibody, a portion of the linker is coupled to the drug, and then the Ab-(linker moiety) a Drug-(linker moiety) b to form the ADC of Formula I.

[0233] The compounds of the present invention expressly contemplate ADCs prepared using, but not limited to, the following linker reagents: bis-maleimido-trioxyethylene glycol (BMPEO), N-(β-maleimidopropyloxy)-N-hydroxysuccinimide ester (BMPS), N-(ε-maleimidocaproyloxy)succinimide ester (EMCS), N-[γ-maleimidobutyryloxy]succinimide ester (GMBS), 1,6-hexane-bis-vinylsulfone (HBV), and 1,6-hexane-bis-vinylsulfone (HBV). S), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate) (LC-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl 3-(bromoacetamido)propionate (SBAP), succinimidyl iodoacetate (SIA), succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-Succinimidyl-4-(2-pyridylthio)pentanoate (SPP), Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), Succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), Succinimidyl 6-[(beta-maleimidopropionamido)hexanoate] (SMPH), Iminothiolane (IT), Sulfo-EMCS, Sulfo Ho-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, as well as succinimidyl-(4-vinylsulfone)benzoate (SVSB), and, for example, bis-maleimide reagents: dithiobismaleimidoethane (DTME), 1,4-bismaleimidobutane (BMB), 1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG). 2 (shown below), and BM(PEG) 3(shown below); bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). In some embodiments, bis-maleimide reagents allow for the attachment of the thiol group of a cysteine ​​in an antibody to a thiol-containing drug moiety, linker, or linker-drug intermediate. Other functional groups that are reactive with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfides, pyridyl disulfides, isocyanates, and isothiocyanates.

[0234] Certain useful linker reagents are available from a variety of commercial sources, e.g., Pierce Biotechnology, Inc. (Rockford, Ill.), Molecular Biosciences Inc. (Boulder, Colo.), or can be found in the art, e.g., in Toki et al., J. Org. Chem., vol. 67, pp. 1866-1872, 2002; Dubowchik, et al., Tetrahedron Letters, vol. 38, pp. 5257-60, 1997; Walker, J. Org. Chem., vol. 60, pp. 5352-5355, 1995; Frisch et al., Bioconjugate Chem., vol. 7, pp. 180-186, 1995; U.S. Pat. No. 6,214,345; WO 02 / 088172; U.S. Patent Application Publication No. 2003130189; U.S. Patent Application Publication No. 2003096743; WO 03 / 026577; WO 03 / 043583; and WO 04 / 032828.

[0235] Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies. See, e.g., WO 94 / 11026.

[0236] b) Exemplary Drug Moieties 1) Maytansine and maytansinoids In some embodiments, the immunoconjugate comprises an antibody conjugated to one or more maytansinoid molecules. Maytansinoids are derivatives of maytansine, which are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; and 4,309,428. and 4,371,533.

[0237] Maytansinoid drug moieties are attractive drug moieties for antibody-drug conjugates because they are (i) relatively accessible to prepare by fermentation or chemical modification, or derivatization of fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation to antibodies via non-disulfide linkers, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.

[0238] Certain maytansinoids suitable for use as maytansinoid drug moieties are known in the art and can be isolated from natural sources according to known methods or can be produced using genetic engineering techniques (see, e.g., Yu et al., PNAS, vol. 99, pp. 7968-7973, 2002). Maytansinoids can also be prepared synthetically according to known methods.

[0239] Exemplary maytansinoid drug moieties include, but are not limited to, those with modified aromatic rings, such as C-19-dechloro (U.S. Pat. No. 4,256,746) (e.g., prepared by lithium aluminum hydride reduction of ansamitocin P2); C-20-hydroxy (or C-20-demethyl) + / -C-19-dechloro (U.S. Pat. Nos. 4,361,650 and 4,307,016) (e.g., prepared by demethylation using Streptomyces or Actinomyces or dechlorination using LAH); and C-20-demethoxy, C-20-acyloxy (-OCOR), + / -dechloro (U.S. Pat. No. 4,294,757) (e.g., prepared by acylation using acyl chlorides), as well as those with modifications at other positions on the aromatic ring.

[0240] Exemplary maytansinoid drug moieties include those having modifications, such as C-9-SH (U.S. Pat. No. 4,424,219) (e.g., maytansinol and H 2 S or P 2 S 5(prepared by reaction with C-14-alkoxymethyl (demethoxy / CH 2 OR) (U.S. Pat. No. 4,331,598); C-14-hydroxymethyl or acyloxymethyl (CH 2 OH or CH 2 OAc) (U.S. Pat. No. 4,450,254) (prepared, for example, from Nocardia); C-15-hydroxy / acyloxy (U.S. Pat. No. 4,364,866) (prepared, for example, by conversion of maytansinol by Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (prepared, for example, from Trewia nudolflora); nudlflora); C-18-N-demethyl (e.g., U.S. Pat. Nos. 4,362,663 and 4,322,348) (prepared by demethylation of maytansinol with Streptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared, for example, by titanium trichloride / LAH reduction of maytansinol).

[0241] Many positions on a maytansinoid compound are useful as attachment positions. For example, ester bonds can be formed by reaction with a hydroxyl group using conventional coupling techniques. In some embodiments, reaction can occur at the C-3 position, which bears a hydroxyl group, the C-14 position, which is modified with a hydroxymethyl, the C-15 position, which is modified with a hydroxyl group, and the C-20 position, which bears a hydroxyl group. In some embodiments, the attachment is formed at the C-3 position of maytansinol or a maytansinol analog.

[0242] Maytansinoid drug moieties include those having the structure: [ka] where the wavy line indicates the covalent attachment of the sulfur atom of the maytansinoid drug moiety to the linker of the ADC. Each R is independently H or C 1 -C 6 The alkylene chain attaching the amide group to the sulfur atom can be methanyl, ethanyl, or propyl, i.e., m is 1, 2, or 3 (U.S. Pat. No. 633,410; U.S. Pat. No. 5,208,020; Chari et al., Cancer Res., vol. 52, pp. 127-131, 1992; Liu et al., Proc. Nall. Acad. Sci. USA, vol. 93, pp. 8618-8623, 1996).

[0243] All stereoisomers of the maytansinoid drug moiety, i.e., any combination of R and S configurations at the chiral carbon, are contemplated for the ADCs of the invention (U.S. Pat. No. 7,276,497; U.S. Pat. No. 6,913,748; U.S. Pat. No. 6,441,163; U.S. Pat. No. 633,410 (RE39151); U.S. Pat. No. 5,208,020; Widdison et al (2006) J. Med. Chem. 49:4392-4408). In some embodiments, the maytansinoid drug moiety has the following stereochemistry: [ka]

[0244] Exemplary embodiments of maytansinoid drug moieties include, but are not limited to, those having the structure: [ka] (wherein the wavy line indicates the covalent attachment of the sulfur atom of the drug to the linker (L) of the antibody-drug conjugate).

[0245] An exemplary antibody-drug conjugate in which DM1 is attached to a thiol group of an antibody via a BMPEO linker has the structure and abbreviation: [ka] where Ab is an antibody; n is 0, 1, or 2; and p is 1 to about 20. In some embodiments, p is 1 to 10, p is 1 to 7, p is 1 to 5, or p is 1 to 4.

[0246] Immunoconjugates containing maytansinoids, methods for their preparation and their therapeutic uses are disclosed, for example, in U.S. Patent Nos. 5,208,020 and 5,416,064; U.S. Patent Application Publication No. 2005 / 0276812A1; and European Patent No. 0425235B1. See also Liu et al., Proc. Natl. Acad. Sci. USA, vol. 93, pp. 8618-8623, 1996; and Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992.

[0247] In some embodiments, antibody-maytansinoid conjugates can be prepared by chemically linking an antibody to a maytansinoid molecule without substantially diminishing the biological activity of either the antibody or the maytansinoid molecule. See, e.g., U.S. Pat. No. 5,208,020. In some embodiments, ADCs with an average of 3-4 maytansinoid molecules conjugated per antibody molecule have shown efficacy in improving target cell cytotoxicity without adversely affecting antibody function or solubility. In some instances, even a single molecule of toxin / antibody is expected to improve cytotoxicity compared to the use of naked antibodies.

[0248] Exemplary linking groups for producing antibody-maytansinoid conjugates include, for example, those described herein and those disclosed in U.S. Pat. No. 5,208,020; European Patent No. 0425235 B1; Chari et al., Cancer Research, vol. 52, pp. 127-131, 1992; U.S. Patent Application Publication No. 2005 / 0276812 A1; and U.S. Patent Application Publication No. 2005 / 016993 A1.

[0249] (2) Auristatins and dolastatins Drug moieties include dolastatins, auristatins, and analogs and derivatives thereof (U.S. Pat. Nos. 5,635,483; 5,780,588; 5,767,237; 6,124,431). Auristatins are derivatives of the marine mollusc compound dolastatin-10. Without being bound by any particular theory, dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al., Antimicrob. Agents and Chemother., vol. 45, pp. 3580-3584, 2001), and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al., Antimicrob. Agents Chemother., vol. 42, pp. 2961-2965, 1998). The dolastatin / auristatin drug moiety can be attached to the antibody through the N-terminus or the C-terminus of the peptidic drug moiety (WO 02 / 088172; Doronina et al., Nature Biotechnology, vol. 21, pp. 778-784, 2003; Francisco et al., Blood, vol. 102, pp. 1458-1465, 2003).

[0250] Exemplary auristatin embodiments include the N-terminally linked monomethyl auristatin drug moiety D disclosed in U.S. Pat. Nos. 7,498,298 and 7,659,241. E and D. F Examples include: [ka] (In the formula, D E and D. F The wavy lines indicate the sites of covalent attachment to the antibody or antibody-linker moiety, and independently at each position: R 2 H and C 1 ~C 8 alkyl; R 3 , H, C 1 ~C 8 Alkyl, C 3 ~C 8 Carbocyclic, Aryl, C 1 ~C 8 Alkyl-aryl, C 1 ~C 8 Alkyl-(C 3 ~C 8 carbocycle), C 3 ~C 8 Heterocycles and C 1 ~C 8 Alkyl-(C 3 ~C 8 heterocycle); R 4 , H, C 1 ~C 8 Alkyl, C 3 ~C 8 Carbocyclic, Aryl, C 1 ~C 8 Alkyl-aryl, C 1 ~C 8 Alkyl-(C 3 ~C 8 carbocycle), C 3 ~C 8 Heterocycles and C 1 ~C 8 Alkyl-(C 3 ~C 8 heterocycle); R 5 is selected from H and methyl; or R 4 and R 5 together form a carbocyclic ring of the formula -(CR a R b ) n -(In the formula, R a and R b , H, C 1 ~C 8 Alkyl and C 3 ~C 8 carbocyclic compounds, and n is selected from 2, 3, 4, 5, and 6; R 6 H and C 1 ~C 8 alkyl; R 7 , H, C 1 ~C 8 Alkyl, C 3 ~C 8 Carbocyclic, Aryl, C 1 ~C 8 Alkyl-aryl, C 1 ~C 8 Alkyl-(C 3 ~C 8 carbocycle), C 3 ~C 8 Heterocycles and C 1 ~C 8 Alkyl-(C 3 ~C 8 heterocycle); Each R 8 is H, OH, C 1 ~C 8 Alkyl, C 3 ~C 8 Carbocyclic and O-(C 1 ~C 8 alkyl); R 9 H and C 1 ~C 8 alkyl; R 10 is aryl or C 3 ~C 8 heterocycle; Z is O, S, NH, or NR 12 and R 12 is C 1 ~C 8 is alkyl; R 11 , H, C 1 ~C 20 Alkyl, aryl, C 3 ~C 8 Heterocycle, -(R 13 O) m -R 14 , or (R 13 O) m -CH(R 15 ) 2 Selected from; m is an integer ranging from 1 to 1000; R 13 is C 2 ~C 8 is alkyl; R 14 is H or C 1 ~C 8 is alkyl; Each occurrence of e is independently H, COOH, -(CH 2 ) n -N(R 16 ) 2 , -(CH 2 ) n -SO 3 H, or (CH 2 ) n -SO 3 -C 1 ~C 8 is alkyl; Each occurrence of e is independently H, C 1 ~C 8 Alkyl, or (CH 2 ) n -COOH; R 18 is -C(R 8 ) 2 -C(R 8 ) 2 -aryl, -C(R 8 ) 2 -C(R 8 ) 2 (C 3 ~C8 heterocycle), and C(R 8 ) 2 -C(R 8 ) 2 (C 3 ~C 8 carbocyclic ring); where n is an integer ranging from 0 to 6.

[0251] In one embodiment, R 3 , R 4 and R 7 are independently isopropyl or sec-butyl; R 5 is -H or methyl. In an exemplary embodiment, R 3 and R 4 are isopropyl, and R 5 is -H, R 7 is sec-butyl.

[0252] In yet another embodiment, R 2 and R 6 are methyl, and R 9 is -H.

[0253] In yet another embodiment, R 8 Each occurrence of -OCH 3 It is.

[0254] In an exemplary embodiment, R 3 and R 4 are isopropyl, and R 2 and R 6 are methyl, and R 5 is -H, R 7 is sec-butyl, R 8 Each occurrence of -OCH 3 and R 9 is -H.

[0255] In one embodiment, Z is -O- or NH-.

[0256] In one embodiment, R10 is aryl.

[0257] In an exemplary embodiment, R 10 is -phenyl.

[0258] In an exemplary embodiment, when Z is -O-, R 11 is -H, methyl or t-butyl.

[0259] In one embodiment, when Z is -NH, R 11 is -CH(R 15 ) 2 and R 15 is -(CH 2 ) n -N(R 16 ) 2 and R 16 -C 1 ~C 8 Alkyl or (CH 2 ) n -COOH.

[0260] In another embodiment, when Z is -NH, R 11 is -CH(R 15 ) 2 and R 15 is -(CH 2 ) n -SO 3 It's H.

[0261] Formula D E An exemplary auristatin embodiment of is MMAE, where the wavy line indicates the covalent attachment to the linker (L) of the antibody-drug conjugate: [ka]

[0262] Formula D E An exemplary auristatin embodiment of is MMAE, where the wavy line indicates the covalent attachment to the linker (L) of the antibody-drug conjugate: [ka]

[0263] Other exemplary embodiments include monomethylvaline compounds with a phenylalanine carboxy modification at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008848) and monomethylvaline compounds with a phenylalanine side chain modification at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008603).

[0264] Non-limiting exemplary embodiments of the ADC of formula I that comprises MMAF and various linker components also include Ab-MC-PAB-MMAF and Ab-PAB-MMAF. It has been shown that immunoconjugates that comprise MMAF attached to an antibody by a non-proteolytically cleavable linker possess activity equivalent to that of immunoconjugates that comprise MMAF attached to an antibody by a proteolytically cleavable linker (see Doronina et al., Bioconjugate Chem., vol.17, pp.114-124, 2006). In some such embodiments, drug release is believed to be influenced by antibody degradation in cells.

[0265] Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, according to liquid phase synthesis methods well known in the field of peptide chemistry (see, for example, E. Schroeder and K. Luebke, "The Peptides", volume 1, pp 76-136, 1965, Academic Press). Auristatin / dolastatin drug moieties, in some embodiments, can be prepared according to the methods of U.S. Pat. No. 7,498,298; U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al., J. Am. Chem. Soc., vol. 111, pp. 5463-5465, 1998; Pettit et al., Anti-Cancer Drug Design, vol. 13, pp. 243-277, 1998; Pettit et al., Synthesis, vol. 6, pp. 719-725, 1996; Pettit et al., J. Chem. Soc. Perkin Trans. vol. 15, pp. 859-863, 1996; and Doronina, Nat. Biotechnol., vol. 21, pp. 778-784, 2003.

[0266] In some embodiments, the compound of formula D E auristatin / dolastatin drug moieties, e.g., MMAE, and D E , e.g., MMAF, as well as their drug-linker intermediates and derivatives, e.g., MC-MMAF, MC-MMAE, MC-vc-PAB-MMAF, and MC-vc-PAB-MMAE, can be prepared using methods described in U.S. Pat. No. 7,498,298; Doronina et al., Bioconjugate Chem., vol. 17, pp. 114-124, 2006; and Doronina et al., Nat. Biotech., vol. 21, pp. 778-784, 2003, and then conjugated to an antibody of interest.

[0267] (3) Calicheamicin In some embodiments, the immunoconjugate comprises an antibody or antibody fragment conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics, and their analogs, can produce double-stranded DNA breaks at sub-picomolar concentrations (Hinman et al., Cancer Research, vol. 53, pp. 3336-3342, 1993; Lode et al., Cancer Research, vol. 58, pp. 2925-2928, 1998). Although calicheamicin has an intracellular site of action, in some instances it does not readily cross cell membranes. Thus, cellular uptake of these agents via antibody-mediated internalization can, in some embodiments, greatly enhance their cytotoxic effects. Non-limiting exemplary methods for preparing antibody-drug conjugates having a calicheamicin drug moiety are described, e.g., in U.S. Pat. Nos. 5,712,374; 5,714,586; 5,739,116; and 5,767,285.

[0268] (4) Pyrrolobenzodiazepines In some embodiments, the ADC comprises a pyrrolobenzodiazepine (PBD). In some embodiments, the PBD dimer recognizes and binds to a specific DNA sequence. The natural product anthramycin, PBD, was first reported in 1965 (Leimgruber et al., J.Am.Chem.Soc., vol.87, pp.5793-5795, 1965; Leimgruber et al., J.Am.Chem.Soc., vol.87, pp.5791-5793, 1965). Since then, numerous PBDs, both naturally occurring and analogs, have been reported (Thurston et al., Chem. Rev. vol. 1994, pp. 433-465 1994), including dimers of tricyclic PBD scaffolds (U.S. Pat. Nos. 6,884,799; 7,049,311; 7,067,511; 7,265,105; 7,511,032; 7,528,126; 7,557,099). Without being bound to any particular theory, it is believed that the dimeric structure confers the proper three-dimensional shape for isohelicity with the minor groove of B-form DNA, resulting in a smooth fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., vol. 19, pp. 230-237, 1986). Dimeric PBD compounds bearing C2 aryl substituents have been shown to be useful as cytotoxic agents (Hartley et al Cancer Res., vol. 70, pp. 6849-6858, 2010; Antonow, J. Med. Chem. vol. 53, pp. 2927-2941, 2010; Howard et al., Bioorganic and Med. Chem. Letters, vol. 19, pp. 6463-6466, 2009).

[0269] PBD dimers have been conjugated to antibodies and the resulting ADCs have been shown to have anti-cancer properties. Non-limiting exemplary binding sites on PBD dimers include the 5-membered pyrrolo ring, the tether between the PBD units, and the N10-C11 imine group (WO 2009 / 016516; US 2009 / 304710; US 2010 / 047257; US 2009 / 036431; US ​​2011 / 0256157; WO 2011 / 130598).

[0270] Non-limiting exemplary PBD dimer components of ADCs include: [ka] and salts and solvates thereof, The wavy line indicates the site of covalent attachment to the linker; The dotted lines indicate the optional presence of a double bond between C1 and C2 or between C2 and C3; R 2 are H, OH, =O, =CH 2 ,CN,R,OR,=CH-R D , =C(R D ) 2 , O-SO 2 -R, CO 2 is independently selected from R and COR, and optionally further selected from halo or dihalo; R D R,CO 2 R, COR, CHO, CO 2 H, and halo; R 6 and R 9 are H, R, OH, OR, SH, SR, NH 2 , NHR, NRR', NO 2 , Me 3 independently selected from Sn and Halo; R 7 are H, R, OH, OR, SH, SR, NH 2 , NHR, NRR', NO 2 , Me3 independently selected from Sn and Halo; Q is independently selected from O, S, and NH; R 11 is either H or R, or, if Q is O, SO 3 M, where M is a metal cation; R and R' are optionally substituted C 1~8 Alkyl, C 1~12 Alkyl, C 3~8 Heterocyclyl, C 3~20 Heterocycles, and C 5~20 aryl groups, optionally in conjunction with the group NRR′, R and R′ together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6-, or 7-membered heterocyclic ring; R 12 , R 16 , R 19 , and R 17 are R 2 , R 6 , R 9 , and R 7 As defined for R'' is C 3~12 an alkylene group, the chain of which may be interrupted by one or more heteroatoms, e.g., O, S, N(H), NMe, and / or aromatic rings, e.g., benzene or pyridine, which rings are optionally substituted; X and X' are independently selected from O, S, and N(H).

[0271] In some embodiments, R and R′ are optionally substituted C 1~12 Alkyl, C 3~20 Heterocycles, and C 5~20 aryl groups, and optionally in conjunction with the group NRR′, R and R′ together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6-, or 7-membered heterocyclic ring. 9 and R 19is H. In some embodiments, R 6 and R 16 is H.

[0272] In some embodiments, R 7 and R 17 are both OR 7A and R 7A is optionally substituted C 1~4 In some embodiments, R 7A is Me. In some embodiments, R 7A Ch 2 Ph, where Ph is a phenyl group. In some embodiments, X is O. In some embodiments, R 11 is H. In some embodiments, there is a double bond between C2 and C3 in each monomer unit.

[0273] In some embodiments, R 2 and R 12 is independently selected from H and R. In some embodiments, R 2 and R 12 is independently R. In some embodiments, R 2 and R 12 independently represents an optionally substituted C 5~20 Aryl or C 5~7 Aryl or C 8~10 In some embodiments, R 2 and R 12 is independently an optionally substituted phenyl, thienyl, naphthyl, pyridyl, quinolinyl, or isoquinolinyl. 2 and R 12 , =O, =CH 2 , =CH-R D , and =C(R D ) 2 In some embodiments, R 2 and R 12 are respectively =CH 2 In some embodiments, R2 and R 12 are each H. In some embodiments, R 2 and R 12 are each =O. 2 and R 12 are respectively =CF 2 In some embodiments, R 2 and / or R 12 are independently = C(R D ) 2 In some embodiments, R 2 and / or R 12 are independently =CH-R D It is.

[0274] In some embodiments, R 2 and / or R 12 =CH-R D When , each group can independently have any of the configurations shown below: [ka] In some embodiments, =CH-R D In some embodiments, R″ is C 3 Alkylene group or C 5 It is an alkylene group.

[0275] The PBD dimer-val-cit-PAB-Ab and PBD dimer-Phe-Lys-PAB-Ab linkers are protease cleavable, whereas the PBD dimer-maleimide-acetal linker is acid labile.

[0276] PBD dimers and ADCs containing PBD dimers can be prepared according to methods known in the art, see, for example, WO 2009 / 016516; U.S. Patent Application Publication No. 2009 / 304710; U.S. Patent Application Publication No. 2010 / 047257; U.S. Patent Application Publication No. 2009 / 036431; U.S. Patent Application Publication No. 2011 / 0256157; WO 2011 / 130598.

[0277] 5) Anthracycline In some embodiments, ADC may include anthracyclines. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. Without being bound by any particular theory, research has shown that anthracyclines can function to kill cells by a number of different mechanisms, for example, 1) intercalation of drug molecules into the DNA of cells, thereby inhibiting DNA-dependent nucleic acid synthesis; 2) drug-induced production of free radicals, which then react with cellular macromolecules and cause damage to cells; and / or 3) drug molecule interaction with cell membranes (see, for example, C. Peterson et al., "Transport And Storage Of Anthracycline In Experimental Systems And Human Leukemia" in Anthracycline Antibiotics In Cancer Therapy; N.R. Bachur, "Free Radical Damage" id.at pp.97-102). Due to their cytotoxic potential, anthracyclines are used in the treatment of many cancers, including leukemia, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcoma (see, for example, PH-Wiernik, in Anthracycline: Current Status And New Developments p11).

[0278] Non-limiting exemplary anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and derivatives thereof. Immunoconjugates and prodrugs of daunorubicin and doxorubicin have been prepared and studied (Kratz et al., Current Med. Chem., vol. 13, pp. 477-523, 2006; Jeffrey et al., Bioorganic & Med. Chem. Letters, vol. 16, pp. 358-362. 1996; Torgov et al., Bioconj. Chem., vol. 16, pp. 717-721, 2005; Nagy et al., Proc. Natl. Acad. Sci. USA, vol. 97, pp. 829-834, 2000; Dubowchik et al., Bioorg. & Med. Chem. Letters, vol. 12, pp. 1529-1532, 2002; King et al., Bioorganic & Med. Chem. Letters, vol. 16, pp. 358-362, ... al., J.Med.Chem., vol.45, pp.4336-4343, 2002; EP 0328147; U.S. Patent 6,630,579). The antibody-drug conjugate BR96-doxorubicin reacts specifically with the tumor-associated antigen Lewis-Y and has been evaluated in phase I and phase II trials (Saleh et al., J.Clin.Oncology, vol.18, pp.2282-2292, 2000; Ajani et al., Cancer Jour., vol.6, pp.78-81, 2000; Tolcher et al., J.Clin.Oncology, vol.17, pp.478-484, 1999).

[0279] PNU-159682 is a potent metabolite (or derivative) of nemorubicin (Quintieri et al., Clinical Cancer Research, vol. 11, pp. 1608-1617, 2005). Nemorubicin is a semisynthetic analog of doxorubicin with a 2-methoxymorpholino group on the glycosidic amino of doxorubicin and is under clinical evaluation (Grandi et al. Cancer Treat. Rev. vol. 17, pp. 133-138, 1990; Ripamonti et al. Brit. J. Cancer, vol. 65, pp. 703-707, 1992), including Phase II / Phase III trials (Sun et al., Proceedings of the American Society for Clinical Oncology, vol. 22, Abs 1448, 2003; Quintieri, Proceedings of the American Association of Cancer Research, vol. 44: 1st Ed, Abs 4649, 2003; Pacciarini et al., Jour. Clin. Oncology, vol. 24, p. 14116, 2006).

[0280] Anthracyclines, such as PNU-159682, can be conjugated to antibodies via several binding sites and a variety of linkers (U.S. Patent Application Publication No. 2011 / 0076287; WO 2009 / 099741; U.S. Patent Application Publication No. 2010 / 0034837; WO 2010 / 009124), such as the linkers described herein.

[0281] The linker of PNU-159682-maleimide acetal-Ab is acid labile, whereas PNU-159682-val-cit-PAB-Ab, PNU-159682-val-cit-PAB-spacer-Ab, and PNU-159682-val-cit-PAB-spacer (R 1 R 2The linker in the )-Ab is protease cleavable.

[0282] (6) Other drug moieties Drug moieties include geldanamycin (Mandler et al., J. Nat. Cancer Inst., vol. 92, pp. 1573-1581, 2000; Mandler et al., Bioorganic & Med. Chem. Letters, vol. 10, pp. 1025-1028, 2000; Mandler et al., Bioconjugate Chem., vol. 13, pp. 786-791, 2002); and enzymatically active toxins and fragments thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments 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, Phytolacca Also included are the Momordica americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes. See, e.g., WO 93 / 21232.

[0283] Drug moieties also include compounds that have nucleolytic activity (eg, ribonucleases or DNA endonucleases).

[0284] In certain embodiments, the immunoconjugate may contain 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. For example, zirconium-89 can be complexed to various metal chelators and conjugated to antibodies for PET imaging (WO 2011 / 056983).

[0285] Radiolabels or other labels can be incorporated into the immunoconjugates by known techniques. For example, peptides can be biosynthesized or chemically synthesized using suitable amino acid precursors that contain, for example, one or more fluorine-19 atoms in place of one or more hydrogens. In some embodiments, the label, e.g., Tc 99 , I 123 , Re 186 , Re 188 , and In 111 can be attached via a cysteine ​​residue in the antibody. In some embodiments, yttrium-90 can be attached via a lysine residue in the antibody. In some embodiments, iodine-123 can be incorporated using the IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun., vol. 80, pp. 49-57, 1978). "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes certain other methods.

[0286] In certain embodiments, the immunoconjugate may include an antibody conjugated to a prodrug-activating enzyme. In some such embodiments, the prodrug-activating enzyme converts a prodrug (e.g., a peptidyl chemotherapeutic agent, see WO 81 / 01145) into an active drug, e.g., an anticancer 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 anticancer drug 5-fluorouracil; proteases, such as Serratia protease, thermocin, subtilisin, carboxypeptidase, and cathepsins (e.g., cathepsins B and L), useful for converting peptide-containing prodrugs to free drugs. D-alanyl carboxypeptidase is useful for converting prodrugs containing D-amino acid substitution; carbohydrate cleavage enzymes, such as β-galactosidase and neuraminidase, are useful for converting glycosylated prodrugs into free drugs; β-lactamase is useful for converting drugs that are derivatized with β-lactams into free drugs; and penicillin amidases, such as penicillin V amidase and penicillin G amidase, are useful for converting drugs that are derivatized with phenoxyacetyl or phenylacetyl groups at amine nitrogen into free drugs, respectively.In some embodiments, enzymes can be covalently bound to antibody by recombinant DNA techniques well known in the art.See, for example, Neuberger et al., Nature, vol.312, pp.604-608, 1984.

[0287] c) Drug Load Drug loading is represented by the average number p of drug moieties per antibody in a molecule of formula I. Drug loading can range from 1 to 20 drug moieties (D) per antibody. ADCs of formula I include a population of antibodies conjugated with drug moieties ranging from 1 to 20. The average number of drug moieties per antibody used in preparing ADCs from conjugation reactions can be characterized by conventional means, such as mass spectrometry, ELISA assays, and HPLC. Quantitative distribution of ADCs in units of p can also be measured. In some cases, separation, purification, and characterization of homogeneous ADCs with a certain value of p from ADCs with other drug loadings can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0288] For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, when the attachment is a cysteine ​​thiol, as in certain exemplary embodiments above, the antibody may have only one or a few cysteine ​​thiol groups, or only one or a few sufficiently reactive thiol groups to allow for the attachment of a linker. 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 for 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, about 2 to about 5 (U.S. Pat. No. 7,498,298).

[0289] In some embodiments, less than the theoretical maximum number of drug moieties are conjugated to the antibody during the conjugation reaction. The antibody may contain lysine residues that do not react with, for example, drug-linker intermediates or linker reagents, 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 some embodiments, the antibody can be reduced under partial or complete reducing conditions with a reducing agent, for example, dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), to generate reactive cysteine ​​thiol groups. In some embodiments, the antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups, for example, lysine or cysteine.

[0290] The loading (drug / antibody ratio) of ADCs can be controlled in a variety of 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 reducing conditions for cysteine ​​thiol modification.

[0291] It is understood that when two or more nucleophilic groups react with a drug-linker intermediate or linker reagent, the resulting product is a mixture of ADCs with a distribution of one or more drug moieties attached to the antibody. The average number of drugs per antibody can be calculated from the mixture by a double ELISA antibody assay that is specific for the antibody and specific for the drug. Individual ADCs can be identified in the mixture by mass spectrometry and separated by HPLC, for example, hydrophobic interaction chromatography (see, for example, McDonagh et al., Prot.Engr.Design & Selection, vol.19,pp.299-307,2006; Hamblett et al., Clin.Cancer Res.,vol.10,pp.7063-7070,2004). In some embodiments, homogeneous ADCs with a single loading value can be isolated from the conjugation mixture by electrophoresis or chromatography.

[0292] d) Certain Methods of Preparing Immunoconjugates Immunoconjugates that are ADCs of formula I can be prepared by several routes using organic chemical reactions, conditions, and reagents known to those of skill in the art, such as, for example, (1) reacting a nucleophilic group of an antibody with a bivalent linker reagent to form Ab-L via a covalent bond, which is then reacted with a drug moiety, D; and (2) reacting a nucleophilic group of a drug moiety with a bivalent linker reagent to form DL via a covalent bond, which is then reacted with a nucleophilic group of an antibody. An exemplary method of preparing ADCs of formula I via the latter route is described in U.S. Pat. No. 7,498,298.

[0293] Nucleophilic groups on antibodies include, but are not limited to, (i) N-terminal amine groups, (ii) side chain amine groups, such as lysine, (iii) side chain thiol groups, such as cysteine, and (iv) sugar hydroxyl or amino groups on which the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents, such as (i) active esters, such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyls, and maleimide groups to form covalent bonds. Some antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be made reactive for conjugation with linker reagents by treatment with a reducing agent, such as DTT (dithiothreitol) or tricarbonylethylphosphine (TCEP), so that the antibody is fully or partially reduced. Thus, each cysteine ​​bridge theoretically forms two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody through modification of lysine residues, for example by reacting the lysine residues with 2-iminothiolane (Traut's reagent) to convert the amine to a thiol. Reactive thiol groups can also be introduced into the antibody by introducing one, two, three, four or more cysteine ​​residues (e.g., by preparing a variant antibody containing one or more non-natural cysteine ​​amino acid residues).

[0294] The antibody-drug conjugates of the present invention can also be produced by the reaction between an electrophilic group on an antibody or antibody fragment, such as an aldehyde or ketone carbonyl group, and a nucleophilic group on a linker reagent or drug. Useful nucleophilic groups on a linker reagent include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide. In one embodiment, an antibody is modified to introduce an electrophilic moiety that can react with a nucleophilic substituent on a linker reagent or drug. In another embodiment, the sugar of a glycosylated antibody can be oxidized, for example, by a periodate oxidation reagent to form an aldehyde or ketone group that can react with an amine group on a linker reagent or drug moiety. The resulting imine Schiff base group can form a stable bond or can be reduced, for example, by a borohydride reagent to form a stable amine bond. In one embodiment, the reaction of carbohydrate moieties of glycosylated antibodies with either galactose oxidase or sodium metaperiodate can generate carbonyl (aldehyde and ketone) groups in the antibody that can react with appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In another embodiment, antibodies containing N-terminal serine or threonine residues can be reacted with sodium metaperiodate to generate an aldehyde in place of the first amino acid (Geoghegan & Stroh, Bioconjugate Chem., vol. 3, pp. 138-146, 1992; U.S. Patent No. 5,362,852). Such aldehydes can be reacted with drug moieties or linker nucleophiles.

[0295] Exemplary nucleophilic groups on the drug moiety include, but are not limited to, electrophilic groups on the linker moiety and linker reagents, such as (i) active esters, e.g., NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides, e.g., haloacetamides; (iii) amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide groups that can react with aldehyde, ketone, carboxyl, and maleimide groups to form covalent bonds.

[0296] Non-limiting exemplary cross-linking reagents that can be used to prepare ADCs are described in the section entitled "Exemplary Linkers" herein.Methods for using such cross-linking reagents to link two moieties, such as proteinaceous and chemical moieties, are known in the art.In some embodiments, fusion proteins comprising antibodies and cytotoxic agents can be produced, for example, by recombinant techniques or peptide synthesis.Recombinant DNA molecules can comprise regions that code for the antibody and cytotoxic moieties of the conjugate, adjacent to each other or separated by regions that code for linker peptides that do not destroy the desired properties of the conjugate.

[0297] In yet another embodiment, the antibody or antibody fragment can be conjugated to a "receptor" (e.g., streptavidin) for use in tumor pretargeting, in which the antibody / antibody fragment-receptor conjugate is administered to a patient, followed by removal of unbound conjugate from the blood circulation using a clearing agent, followed by administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a drug or radionucleotide).

[0298] C. Methods and Compositions for Diagnosis and Detection In some embodiments, any of the anti-ROR2 antibodies or antibody fragments provided herein can be used to detect the presence of ROR2 in a biological sample. As used herein, the term "detecting" includes quantitative or qualitative detection. In some embodiments, the biological sample includes cells or tissues, such as breast, pancreatic, esophageal, lung and / or brain cells or tissues.

[0299] A further aspect of the invention relates to an anti-ROR2 antibody or antibody fragment of the invention for diagnosing and / or monitoring cancer or another disease in which ROR2 expression levels are increased or decreased from normal physiological levels in at least one location in the body.

[0300] In a preferred embodiment, the antibody or antibody fragment of the present invention can be labeled with a detectable molecule or substance, such as a fluorescent molecule as described above, a 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, radioactive atoms used in scintigraphy studies, such as 123 I, 124 I, 111 In, 186 Re, and 188 The antibody or antibody fragment of the present invention can also be labeled with spin labels for nuclear magnetic resonance (NMR) imaging, such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron. After administration of the antibody, the distribution of the radiolabeled antibody in 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 diagnosis.

[0301] The antibody or antibody fragment of the present invention can be useful for the diagnosis and staging of cancers and diseases associated with ROR2 overexpression.Cancers associated with ROR2 overexpression can include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, glial cell tumors, such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, sarcoma (e.g., fibrosarcoma and osteosarcoma), blood cancer (leukemia), astrocytoma, and various types of head and neck cancer or other hyperproliferative diseases of ROR2 expression or overexpression.

[0302] The antibodies or antibody fragments of the present invention may be useful in the diagnosis of diseases other than cancer in which ROR2 expression is increased or decreased. (Both soluble or cellular ROR2 forms can be used for such diagnosis. Typically, such diagnostic methods involve the use of a biological sample obtained from a patient. As used herein, the term "biological sample" encompasses 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 collected from individuals suspected of having cancers associated with ROR2 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.

[0303] In a particular embodiment, the present invention is a method for diagnosing a cancer associated with ROR2 overexpression in a subject by detecting ROR2 on cells from the subject using an antibody of the present invention. In particular, the method comprises: (a) contacting a biological sample of a subject with an antibody or antibody fragment according to the invention under conditions suitable for the antibody or antibody fragment to form a complex with cells of the biological sample expressing ROR2; and (b) detecting and / or quantifying said complex, wherein detection of said complex is indicative of a cancer associated with ROR2 overexpression. may include.

[0304] 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 from that, it can be determined whether the cancer is progressing, regressing or stable.

[0305] In a particular embodiment, the present invention is a method for diagnosing diseases associated with expression or overexpression of ROR2 or a decrease or increase in the soluble form of ROR2. Examples of such diseases include human immune disorders, thrombotic diseases (thrombosis and atherothrombosis), and cardiovascular diseases.

[0306] In one embodiment, an anti-ROR2 antibody or antibody fragment is provided for use in a method of diagnosis or detection. In a further embodiment, a method for detecting the presence of ROR2 in a biological sample is provided. In a further embodiment, a method for quantifying the amount of ROR2 in a biological sample is provided. In an embodiment, the method comprises contacting a biological sample with an anti-ROR2 antibody or antibody fragment described herein under conditions that allow binding of the anti-ROR2 antibody or antibody fragment to ROR2, and detecting whether a complex is formed between the anti-ROR2 antibody or antibody fragment and ROR2. Such a method can be performed in vitro or in vivo. In one embodiment, the anti-ROR2 antibody or antibody fragment is used to select subjects eligible for a therapy. In some embodiments, the therapy comprises administering an anti-ROR2 antibody or antibody fragment to a subject.

[0307] In certain embodiments, a labeled anti-ROR2 antibody or antibody fragment is provided. Labels include, but are not limited to, labels or moieties that are directly detected (e.g., fluorescent labels, chromophores, electron-dense, chemiluminescent, and radioactive labels) and moieties that are indirectly detected, e.g., via enzymatic reactions or molecular interactions, such as enzymes or ligands. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C. 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as 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, saccharide oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, enzymes that oxidize dye precursors with hydrogen peroxide such as heterocyclic oxidases coupled with HRP, lactoperoxidase, or microperoxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0308] D. Pharmaceutical Formulations Anti-ROR2 antibodies or antibody fragments have cell killing activity. This cell killing activity extends to multiple different cell lineage types. Furthermore, these antigens or antibody fragments can reduce tumor size and exhibit reduced toxicity when conjugated to cytotoxic agents. See Example 2 of the present application. Thus, anti-ROR2 antibodies, fragments thereof or immunoconjugates can be useful for treating proliferative diseases associated with ROR2 expression. The antibodies, fragments or immunoconjugates can be used alone or in combination with any suitable agent or other conventional treatment.

[0309] Anti-ROR2 antibody or antibody fragment can be used to treat diseases associated with ROR2 expression, overexpression or activation. Other than the requirement for ROR2 expression, there is no specific limitation on the type or tissue of cancer that can be treated. Examples include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer (NSCLC), gastric cancer, pancreatic cancer, glial cell tumors, such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, melanoma, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, renal cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, sarcoma (e.g., fibrosarcoma and osteosarcoma), blood cancer (leukemia), astrocytoma, and various types of head and neck cancer. More preferred cancers are glioblastoma, gastric cancer, lung cancer, pancreatic cancer, breast cancer, prostate cancer, kidney cancer, liver cancer and endometrial cancer.

[0310] Anti-ROR2 antibodies or antibody fragments are potent activators of innate immune responses and can therefore be used in the treatment of human immune disorders, such as sepsis. Anti-ROR2 antibodies or antibody fragments of the present invention can also be used as adjuvants for immunization, such as vaccines, and as anti-infective agents, such as against bacteria, viruses and parasites.

[0311] Anti-ROR2 antibodies or antibody fragments can be used to protect against, prevent, or treat thrombotic diseases, such as venous and arterial thrombosis and atherothrombosis. Anti-ROR2 antibodies or antibody fragments can also be used to protect against, prevent, or treat cardiovascular diseases, as well as to prevent or inhibit the entry of viruses, such as Lassa and Ebola viruses, and to treat viral infections.

[0312] In each of the embodiments of the method of treatment described herein, the anti-ROR2 antibody, antibody fragment, or anti-ROR2 antibody or antibody fragment immunoconjugate can 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 period of time and under conditions sufficient to prevent or treat the disease or disorder. Thus, one aspect of the present invention relates to a method of treating a disease associated with expression of ROR2, 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 a disease associated with expression of ROR2.

[0313] For administration, the anti-ROR2 antibody, antibody fragment or immunoconjugate can be formulated as a pharmaceutical composition. The pharmaceutical composition containing the anti-ROR2 antibody, antibody fragment or immunoconjugate can be formulated according to known methods for preparing pharmaceutical compositions. In such methods, the therapeutic molecule is typically combined with a mixture, solution or composition containing a pharma- ceutically acceptable carrier.

[0314] A pharmaceutically acceptable carrier is a material 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 include one or more excipients, preservatives, stabilizers, buffers, albumin to prevent protein loss on the vial surface, etc.

[0315] The form, route of administration, dosage and regimen of the pharmaceutical composition usually depend on the pathology 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, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration, etc.

[0316] Preferably, the pharmaceutical composition contains a pharma- ceutically acceptable vehicle for the formulation to be injected. These may be present in a particular isotonic sterile saline solution (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, etc. or mixtures 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.

[0317] In some embodiments, tonicity agents, sometimes known as "stabilizers", are present to adjust or maintain the isotonicity 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, thereby reducing the potential for inter- and intra-molecular interactions. Tonicity agents can be present in any amount between 0.1% and 25% by weight of the pharmaceutical composition, preferably between 1% and 5%. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0318] Additional excipients include agents that may 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 such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; organic sugars or sugar alcohols such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol), and the like. ), polyethylene glycol; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers, such as 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).

[0319] Non-ionic surfactants or detergents (also known as "wetting agents") can be used to help dissolve the therapeutic agent and to protect the therapeutic protein from agitation-induced aggregation, thereby allowing exposure of the formulation to shear surface stresses without causing denaturation of the active therapeutic protein or antibody. The non-ionic surfactants can 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.

[0320] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid esters, methylcellulose and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0321] The doses 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 pharma- ceutically acceptable carrier or aqueous medium.

[0322] Suitable dosage forms for injection use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions.In all cases, the dosage form must be sterile and fluid to the extent that easy syringeability 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.

[0323] The solution of active compound as free base or pharmacologically acceptable salt can be prepared in water mixed with surfactant appropriately.Dispersion can also be prepared in glycerol, liquid polyethylene glycol, and its mixture, and in oil.Under normal storage and use conditions, these preparations contain preservatives to prevent the growth of microorganisms.

[0324] Anti-ROR2 antibodies or antibody fragments can be formulated in the composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) that are formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as potassium, ammonium, calcium or iron hydroxide, and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0325] The carrier may be a solvent or dispersion medium containing, for example, 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 the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about 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 the injectable composition can be brought about by using an agent that delays absorption in the composition, for example, aluminum monostearate and gelatin.

[0326] Sterile injectable solution is prepared by incorporating the required amount of active compound in a suitable solvent, if necessary, together with one or more of the other components listed above, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating various sterilized active components into a sterile vehicle that contains a basic dispersion medium and other components required from those listed above.In the case of sterile powder for preparing sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of active component and any additional desired component from its solution that has been previously sterilized and filtered.

[0327] The preparation of more concentrated or highly concentrated solutions for direct injection is also contemplated, with the use of dimethyl sulfoxide (DMSO) as a solvent expected to result in extremely rapid penetration, delivering high concentrations of active agent to small tumor areas.

[0328] 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 types of injectable solutions described above, but drug release capsules and the like can also be used.

[0329] For parenteral administration in aqueous solution, for example, 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, the sterile aqueous media that can be used will be known to those skilled in the art in light of this disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution, added to 1000 ml of subcutaneous infusion therapy liquid, or injected at the proposed injection site (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will in any event determine the appropriate dose for the individual subject.

[0330] The antibody or antibody fragment can be formulated to deliver between 0.0001 and 10.0 milligrams, or about 0.001 and 5 milligrams, or about 0.001 and 1 milligrams, or about 0.001 and 0.1 milligrams, or about 0.1 and 1.0 or even about 10 milligrams per dose within the therapeutic mixture. Multiple doses can also be administered at selected time intervals.

[0331] In addition to compounds formulated for parenteral administration, e.g., intravenous or intramuscular injection, other pharma- ceutically acceptable forms include, for example, tablets or other solid forms for oral administration; sustained release capsules; and any other form currently in use.

[0332] In certain embodiments, the use of liposomes and / or nanoparticles is contemplated for the introduction of antibodies or antibody fragments into host cells. The formation and use of liposomes and / or nanoparticles are known to those of skill in the art.

[0333] Nanocapsules can generally capture compounds in a stable and reproducible manner.To avoid side effects caused by polymer overloading in cells, such ultrafine particles (size is about 0.1 μm) are generally designed using polymers that can degrade in vivo.The biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention, and such particles can be easily produced.

[0334] Liposomes are formed from phospholipids dispersed in aqueous media, which spontaneously form multilamellar concentric bilayer vesicles, also called multilamellar vesicles (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 in the range of 200-500 Å that contain aqueous solution in their core. The physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations.

[0335] Pharmaceutical formulations containing the anti-ROR2 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 pharma- ceutically 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 employed, and include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (approximately 10 residues) soluble ... (less than 100%) polypeptides; 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, such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0336] Exemplary pharma- ceutically acceptable carriers herein further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), such as human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, such as rHuPH20, are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0337] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0338] The formulation herein may also contain two or more active ingredients, as required for the particular indication being treated. Preferably, ingredients with complementary activities that do not adversely affect each other can be combined in a single formulation. For example, it may be desirable to provide an EGFR antagonist (e.g., erlotinib), an anti-androgen agent (e.g., a VEGF antagonist, which may be an anti-VEGF antibody), or a chemotherapeutic agent (e.g., a taxoid or a platinum agent) in addition to the anti-ROR2 antibody, antibody fragment, or immunoconjugate of the present invention. Such active ingredients are suitably present in a combination in amounts that are effective for the intended purpose.

[0339] The active ingredient can be encapsulated in microcapsules, which are prepared, for example, by coacervation technology or by interfacial polymerization.For example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules can be used in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, respectively.Such technology is disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0340] 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 may be in the form of shaped articles, e.g., films, or microcapsules.

[0341] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0342] E. Therapeutic Methods and Compositions Any of the anti-ROR2 antibodies or antibody fragments provided herein can be used in a method of treatment. In one aspect, an anti-ROR2 antibody or antibody fragment is provided for use as a medicament. In a further aspect, an anti-ROR2 antibody or antibody fragment is provided for use in the treatment of cancer (e.g., breast cancer, non-small cell lung cancer, pancreatic cancer, brain tumor, pancreas, brain, kidney, ovarian, stomach, leukemia, endometrial, colon, prostate, thyroid, liver cancer, sarcoma, such as fibrosarcoma and osteosarcoma melanoma, and / or various head and neck cancers). In one embodiment, an anti-ROR2 antibody or antibody fragment is provided for use in a method of treatment. In one embodiment, the present invention provides an anti-ROR2 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-ROR2 antibody or antibody fragment. In certain embodiments, the invention provides an anti-ROR2 antibody or antibody fragment for use in a method of 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-ROR2 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 those described below. In further embodiments, the invention provides an anti-ROR2 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 or tumor-associated vasculature), and / or inhibiting tumor stromal function.

[0343] In certain embodiments, the invention provides an anti-ROR2 antibody or antibody fragment for use in a method of inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumor or tumor-associated vasculature), and / or inhibiting tumor stromal function in an individual, comprising administering to the individual an effective anti-ROR2 antibody or antibody fragment to inhibit angiogenesis, inhibit cell proliferation, inhibit immune function, inhibit 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. An "individual" according to any of the above embodiments is preferably a human.

[0344] In a further aspect, the present invention provides the use of an anti-ROR2 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, sarcoma, such as fibrosarcoma and osteosarcoma, melanoma and / or various head and neck cancers). 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-ROR2 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 further embodiments, the medicament is for inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral or tumor-associated vasculature), and / or inhibiting tumor stromal function. In further embodiments, the medicament is used in a method of inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral or tumor-associated vasculature), and / or inhibiting tumor stromal function in an individual, 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., intratumoral or tumor-associated vasculature), and / or inhibit tumor stromal function. An "individual" according to any of the above embodiments may be a human.

[0345] In a further aspect, the invention provides a method of treating a ROR2-expressing tumor in an individual with cancer. In one embodiment, the method comprises administering to such an individual with cancer an effective amount of an anti-ROR2 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.

[0346] In this aspect of the invention, methods of treating ROR2-expressing tumors are provided. In some embodiments, the methods comprise administering an immunoconjugate comprising an anti-ROR2 antibody or antibody fragment of the invention.

[0347] In one embodiment of this aspect, the method of treating ROR2-expressing tumors comprises administering an immunoconjugate comprising an antibody or antibody fragment of the invention conjugated to an agent selected from a chemotherapeutic agent, a radioactive atom, a cytostatic agent, and a cytotoxic agent. The immunoconjugate is an antibody drug conjugate (ADC) in which a conditionally active biological (CAB) anti-ROR2 antibody or antibody fragment is linked to one or more drug moieties via a cleavable linker (CAB-ROR2-ADC). For example, a CAB-ROR2-ADC may be composed of BA3021-cleavable linker-MMAE. (n) where the drug moiety is monomethylauristatin E (MMAE) and (n) is an integer from 1 to 4, inclusive.

[0348] In certain embodiments, the method of treating a ROR2-expressing tumor comprises administering to a human subject in need of such treatment BA3021-cleavable linker-MMAE. (n) wherein BA3021 is an antibody or antibody fragment having a heavy chain variable region comprising CDRs H1, H2, and H3 having the amino acid sequence of SEQ ID NO: 16 and a light chain variable region comprising CDRs L1, L2, and L3 having the amino acid sequence of SEQ ID NO: 21; MMAE is monomethylauristatin E (MMAE), and (n) is an integer between 1 and 4 (inclusive).

[0349] In some embodiments, the present invention provides a BA3021-cleavable linker-MMAE (n) The present invention provides a treatment regimen in which a CAB-ROR2-ADC such as BA3021-cleavable linker-MMAE is administered at a dose of about 0.3 mg / kg to about 3.3 mg / kg once or twice every 21 days or 3 weeks, or once or twice every 14 days or 2 weeks. (n) It can be administered once every three weeks (Q3W) at a dosage of about 0.3 mg / kg to about 3.3 mg / kg, or twice every three weeks (2Q3W), such as on days 1 and 8, at a dosage ranging from 1.5 mg / kg to 1.8 mg / kg.

[0350] In a particular embodiment, the present invention provides a BA3021-cleavable linker-MMAE (n) The present invention provides a treatment regimen in which a CAB-ROR2-ADC such as BA3021-cleavable linker-MMAE is administered at a dose of about 0.3 mg / kg to about 1.8 mg / kg once or twice every 21 days or 3 weeks, or once or twice every 14 days or 2 weeks. (n) can be administered at a dosage of about 0.3 mg / kg to about 1.8 mg / kg, twice every 14 days or every two weeks, such as on days 1 and 8 of every 14 days or every two weeks. (n) is administered at a dosage of about 0.8 mg / kg to about 1.8 mg / kg, once or twice every 21 days (e.g., days 1 and 8 of a 21 day period), or once or twice every 14 days (e.g., days 1 and 8 of a 14 day period). In one embodiment, CAB-ROR2-ADC is administered at 1.8 mg / kg on days 1 and 8 of each 21 day period for one or more consecutive 21 days.

[0351] Such treatment regimens are surprisingly effective, because the antibody drug conjugates of the present invention administered at such doses and intervals provide surprisingly high response rates and acceptable toxicity or tolerability profiles. Thus, the present method provides a safe and effective dosing regimen for administering CAB-ROR2-ADC antibody drug conjugates to a subject. In some embodiments, the dosing regimen increases the probability that the subject will respond to the therapy compared to other dosing regimens. In some embodiments, the dosing regimen does not increase the probability that the subject will suffer from adverse events (including dose-limiting toxicity) compared to other dosing regimens. The present invention also provides a maintenance therapy following the dosing regimen.

[0352] In yet another embodiment, the method of treating ROR2-expressing tumors comprises administering to a human subject in need of such treatment BA3021-cleavable linker-MMAE. (n) and a pharmaceutical acceptable carrier, wherein the pharmaceutical composition is administered at a dosage of 1.8 mg / kg of body weight of the human subject by intravenous infusion every 14 days.

[0353] BBA3021-cleavable linker-MMAE of the present invention (n) CAB-ROR2-ADCs such as those described above preferentially bind under defined physiological conditions associated with different diseases and tissues. For example, in cancer, the unique cellular metabolism proposed by Warburg creates a characteristic tumor microenvironment (TME) with low pH and high lactic acid (Warburg 1924; Warburg 1956). The BA3021-cleavable linker-MMAE of the present invention. (n) utilizes a specific TME and selectively binds to its target when in close proximity to tumors expressing ROR2. BA3021-cleavable linker-MMAE (n) The activated binding properties of are reversible and are not permanently altered upon transfer from pathological normal tissue to pathological tissue microenvironments.

[0354] In yet another embodiment, ROR2-expressing tumors have a Tumor Membrane Percentage Score (TmPS) of at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, or at least 95. In one embodiment, ROR2-expressing tumors have a Tumor Membrane P Score of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0355] In a further aspect, the 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.

[0356] In a further aspect, the invention provides a method of inhibiting angiogenesis, inhibiting cell proliferation, inhibiting immune function, inhibiting inflammatory cytokine secretion (e.g., from tumor-associated macrophages), inhibiting tumor vasculature (e.g., intratumoral or tumor-associated vasculature), and / or inhibiting tumor stromal function in an individual. In one embodiment, the method comprises administering to the individual an effective amount of an anti-ROR2 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., intratumoral or tumor-associated vasculature), and / or inhibit tumor stromal function. In one embodiment, the "individual" is a human.

[0357] In a further aspect, the invention provides pharmaceutical formulations comprising any of the anti-ROR2 antibodies or antibody fragments provided herein, e.g., for use in any of the above methods of treatment. In one embodiment, the pharmaceutical formulation comprises any of the anti-ROR2 antibodies or antibody fragments provided herein and a pharma- ceutical acceptable carrier. In another embodiment, the pharmaceutical formulation comprises any of the anti-ROR2 antibodies or antibody fragments provided herein and at least one additional therapeutic agent, e.g., as described below.

[0358] In each and every of the above treatments, the antibody or antibody fragment of the present invention can be used in the therapy alone, as an immunoconjugate, or in combination with other agents. For example, the antibody of the present invention can be co-administered with at least one additional therapeutic agent. In some embodiments, the additional therapeutic agent is an anti-angiogenic agent. In some embodiments, the additional therapeutic agent is a VEGF antagonist (in some embodiments, an anti-VEGF antibody, e.g., bevacizumab). In some embodiments, the additional therapeutic agent is an EGFR antagonist (in some embodiments, erlotinib). In some embodiments, the additional therapeutic agent is a chemotherapeutic agent and / or a cytostatic agent. In some embodiments, the additional therapeutic agent is a taxoid (e.g., paclitaxel) and / or a platinum agent (e.g., carboplatinum). In some embodiments, the additional therapeutic agent is an agent that enhances the patient's immunity or immune system.

[0359] Such combination therapy as described above includes combined administration (two or more therapeutic agents in the same or separate formulations) and separate administration, where administration of the antibody or antibody fragment can occur before, simultaneously with, and / or after administration of the additional therapeutic agent and / or adjuvant. The antibody or antibody fragment can also be used in combination with radiation therapy.

[0360] The antibody or antibody fragment may be formulated, dosed, and administered in a manner consistent with good medical practice. Factors of consideration in this regard 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 delivery of the agent, the method of administration, the scheduling of administration, and other factors known to physicians. The antibody or antibody fragment is optionally, but need not be, 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 in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and routes of administration as described herein, or at about 1-99% of the dosages described herein, or at any dosage and by any route that is deemed experimentally / clinically appropriate.

[0361] For the prevention or treatment of a disease, the appropriate dosage of the antibody or antibody fragment (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease to be treated, the type of antibody or antibody fragment, the severity and course of the disease, whether the antibody or antibody fragment is administered for prophylactic or therapeutic purposes, prior treatment, 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, for example, about 1 μg / kg to 40 mg / kg of the antibody or antibody fragment may be an initial candidate dosage for administration to a patient, whether by one or more separate administrations or by continuous infusion. One typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or more, depending on the condition, treatment is generally sustained until a desired suppression of disease symptoms occurs. Such dosages can be administered intermittently, e.g., weekly or every three weeks (e.g., so that the patient receives from about 2 to about 20, or, e.g., 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.

[0362] It will be understood that any of the above formulations or methods of treatment can be practiced using an antibody fragment or immunoconjugate of the invention in place of, or in addition to, an anti-ROR2 antibody.

[0363] Improving the host's immune function to eradicate tumors is a subject of increasing interest. Conventional methods include (i) APC enhancement, such as (a) injection of DNA encoding foreign MHC alloantigens into the tumor, or (b) transfection of biopsy-obtained tumor cells with genes that increase the probability of immune antigen recognition of the tumor (e.g., immune stimulatory 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, via stimulation with IL-2 or tumor or both. In addition, dysfunctional isolated T cells can also be activated by in vitro application of the anti-PD-L1 antibodies of the invention. The T cells thus activated 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.

[0364] Conventional treatments 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 externally via external beam radiotherapy (EBRT) or internally via brachytherapy; (ii) chemotherapy, or application of cytotoxic drugs, which generally affect rapidly dividing cells; (iii) targeted therapy, or drugs that specifically affect dysregulated proteins in cancer cells (e.g., tyrosine kinase inhibitors imatinib, gefitinib; monoclonal antibodies, photodynamic therapy); (iv) immunotherapy, or enhancement of host immune response (e.g., vaccines); (v) hormone therapy, or hormone blockade (e.g., when tumors are hormone sensitive); (vi) angiogenesis inhibitors, or blockade of angiogenesis and growth, and (vii) palliative care, or treatments directed at improving the quality of care to reduce pain, nausea, vomiting, diarrhea, and bleeding. Painkillers, such as morphine and oxycodone, and antiemetics, such as ondansetron and aprepitant, may allow for more aggressive treatment regimens.

[0365] In treating cancer, any of the conventional therapies already described for the treatment of cancer immunity can be administered before, after, or simultaneously with the administration of the anti-ROR2 antibody or antibody fragment. Furthermore, the anti-ROR2 antibody or antibody fragment can be administered before, after, or simultaneously with conventional cancer therapies, such as tumor-binding antibodies (e.g., monoclonal antibodies, toxin-conjugated monoclonal antibodies) and / or chemotherapeutic agents.

[0366] F. Dosing Regimen The present invention provides dosing regimens for the treatment of ROR2-expressing tumors. The dosing regimens include doses of the polypeptide, antibody or antibody fragment, or immunoconjugate of the present invention described herein of about 0.3 mg / kg body weight to about 3.3 mg / kg body weight, about 0.4 mg / kg body weight to about 3.3 mg / kg body weight, about 0.5 mg / kg body weight to about 3.3 mg / kg body weight, about 0.6 mg / kg body weight to about 3.3 mg / kg body weight, about 0.7 mg / kg body weight to about 3.3 mg / kg body weight, about 0.8 mg / kg body weight to about 3.3 mg / kg body weight, about 0.9 mg / kg body weight to about 3.3 mg / kg body weight, about 1.0 mg / kg body weight to about 3.3 mg / kg body weight, about 2.0 mg / kg body weight to about 3.3 mg / kg body weight, about 3.0 mg / kg body weight to about 3.3 mg / kg body weight, about 4.0 mg / kg body weight to about 3.3 mg / kg body weight, about 4.0 mg / kg body weight to about 3.3 mg / kg body weight, about 5.0 mg / kg body weight to about 3.3 mg / kg body weight, about 6.0 mg / kg body weight to about 3.3 mg / kg body weight, about 7.0 mg / kg body weight to about 3.3 mg / kg body weight, about 8.0 mg / kg body weight to about 3.3 mg / kg body weight, about 9.0 mg / kg body weight to about 3.3 mg / kg body weight, about 10.0 mg / kg body weight to about 3.3 mg / kg body weight, about 12.0 mg / kg body weight to about 3.3 mg / kg body weight, about 16.0 mg / kg body weight to about 3.3 mg / kg body weight, about 18.0 mg / kg body weight to about 3. mg / kg body weight, about 1.1 mg / kg body weight to about 3.3 mg / kg body weight, about 1.2 mg / kg body weight to about 3.3 mg / kg body weight, about 1.3 mg / kg body weight to about 3.3 mg / kg body weight, about 1.4 mg / kg body weight to about 3.3 mg / kg body weight, about 1.5 mg / kg body weight to about 3.3 mg / kg body weight, about 1.6 mg / kg body weight to about 3.3 mg / kg body weight, about 1.7 mg / kg body weight to about 3.3 mg / kg body weight, about 1.8 mg / kg body weight to about 3.3 mg / kg body weight, about 1.9 mg / kg body weight to about 3.3 mg / kg body weight, about 2.0 mg / kg body weight to about 3.3 mg / kg body weight, about 2.1 mg / kg body weight to about 3.3 mg / kg body weight, about 2.2 mg / kg body weight to about 3.3 mg / kg body weight, about 2.3 mg / kg body weight to about 3.3 mg / kg body weight, about 2.4 mg / kg body weight to about 3.3 mg / kg body weight, about 2.5 mg / kg body weight to about 3.3 mg / kg body weight, about 2.6 mg / kg body weight to about 3.3 mg / kg body weight, about 2.7 mg / kg body weight to about 3.3 mg / kg body weight, about 2.8 mg / kg body weight to about 3.3 mg / kg body weight, about 2.9 mg / kg body weight to about 3.3 mg / kg body weight, about 3.0 mg / kg body weight to about 3. 3mg / kg body weight, about 3.1mg / kg body weight to about 3.3mg / kg body weight, about 3.2mg / kg body weight to about 3.3mg / kg body weight, about 0.3mg / kg body weight to about 1.8mg / kg body weight, about 0.4mg / kg body weight to about 1.8mg / kg body weight, about 0.5mg / kg body weight to about 1.8mg / kg body weight, about 0.6mg / kg body weight to about 1.8mg / kg body weight, about 0.7mg / kg body weight to about 1.8mg / kg body weight, about 0.8mg / kg body weight to about 1.8mg / kg body weight, about 0.9mg / kg body weight to about 1.8mg / kg body weight, about 1.0mg / kg body weight to about 1.The present invention includes administering about 8 mg / kg body weight, about 1.1 mg / kg body weight to about 1.8 mg / kg body weight, about 1.4 mg / kg body weight to about 1.8 mg / kg body weight, about 1.5 mg / kg body weight to about 1.8 mg / kg body weight, about 1.6 mg / kg body weight to about 1.8 mg / kg body weight, or about 1.7 mg / kg body weight to about 1.8 mg / kg body weight for at least 2 weeks (e.g., 14 days) or 3 weeks (e.g., 21 days). More preferably, about 1.5 mg / kg body weight to about 1.8 mg / kg body weight is administered for a period of at least 2 weeks (e.g., 14 days) or 3 weeks (e.g., 21 days). Most preferably, about 1.5 mg / kg body weight to about 1.8 mg / kg body weight is administered for at least 2 weeks (e.g., 14 days). The (bi)weekly dosage can also be administered as a once (bi)weekly administration (once a week) or by split delivery (e.g., two or more times per (bi)week). In some embodiments, the (bi)weekly dosage of the polypeptide, antibody or antibody fragment, or immunoconjugate of the invention described herein is 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3 mg / kg of the subject's body weight.

[0367] In certain embodiments, the dosing regimen comprises a dosage of about 0.3 mg / kg body weight to about 3.3 mg / kg body weight, about 0.4 mg / kg body weight to about 3.3 mg / kg body weight, about 0.5 mg / kg body weight to about 3.3 mg / kg body weight, about 0.6 mg / kg body weight to about 3.3 mg / kg body weight, about 0.7 mg / kg body weight to about 3.3 mg / kg body weight, about 0.8 mg / kg body weight to about 3.3 mg / kg body weight, about 0.9 mg / kg body weight to about 3.3 mg / kg body weight, about 0.10 mg / kg body weight to about 3.3 mg / kg body weight, about 0.12 mg / kg body weight to about 3.3 mg / kg body weight, about 0.14 mg / kg body weight to about 3.3 mg / kg body weight, about 0.16 mg / kg body weight to about 3.3 mg / kg body weight, about 0.18 mg / kg body weight to about 3.3 mg / kg body weight, about 0.19 mg / kg body weight to about 3.3 mg / kg body weight, about 0.20 mg / kg body weight to about 3.3 mg / kg body weight, about 0.21 mg / kg body weight to about 3.3 mg / kg body weight, about 0.22 mg / kg body weight to about 3.3 mg / kg body weight, about 0.23 mg / kg body weight to about 3.3 mg / kg body weight, about 0.24 mg / kg body weight to about 3.3 mg / kg body weight, about 0.25 mg / kg body weight to about 3.3 mg / kg body weight, about 0.26 mg / kg body weight to about 3.3 mg / kg body weight, about 0.27 mg / kg body weight to about 3.3 mg / kg body weight, about 0.28 mg / kg body weight to about 3.3 mg / kg body 3mg / kg body weight, approximately 1.0mg / kg body weight ~ approximately 3.3mg / kg body weight, approximately 1.1mg / kg body weight ~ approximately 3.3mg / kg body weight, approximately 1.2mg / kg body weight ~ approximately 3.3mg / kg body weight, approximately 1.3mg / kg body weight ~ approximately 3.3mg / kg body weight, approximately 1.4mg / kg body weight ~ approximately 3.3mg / kg body weight kg body weight, approximately 1.5 mg / kg body weight ~ approximately 3.3 mg / kg body weight, approximately 1.6 mg / kg body weight ~ approximately 3.3 mg / kg body weight, approximately 1.7 mg / kg body weight ~ approximately 3.3 mg / kg body weight, approximately 1.8 mg / kg body weight ~ approximately 3.3 mg / kg body weight, approximately 1.9 mg / kg body weight ~ approximately 3.3 mg / kg body weight , about 2.0 mg / kg body weight to about 3.3 mg / kg body weight, about 2.1 mg / kg body weight to about 3.3 mg / kg body weight, about 2.2 mg / kg body weight to about 3.3 mg / kg body weight, about 2.3 mg / kg body weight to about 3.3 mg / kg body weight, about 2.4 mg / kg body weight to about 3.3 mg / kg body weight, about 2.5 mg / kg body weight to about 3.3 mg / kg body weight, about 2.6 mg / kg body weight to about 3.3 mg / kg body weight, about 2.7 mg / kg body weight to about 3.3 mg / kg body weight, about 2.8 mg / kg body weight to about 3.3 mg / kg body weight, about 2.9 mg / kg body weight to about 3.3 mg / kg body weight, about 3.0 mg / kg body weight to about 3.3 mg / kg body weight, about 3.1 mg / kg body weight to about 3.3 mg / kg body weight, about 3.2 mg / kg body weight to about 3.3 mg / kg body weight, about 0.3 mg / kg body weight to about 1.8 mg / kg body weight, about 0.4 mg / kg body weight to about 1.8 mg / kg body weight, about 0.5 mg / kg body weight to about 1.8 mg / kg body weight, about 0.6 mg / kg body weight to about 1.8 mg / kg body weight, about 0.7 mg / kg body weight to about 1.8 mg / kg body weight, about 0.8 mg / kg body weight to about 1.8 mg / kg body weight, about 0.9 mg / kg body weight to about 1.8 mg / kg body weight, about 1.0 mg / kg body weight to about 1.The present invention includes administering about 8 mg / kg body weight, about 1.1 mg / kg body weight to about 1.8 mg / kg body weight, about 1.4 mg / kg body weight to about 1.8 mg / kg body weight, about 1.5 mg / kg body weight to about 1.8 mg / kg body weight, about 1.6 mg / kg body weight to about 1.8 mg / kg body weight, or about 1.7 mg / kg body weight to about 1.8 mg / kg body weight for at least 2 weeks (e.g., 14 days) or 3 weeks (e.g., 21 days). More preferably, about 1.5 mg / kg body weight to about 1.8 mg / kg body weight is administered for a period of at least 2 weeks (e.g., 14 days) or 3 weeks (e.g., 21 days). Most preferably, about 1.5 mg / kg body weight to about 1.8 mg / kg body weight is administered for at least 2 weeks (e.g., 14 days). The (bi)weekly dosage can also be administered as a once (bi)weekly administration (once a week) or by split delivery (e.g., two or more times per (bi)week). In some embodiments, the (bi)weekly dosage of the anti-ROR2 antibody or antibody fragment of the invention, or immunoconjugate comprising an anti-ROR2 antibody or antibody fragment, described herein, is 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3 mg / kg of subject body weight.

[0368] In certain embodiments, the dosing regimen comprises a dose of about 0.3 mg / kg to about 3.3 mg / kg body weight, about 0.4 mg / kg to about 3.3 mg / kg body weight, about 0.5 mg / kg to about 3.3 mg / kg body weight, about 0.6 mg / kg to about 3.3 mg / kg body weight, about 0.7 mg / kg to about 3.3 mg / kg body weight, about 0.8 mg / kg to about 3.3 mg / kg body weight, about 0.9 mg / kg to about 3.3 mg / kg body weight, about 1.0 mg / kg to about 3.3 mg / kg body weight, about 1.1 mg / kg body weight, or about 1.2 mg / kg body weight of the antibody drug conjugate described herein. Weight ~ about 3.3 mg / kg body weight, about 1.2 mg / kg body weight - about 3.3 mg / kg body weight, about 1.3 mg / kg body weight - about 3.3 mg / kg body weight, about 1.4 mg / kg body weight - about 3.3 mg / kg body weight, about 1.5 mg / kg body weight - about 3.3 mg / kg body weight, about 1.6 mg / kg body weight - about 3. 3mg / kg body weight, approximately 1.7mg / kg body weight ~ approximately 3.3mg / kg body weight, approximately 1.8mg / kg body weight ~ approximately 3.3mg / kg body weight, approximately 1.9mg / kg body weight ~ approximately 3.3mg / kg body weight, approximately 2.0mg / kg body weight ~ approximately 3.3mg / kg body weight, approximately 2.1mg / kg body weight ~ approximately 3.3mg / k g body weight, approximately 2.2 mg / kg body weight ~ approximately 3.3 mg / kg body weight, approximately 2.3 mg / kg body weight ~ approximately 3.3 mg / kg body weight, approximately 2.4 mg / kg body weight ~ approximately 3.3 mg / kg body weight, approximately 2.5 mg / kg body weight ~ approximately 3.3 mg / kg body weight, approximately 2.6 mg / kg body weight ~ approximately 3.3 mg / kg body weight, Approx. 2.7 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.8 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.9 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 3.0 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 3.1 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 3.2 m g / kg body weight to about 3.3 mg / kg body weight, about 0.3 mg / kg body weight to about 1.8 mg / kg body weight, about 0.4 mg / kg body weight to about 1.8 mg / kg body weight, about 0.5 mg / kg body weight to about 1.8 mg / kg body weight, about 0.6 mg / kg body weight to about 1.8 mg / kg body weight, about 0.7 mg / kg body weight to about 1.8 mg / kg body weight, about 0.8 mg / kg body weight to about 1.8 mg / kg body weight, about 0.9 mg / kg body weight to about 1.8 mg / kg body weight, about 1.0 mg / kg body weight to about 1.8 mg / kg body weight, about 1.1 mg / kg body weight to about 1.8 mg / kg body weight, about 1.4 mg / kg body weight to about 1.This includes administering about 8 mg / kg body weight, about 1.5 mg / kg body weight to about 1.8 mg / kg body weight, about 1.6 mg / kg body weight to about 1.8 mg / kg body weight, or about 1.7 mg / kg body weight to about 1.8 mg / kg body weight for at least 2 weeks (e.g., 14 days) or 3 weeks (e.g., 21 days). More preferably, about 1.5 mg / kg body weight to about 1.8 mg / kg body weight is administered for a period of at least 2 weeks (e.g., 14 days) or 3 weeks (e.g., 21 days). Most preferably, about 1.5 mg / kg body weight to about 1.8 mg / kg body weight is administered for at least 2 weeks (e.g., 14 days). The (bi)weekly dosage can also be administered as a once (bi)weekly administration (once (bi)weekly) or by split delivery (e.g., two or more times per (bi)weekly). In some embodiments, the (bi)weekly dosage of the antibody drug conjugate is 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3 mg / kg of the subject's body weight.

[0369] In certain embodiments, the dosage is administered as a split delivery or as a weekly administration over at least one 2-week (e.g., 14-day) treatment cycle, or at least one 3-week (e.g., 21-day) treatment cycle. In some embodiments, the dosage is administered as a single dose on days 1 and 8 of a 14-day treatment cycle. Preferably, the dosage is administered as a split delivery or as a single dose over two or more 14-day treatment cycles, more preferably over three or more, four or more, five or more, or six or more treatment cycles. In some embodiments, the weekly dosage is administered for no more than three, no more than four, no more than five, or no more than six treatment cycles. In some embodiments, the dosage is administered as a weekly administration on days 1 and 8 of a 21-day treatment cycle. Preferably, the dosage is administered as a split delivery or as a single dose over two or more 21-day treatment cycles, more preferably over three or more, four or more, five or more, or six or more treatment cycles. In some embodiments, the weekly dosage is administered for no more than 3, no more than 4, no more than 5, or no more than 6 treatment cycles. In some embodiments, there are drug holidays between the treatment cycles.

[0370] For example, in some embodiments, the dosing regimen may comprise, for at least one weekly (e.g., 7 day) treatment cycle, a total weekly dose of antibody drug conjugate of about 0.3 mg / kg body weight to about 3.3 mg / kg body weight, about 0.4 mg / kg body weight to about 3.3 mg / kg body weight, about 0.5 mg / kg body weight to about 3.3 mg / kg body weight, about 0.6 mg / kg body weight to about 3.3 mg / kg body weight, about 0.7 mg / kg body weight to about 3.3 mg / kg body weight, about 0.8 mg / kg body weight to about 3.3 mg / kg body weight, about 0.9 mg / kg body weight to about 3.3 mg / kg body weight, about 1.0 mg / kg body weight to about 3.3 mg / kg body weight, about 1.1 mg / kg body weight to about 3.3 mg / kg body weight, About 1.2 mg / kg body weight to about 3.3 mg / kg body weight, about 0.3 mg / kg body weight to about 1.8 mg / kg body weight, about 0.4 mg / kg body weight to about 1.8 mg / kg body weight, about 0.5 mg / kg body weight to about 1.8 mg / kg body weight, about 0.6 mg / kg body weight to about 1.8 mg / kg body weight, about 0.7 mg / kg body weight to about 1.8 mg / kg body weight, about 0.8 mg / kg body weight to about 1.8 mg / kg body weight, about 0.9 mg / kg body weight to about 1.8 mg / kg body weight, about 1.0 mg / kg body weight to about 1.8 mg / kg body weight, about 1.1 mg / kg body weight to about 1.8 mg / kg body weight, about 1.4 mg / kg body weight to about 1.8 mg / kg body weight, about 1.5 mg / kg body weight to about 1.8 mg / kg body weight. In some embodiments, the treatment cycle is 7 days or more. In some embodiments, the treatment is for at least two treatment cycles, with a one-week rest period between each treatment cycle. In some embodiments, the treatment is for 21 days or more. The weekly dosage can be administered as a weekly administration (once a week) or by split delivery (e.g., two or more times a week).

[0371] In some embodiments, the dosing regimen includes at least one biweekly (e.g., 14-day) treatment cycle, with a biweekly total dose of antibody drug conjugate of 0.8 mg / kg to about 1.8 mg / kg body weight, about 0.9 mg / kg to about 1.8 mg / kg body weight, about 1.0 mg / kg to about 1.8 mg / kg body weight, about 1.1 mg / kg to about 1.8 mg / kg body weight, about 1.2 mg / kg to about 1.8 mg / kg body weight, about 1.3 mg / kg to about 1.8 mg / kg body weight, about 1.4 mg / kg to about 1.8 mg / kg body weight, or about 1.5 mg / kg to about 1.8 mg / kg body weight. In some embodiments, the treatment cycle is 14 days or longer. In some embodiments, the treatment is for at least two treatment cycles, with a two-week break between each treatment cycle (e.g., six weekly doses over an eight-week period). In some embodiments, the treatment cycle is 21 days or longer. The biweekly dose can be administered as a single biweekly dose (once every two weeks) or by divided delivery (eg, two or more times every other week).

[0372] In some embodiments, the weekly dosage of the antibody drug conjugate is about 0.3 mg / kg body weight to about 1.8 mg / kg body weight administered at least once every two weeks as a weekly administration (once a week) or by split delivery (e.g., twice or more per week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every two weeks is about 0.4 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more per week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every two weeks is about 0.5 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more per week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every two weeks is about 0.6 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more per week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every two weeks is about 0.7 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., two or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every two weeks is about 0.8 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., two or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every two weeks is about 0.9 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., two or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every two weeks is about 1.0 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., two or more times a week). In some embodiments, the weekly dosage of an antibody drug conjugate administered at least once every two weeks will be about 1.1 mg / kg body weight administered as a weekly dose (once per week) or by split delivery (e.g., two or more times per week).In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every two weeks is about 1.2 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., two or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every two weeks is about 1.3 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., two or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every two weeks is about 1.4 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., two or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every two weeks is about 1.5 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., two or more times a week). In some embodiments, the weekly dosage of an antibody drug conjugate administered at least once every two weeks is about 1.6 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., two or more times a week). In some embodiments, the weekly dosage of an antibody drug conjugate administered at least once every two weeks is about 1.7 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., two or more times a week). In some embodiments, the weekly dosage of an antibody drug conjugate administered at least once every two weeks is about 1.8 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., two or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every two weeks will be 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3 mg / kg of the subject's body weight.

[0373] In some embodiments, the weekly dosage of the antibody drug conjugate is about 0.3 mg / kg body weight to about 3.3 mg / kg body weight administered at least once every three weeks as a weekly administration (once a week) or as a split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 0.4 mg / kg body weight administered at least once a week (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 0.5 mg / kg body weight administered at least once a week (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 0.6 mg / kg body weight administered at least once a week (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 0.7 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 0.8 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 0.9 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 1.0 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks will be about 1.1 mg / kg body weight administered as a weekly dose (once per week) or by divided delivery (e.g., two or more times per week).In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 1.2 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 1.3 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 1.4 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 1.5 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 1.6 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 1.7 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 1.8 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 1.9 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of an antibody drug conjugate administered at least once every three weeks will be about 2.0 mg / kg body weight administered as a weekly dose (once per week) or by divided delivery (e.g., two or more times per week).In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 2.1 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 2.2 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 2.3 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 2.4 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 2.5 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 2.6 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 2.7 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks is about 2.8 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks will be about 2.9 mg / kg body weight administered as a weekly dose (once per week) or by divided delivery (e.g., two or more times per week).In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks will be about 3.0 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks will be about 3.1 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks will be about 3.2 mg / kg body weight administered as a weekly administration (once a week) or by split delivery (e.g., twice or more times a week). In some embodiments, the weekly dosage of the antibody drug conjugate administered at least once every three weeks will be 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.71.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3 mg / kg of the subject's body weight.

[0374] A treatment cycle with a one-week break between two-week (14-day) treatment cycles may also be referred to as a three-week (21-day) treatment cycle, in which the antibody drug conjugate is administered for two out of three weeks of the three-week treatment cycle. Similarly, a treatment cycle with a one-week break between three-week (21-day) treatment cycles may also be referred to as a four-week (28-day) treatment cycle, in which the antibody drug conjugate is administered for three out of four weeks of the four-week treatment cycle. Thus, in some embodiments, the dosage is administered weekly, either as a split delivery or as a weekly administration, for two out of three weeks of the three-week treatment cycle, or the dosage is administered weekly, either as a split delivery or as a weekly administration, for three out of four weeks of the four-week treatment cycle. In some embodiments, the dosage is administered as a weekly administration on days 1 and 8 of a 14 day treatment cycle, the dosage is administered as a weekly administration on days 1 and 8 of a 21 day treatment cycle, or the dosage is administered as a weekly administration on days 1 and 8 of a 28 day treatment cycle. Preferably, the weekly dosage is administered as a split delivery or as a weekly administration for two or more four week treatment cycles, more preferably for three or more, four or more, five or more, or even six or more four week treatment cycles (e.g., 2, 3, 4, 5, or 6 consecutive treatment cycles). In some embodiments, the weekly dosage is administered for no more than three, no more than four, no more than five, or no more than six treatment cycles.

[0375] For example, in some embodiments, the dosing regimen is a weekly dose, either as divided deliveries or as a once weekly administration, with a total weekly dose of about 0.8 mg / kg body weight to about 1.8 mg / kg body weight, about 0.9 mg / kg body weight to about 1.8 mg / kg body weight, about 1.0 mg / kg body weight to about 1.8 mg / kg body weight, about 1.1 mg / kg body weight to about 1.8 mg / kg body weight, about 1.2 mg / kg body weight to about 1.8 mg / kg body weight, about 1.3 mg / kg body weight to about 1.8 mg / kg body weight, about 1.5 mg / kg body weight to about 1.8 mg / kg body weight, about 1.6 mg / kg body weight to about 1.8 mg / kg body weight, about 1.7 mg / kg body weight to about 1.8 mg / kg body weight, about 1.8 ... mg / kg body weight, about 1.4 mg / kg body weight to about 1.8 mg / kg body weight, about 1.5 mg / kg body weight to about 1.8 mg / kg body weight, about 1.6 mg / kg body weight to about 1.8 mg / kg body weight, about 1.7 mg / kg body weight to about 1.8 mg / kg body weight, or about 1.8 mg / kg body weight of the antibody drug conjugate is administered one week out of two weeks for at least one two week treatment cycle, or two weeks out of three weeks for at least two three week treatment cycles.

[0376] For example, in some embodiments, the dosing regimen is a weekly dose, either as divided deliveries or as a once weekly administration, with a total weekly dose of 0.8 mg / kg body weight to about 3.3 mg / kg body weight, about 0.9 mg / kg body weight to about 3.3 mg / kg body weight, about 1.0 mg / kg body weight to about 3.3 mg / kg body weight, about 1.1 mg / kg body weight to about 3.3 mg / kg body weight, about 1.2 mg / kg body weight to about 3.3 mg / kg body weight, About 1.3 mg / kg body weight to about 3.3 mg / kg body weight, about 1.4 mg / kg body weight to about 3.3 mg / kg body weight, about 1.5 mg / kg body weight to about 3.3 mg / kg body weight, about 1.6 mg / kg body weight to about 3.3 mg / kg body weight, about 1.7 mg / kg body weight to about 3.3 mg / kg body weight, about 1.8 mg / kg body weight to about 3.3 mg / kg body weight, about 1.9 mg / kg body weight to about 3.3 mg / kg body weight, about 2.0 mg / kg body weight to about 3.3 mg / kg body weight Approx. 3.3 mg / kg body weight, approx. 2.1 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.2 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.3 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.4 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.5 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.6 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.7 mg / kg body weight ~ approx. 3.3 mg / kg body weight, About 2.8 mg / kg body weight to about 3.3 mg / kg body weight, about 2.9 mg / kg body weight to about 3.3 mg / kg body weight, about 3.0 mg / kg body weight to about 3.3 mg / kg body weight, about 3.1 mg / kg body weight to about 3.3 mg / kg body weight, about 3.2 mg / kg body weight to about 3.3 mg / kg body weight, or about 3.3 mg / kg body weight of the antibody drug conjugate is administered 2 weeks out of 3 weeks for at least one 3 week treatment cycle. In some embodiments, the weekly dosage of the antibody drug conjugate administered two weeks out of three will be 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3 mg / kg of the subject's body weight.

[0377] In some embodiments, the dosing regimen is a weekly dose, either as divided deliveries or as a once weekly administration, with a total weekly dose of 0.8 mg / kg body weight to about 3.3 mg / kg body weight, about 0.9 mg / kg body weight to about 3.3 mg / kg body weight, about 1.0 mg / kg body weight to about 3.3 mg / kg body weight, about 1.1 mg / kg body weight to about 3.3 mg / kg body weight, about 1.2 mg / kg body weight to about 3.3 mg / kg body weight, about 1. 3mg / kg body weight to about 3.3mg / kg body weight, about 1.4mg / kg body weight to about 3.3mg / kg body weight, about 1.5mg / kg body weight to about 3.3mg / kg body weight, about 1.6mg / kg body weight to about 3.3mg / kg body weight, about 1.7mg / kg body weight to about 3.3mg / kg body weight, about 1.8mg / kg body weight to about 3.3mg / kg body weight, about 1.9mg / kg body weight to about 3.3mg / kg body weight, about 2.0mg / kg body weight to about 3 .3mg / kg body weight, approx. 2.1mg / kg body weight ~ approx. 3.3mg / kg body weight, approx. 2.2mg / kg body weight ~ approx. 3.3mg / kg body weight, approx. 2.3mg / kg body weight ~ approx. 3.3mg / kg body weight, approx. 2.4mg / kg body weight kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.5 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.6 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.7 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.8 mg / kg body weight to about 3.3 mg / kg body weight, about 2.9 mg / kg body weight to about 3.3 mg / kg body weight, about 3.0 mg / kg body weight to about 3.3 mg / kg body weight, about 3.1 mg / kg body weight to about 3.3 mg / kg body weight, about 3.2 mg / kg body weight to about 3.3 mg / kg body weight, or about 3.3 mg / kg body weight of the antibody drug conjugate is administered 3 weeks out of 4 weeks for at least 2 4-week treatment cycles.

[0378] In some embodiments, the dosing regimen is a weekly dose, either as divided deliveries or as a once weekly administration, with a total weekly dose of 0.8 mg / kg body weight to about 3.3 mg / kg body weight, about 0.9 mg / kg body weight to about 3.3 mg / kg body weight, about 1.0 mg / kg body weight to about 3.3 mg / kg body weight, about 1.1 mg / kg body weight to about 3.3 mg / kg body weight, about 1.2 mg / kg body weight to about 3.3 mg / kg body weight, about 1.3 mg / kg body weight to about 3.3 mg / kg body weight, about 1.4 mg / kg body weight to about 3.3 mg / kg body weight, about 1.5 mg / kg body weight to about 3.3 mg / kg body weight, about 1.6 mg / kg body weight to about 3.3 mg / kg body weight, about 1.7 mg / kg body weight to about 3.3 mg / kg body weight, about 1.8 mg / kg body weight to about 3.3 mg / kg body weight, about 1.9 mg / kg body weight to about 3.3 mg / kg body weight, about 2.0 mg / kg body weight to about 3.3 mg / kg body weight, approx. 2.1 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.2 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.3 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.4 mg / kg body weight Weight ~ approx. 3.3 mg / kg body weight, approx. 2.5 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.6 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.7 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.8 2.9 mg / kg body weight to about 3.3 mg / kg body weight, about 3.0 mg / kg body weight to about 3.3 mg / kg body weight, about 3.1 mg / kg body weight to about 3.3 mg / kg body weight, about 3.2 mg / kg body weight to about 3.3 mg / kg body weight, or about 3.3 mg / kg body weight of the antibody drug conjugate is administered for 2 weeks, 1, 2, 3, 4 or 5 of 3 weeks of 4 week treatment cycles (e.g., 4 weekly doses for 6 weeks, 6 weekly doses for 9 weeks, 8 weekly doses for 12 weeks).

[0379] In some embodiments, the dosing regimen is a weekly dose, either as divided deliveries or as a once weekly administration, with a total weekly dose of 0.8 mg / kg body weight to about 3.3 mg / kg body weight, about 0.9 mg / kg body weight to about 3.3 mg / kg body weight, about 1.0 mg / kg body weight to about 3.3 mg / kg body weight, about 1.1 mg / kg body weight to about 3.3 mg / kg body weight, about 1.2 mg / kg body weight to about 3.3 mg / kg body weight, About 1.3 mg / kg body weight to about 3.3 mg / kg body weight, about 1.4 mg / kg body weight to about 3.3 mg / kg body weight, about 1.5 mg / kg body weight to about 3.3 mg / kg body weight, about 1.6 mg / kg body weight to about 3.3 mg / kg body weight, about 1.7 mg / kg body weight to about 3.3 mg / kg body weight, about 1.8 mg / kg body weight to about 3.3 mg / kg body weight, about 1.9 mg / kg body weight to about 3.3 mg / kg body weight, about 2.0 mg / kg body weight to about 3.3 mg / kg body weight kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.1 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.2 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.3 mg / kg body weight ~ approx. 3.3 mg / kg body weight Weight, approx. 2.4 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.5 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.6 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.7 mg / kg body weight ~ approx. 3 0.3 mg / kg body weight, about 2.8 mg / kg body weight to about 3.3 mg / kg body weight, about 2.9 mg / kg body weight to about 3.3 mg / kg body weight, about 3.0 mg / kg body weight to about 3.3 mg / kg body weight, about 3.1 mg / kg body weight to about 3.3 mg / kg body weight, about 3.2 mg / kg body weight to about 3.3 mg / kg body weight, or about 3.3 mg / kg body weight of the antibody drug conjugate is optionally administered 3 out of every 4 weeks for 1, 2, 3, 4, or 5 4 week treatment cycles (e.g., 6 weekly doses for an 8 week period, 9 weekly doses for a 12 week period, or 12 weekly doses for a 16 week period). In some embodiments, each dose of antibody drug conjugate will be 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3 mg / kg of the subject's body weight.

[0380] In some preferred embodiments, the dosing regimen is a biweekly dose, either as a divided delivery or as a once weekly administration, with a two week total dose of 0.8 mg / kg body weight to about 3.3 mg / kg body weight, about 0.9 mg / kg body weight to about 3.3 mg / kg body weight, about 1.0 mg / kg body weight to about 3.3 mg / kg body weight, about 1.1 mg / kg body weight to about 3.3 mg / kg body weight, about 1.2 mg / kg body weight to about 3.3 mg / kg body weight, about 1.4 mg / kg body weight to about 3.3 mg / kg body weight, about 1.5 mg / kg body weight to about 3.3 mg / kg body weight, about 1.6 mg / kg body weight to about 3.3 mg / kg body weight, about 1.7 mg / kg body weight to about 3.3 mg / kg body weight, about 1.8 mg / kg body weight to about 3.3 mg / kg body weight, about 1.9 ... kg body weight, about 1.3 mg / kg body weight to about 3.3 mg / kg body weight, about 1.4 mg / kg body weight to about 3.3 mg / kg body weight, about 1.5 mg / kg body weight to about 3.3 mg / kg body weight, about 1.6 mg / kg body weight to about 3.3 mg / kg body weight, about 1.7 mg / kg body weight to about 3.3 mg / kg body weight, about 1.8 mg / kg body weight to about 3.3 mg / kg body weight, about 1.9 mg / kg body weight to about 3.3 mg / kg body weight, about 2.0 ... mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.1 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.2 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.3 mg / kg body weight ~ approx. 3.3 mg / k g body weight, approx. 2.4 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.5 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.6 mg / kg body weight ~ approx. 3.3 mg / kg body weight, approx. 2.7 mg / kg body weight ~ approx. 3.3 mg / kg body weight, about 2.8 mg / kg body weight to about 3.3 mg / kg body weight, about 2.9 mg / kg body weight to about 3.3 mg / kg body weight, about 3.0 mg / kg body weight to about 3.3 mg / kg body weight, about 3.1 mg / kg body weight to about 3.3 mg / kg body weight, about 3.2 mg / kg body weight to about 3.3 mg / kg body weight, or about 3.3 mg / kg body weight of the antibody drug conjugate, optionally 3 out of every 4 biweeks, for 1, 2, 3, 4, or 5 biweekly treatment cycles (e.g., 6 biweekly doses in a 14 week period, 9 biweekly doses in a 20 week period, or 12 biweekly doses in a 26 week period).In some embodiments, each dose of antibody drug conjugate administered every other week is administered at 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, or 3.3 mg / kg of the subject's body weight.

[0381] After or during one or more treatment cycles (e.g., days 14-21 of the second treatment cycle or days 21-28 of the second treatment cycle), the subject can be evaluated (e.g., through clinical or diagnostic testing) to determine whether the subject should remain on the treatment schedule. For example, the subject can be evaluated (e.g., clinical and / or diagnostic evaluation) after or during one or more 28-day treatment cycles (e.g., 1, 2, 3, 4, 5, or 6 28-day treatment cycles). Depending on the evaluation, the subject can discontinue treatment, continue treatment with additional treatment cycles, or begin maintenance therapy by continuing treatment until progression or unacceptable toxicity. If the subject continues treatment, the subject can be further evaluated after one or more additional treatment cycles. Depending on the continued evaluation, the subject can discontinue treatment, continue treatment with additional treatment cycles, or begin maintenance therapy by continuing treatment until progression or unacceptable toxicity.

[0382] The invention encompasses embodiments in which a subject remains in a treatment cycle (e.g., a 2-week treatment cycle or a 3-week treatment cycle) following an evaluation that indicates the subject does not have detectable cancer, e.g., following a diagnostic test that is negative for a ROR2-expressing cancer (i.e., the diagnostic test fails to detect cancer in the subject). For example, in some embodiments, the subject continues in a treatment cycle for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more treatment cycles following such an evaluation. In some embodiments, the subject continues in a treatment cycle for at least 2, 3 or less, 4 or less, 5 or less, or 6 or less treatment cycles. One example of a diagnostic test used to determine the presence or severity of cancer is positron emission tomography (PET).

[0383] In some embodiments, a subject begins maintenance therapy after one or more, preferably two or more (e.g., 1, 2, 3, 4, 5, or 6) treatment cycles (e.g., 4-week treatment cycles) by continuing treatment until progression or unacceptable toxicity. In some embodiments, a subject begins maintenance therapy after an assessment indicating little to no detectable cancer, e.g., an assessment indicating the subject has had a complete response, by continuing treatment until progression or unacceptable toxicity. Maintenance therapy, as used herein, refers to treatment with an antibody drug conjugate, but at a reduced dosing schedule, either at the same dose or at a different dose, by continuing treatment until progression or unacceptable toxicity. During maintenance therapy, the antibody drug conjugate is preferably administered at least once per 2-week treatment period, once per 3-week treatment period, on days 1 and 8 of a 2-week treatment period, or on days 1 and 8 of a 3-week treatment period. After these maintenance therapy cycles, the subject can be further evaluated (e.g., by clinical or diagnostic tests) to determine whether the subject should continue maintenance therapy, continue regular treatment, or discontinue treatment. In some embodiments, maintenance therapy is administered once every 2 to 4 weeks, or once every 3 to 6 weeks. The dosage of the antibody drug conjugate administered during maintenance therapy can be, for example, in the range of about 0.3 mg / kg body weight to about 3.3 mg / kg body weight, preferably about 0.6 mg / kg body weight to about 1.8 mg / kg body weight, preferably about 1.2 mg / kg body weight to about 2.0 mg / kg body weight, more preferably about 1.5 mg / kg body weight to about 2.4 mg / kg body weight per administration, with 1.8 mg / kg body weight being an exemplary dosage.

[0384] In some embodiments, after completion of weekly or biweekly treatment and evaluation at a dose of antibody drug conjugate of about 0.3 mg / kg body weight to about 3.3 mg / kg body weight, more preferably about 1.5 mg / kg body weight to about 1.8 mg / kg body weight, the subject is initiated into a maintenance therapy comprising administering the antibody drug conjugate once every 2-4 weeks or once every 3-6 weeks at a dose of about 0.3 mg / kg body weight to about 3.3 mg / kg body weight, preferably about 0.6 mg / kg body weight to about 1.8 mg / kg body weight, preferably about 1.2 mg / kg body weight to about 2.0 mg / kg body weight, more preferably about 1.5 mg / kg body weight to about 2.4 mg / kg body weight, about 1.8 mg / kg body weight, preferably administered biweekly.

[0385] In some embodiments, after completion of treatment (e.g., 1, 2, 3, 4 or 5 treatment cycles), the subject is initiated on a biweekly dosing schedule (e.g., dosing on day 1 of a 2-week maintenance therapy cycle) and the dose of antibody drug conjugate is about 0.3 mg / kg body weight to about 3.3 mg / kg body weight, preferably about 0.6 mg / kg body weight to about 1.8 mg / kg body weight, preferably about 1.2 mg / kg body weight to about 2.0 mg / kg body weight, more preferably about 1.5 mg / kg body weight to about 2.4 mg / kg body weight, with about 1.8 mg / kg body weight being preferred.

[0386] In some embodiments, after completion of treatment (e.g., 1, 2, 3, 4 or 5 treatment cycles), the subject is initiated on a once every 3 weeks dosing schedule (e.g., dosing on day 1 of a 3 week maintenance therapy cycle) and the dose of antibody drug conjugate is about 0.3 mg / kg body weight to about 3.3 mg / kg body weight, preferably about 0.6 mg / kg body weight to about 1.8 mg / kg body weight, preferably about 1.2 mg / kg body weight to about 2.0 mg / kg body weight, more preferably about 1.5 mg / kg body weight to about 2.4 mg / kg body weight, with about 1.8 mg / kg body weight being preferred.

[0387] The invention includes embodiments in which the subject is administered a weekly dose of antibody drug conjugate, either as a divided delivery or as a once weekly administration, for a total weekly dosage of about 0.3 mg / kg body weight to about 3.3 mg / kg body weight, about 0.6 mg / kg body weight to about 1.8 mg / kg body weight, about 1.2 mg / kg body weight to about 2.0 mg / kg body weight, about 1.5 mg / kg body weight to about 2.4 mg / kg body weight, with about 1.8 mg / kg body weight being preferred, with 1, 2, 3, 4, 5, or 6 21 day treatment cycles for 2 weeks out of 3 weeks, followed by 2 or more maintenance therapy cycles in which the antibody drug conjugate is administered at a dosage of about 0.4 mg / kg body weight to about 2 mg / kg body weight, about 0.6 mg / kg body weight to about 2.0 mg / kg body weight, or about 0.8 mg / kg body weight to about 1.8 mg / kg body weight every 2 to 4 weeks, preferably every 2 weeks. In some embodiments, the weekly administration is 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more treatment cycles, and the biweekly administration is 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more maintenance therapy cycles. In some embodiments, the (bi)weekly administration cycle is 2, 3, 4, 5, or 6 or fewer treatment cycles.

[0388] The invention includes embodiments in which a subject is administered a weekly dose of antibody drug conjugate, either as a divided delivery or as a once weekly administration, for a total weekly dosage of about 0.3 mg / kg to about 3.3 mg / kg body weight, about 0.6 mg / kg to about 1.8 mg / kg body weight, about 1.2 mg / kg to about 2.0 mg / kg body weight, or about 1.5 mg / kg to about 2.4 mg / kg body weight, for 3 out of 4 weeks, for 1, 2, 3, 4, 5, or 6 28 day treatment cycles, followed by 2 or more maintenance therapy cycles in which the anti-antibody drug conjugate is administered every 3-6 weeks, preferably every 3 weeks, in a dosage of about 0.3 mg / kg to about 3.3 mg / kg body weight, about 0.6 mg / kg to about 1.8 mg / kg body weight, or about 1.2 mg / kg to about 2.0 mg / kg body weight. In some embodiments, the weekly dosing cycles result in 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more treatment cycles, and the once every three weeks dosing schedule results in 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more maintenance therapy cycles. In some embodiments, the once every three weeks dosing cycle results in no more than 2, 3, 4, 5, or 6 treatment cycles.

[0389] The invention includes embodiments in which a subject is administered a weekly dose of antibody drug conjugate, either as a split delivery or as a weekly administration, with a total weekly dose of about 0.8 mg / kg patient body weight to about 1.8 mg / kg body weight, for 2 out of 3 weeks (e.g., days 1 and 8 of a 21-day treatment cycle) for 1, 2, 3, 4, 5, or 6 21-day treatment cycles, followed by administration of about 1.8 mg / kg body weight of antibody drug conjugate every 2 to 4 weeks, preferably every 2 weeks, for 2 or more maintenance therapy cycles (e.g., about 1.8 mg / kg body weight every 2 weeks for 2 or more 2-week maintenance therapy cycles). Preferably, the biweekly dosage of antibody drug conjugate is about 0.8 mg / kg body weight for 2 or more maintenance therapy cycles (e.g., about 0.8 mg / kg body weight every 2 weeks for 2 or more 2-week maintenance therapy cycles).

[0390] The invention includes embodiments in which a subject is administered a weekly dose of antibody drug conjugate, either as a split delivery or as a weekly administration, with a total weekly dosage of about 0.8 mg / kg to about 1.8 mg / kg of subject body weight, for 1, 2, 3, 4, 5, or 6 28-day treatment cycles on 3 weeks out of 4 weeks (e.g., days 1 and 8 of a 28-day treatment cycle), and then every 3-6 weeks thereafter. The once-every-3-week dose of antibody drug conjugate can be at a dose of about 1.8 mg / kg body weight for 2 or more maintenance cycles (e.g., once-every-3-week dose of about 1.8 mg / kg body weight for 2 or more 3-week maintenance cycles). The once-every-3-week dose of antibody drug conjugate can be at a dose of about 0.8 mg / kg body weight for 2 or more maintenance cycles (e.g., once-every-3-week dose of about 0.8 mg / kg body weight for 2 or more 3-week maintenance cycles).

[0391] The present invention relates to a method for the preparation of a mAb comprising administering to a subject the mAb BA301 cleavable linker-MMAE of the present invention. (n) These include embodiments in which patients are treated with an antibody-drug conjugate, but on a schedule other than a (bi)weekly dosing regimen (e.g., the antibody drug conjugate is administered at a dosage of about 1.8 mg / kg body weight every one or two weeks for one or more than two week treatment cycles, or once every three weeks for three or more than two week treatment cycles), and switch to a weekly dosing regimen as described herein for no more than one, two, three, four, five, or six treatment cycles. Following the weekly dosing regimen, patients can optionally begin maintenance therapy as described herein.

[0392] The antibody drug conjugate is preferably administered as a monotherapy. The term "monotherapy" means that the antibody drug conjugate is the only anti-cancer agent administered to the subject during a treatment cycle. However, other therapeutic agents can also be administered to the subject as described herein. For example, a programmed death receptor-1 (PD-1) blocking antibody, granulocyte colony stimulating factor or analogs thereof can be co-administered with the antibody drug conjugate. In addition, anti-inflammatory or other agents can also be administered to the subject with cancer during the monotherapy period to treat symptoms associated with the cancer, rather than the underlying cancer itself, such as inflammation, pain, weight loss, general malaise, etc. The subject treated with the method of the present invention preferably has completed pretreatment with an anti-cancer agent before administration of the anti-antibody drug conjugate. In some embodiments, the subject has completed pretreatment with an anti-cancer agent at least one week (preferably 2, 3, 4, 5, 6, 7, or 8 weeks) prior to treatment with the antibody drug conjugate. The subject also preferably does not undergo treatment with an additional anti-cancer agent for at least 2 weeks (preferably at least 3, 4, 5, 6, 7, or 8 weeks) after the end of the first treatment cycle with the antibody drug conjugate, preferably at least 2 weeks (preferably at least 3, 4, 5, 6, 7, or 8 weeks) after the end of the final administration of the antibody drug conjugate. The method of the present invention encompasses administering to a subject an anti-ROR2 antibody or antibody fragment of the present invention, or an immunoconjugate comprising an anti-ROR2 antibody or antibody fragment, for the treatment of a ROR2-expressing tumor.

[0393] In some embodiments, an immunoconjugate comprising an anti-ROR2 antibody or antibody fragment of the invention is administered to a subject, and after the anti-ROR2 antibody binds to a ROR2-expressing tumor cell, the antibody drug conjugate is internalized within the cell and the drug is released. For example, the method of the invention includes administering BA3021-cleavable linker-MMAE to a subject for treatment of a ROR2-expressing tumor. (n) and administering the antibody drug conjugate to the subject. After conjugation, the subject is administered BA3021-cleavable linker-MMAE. (n)The antibody-drug conjugate is internalized into tumor cells, where the peptide linker is cleaved by proteases to release MMAE. By specifically delivering MMAE to tumor cells expressing ROR2, further tumor cell proliferation is inhibited, and tumor shrinkage is expected.

[0394] The subject to be treated with the method of the present invention is a subject diagnosed with or suspected of having a ROR2-expressing cancer. Diagnosis can be made by methods known in the art, including, for example, tissue biopsy.

[0395] G. Products and Kits In another aspect of the invention, an article of manufacture is provided that contains an anti-ROR2 antibody or antibody fragment and other materials useful for the treatment, prevention, and / or diagnosis of the above disorders. 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, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds the composition 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 solution bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody or antibody fragment of the invention. The label or package insert indicates that the composition is used to treat a selected condition. Additionally, the article of manufacture can include (a) a first container containing therein a composition comprising an antibody or antibody fragment; and (b) a second container containing therein a composition comprising an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the invention can 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 comprise a second (or third) container containing a pharma- ceutically 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, such as other buffers, diluents, filters, needles, and syringes.

[0396] It is understood that any of the above products may contain an immunoconjugate of the invention instead of, or in addition to, an anti-ROR2 antibody or antibody fragment.

[0397] Finally, the present invention also provides a kit comprising at least one of the antibodies or antibody fragments of the present invention. The kit containing the polypeptide, antibody or antibody fragment, or antibody drug conjugate of the present invention is used in detecting ROR2 expression (increase or decrease) or in therapeutic or diagnostic assays. The kit of the present invention may contain an antibody coupled to a solid support, such as a tissue culture plate or beads (e.g., sepharose beads). A kit containing an antibody for in vitro detection and quantification of ROR2, 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 or radioactive label.

[0398] The kit further contains instructions for its use. In some embodiments, the instructions include those required by the US Food and Drug Administration for in vitro diagnostic kits. In some embodiments, the kit further includes instructions for diagnosing the presence or absence of cerebrospinal fluid in a sample based on the presence or absence of ROR2 in said sample. In some embodiments, the kit includes one or more antibodies or antibody fragments. In other embodiments, the kit further includes one or more enzymes, enzyme inhibitors, or enzyme activators. In still other embodiments, the kit further includes one or more chromatography compounds. In still other embodiments, the kit further includes one or more compounds used to prepare samples for spectrophotometric assays. In further embodiments, the kit further includes a comparative reference material for interpreting the presence or absence of ROR2 according to the intensity, color spectrum, or other physical attributes of the indicator.

[0399] H. Cell membrane scoring of ROR2 in tumors (Tumor Membrane P Score) In tumor cells, ROR2 is primarily localized to the plasma membrane, but is also found in the cytoplasm.

[0400] In the present invention, a scoring method is used to compare ROR2 staining in serial sections of each specimen. Using this method, ROR2 cell membrane staining is scored in tumor cells only, providing a ROR2 tumor score (hereinafter "Tumor Membrane Percent Score" or (TmPS)).

[0401] The approach used to score ROR2 is described below, which may be detected by methods including, but not limited to, immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded (FFPE) tumor specimens. All specimens are also stained with hematoxylin and eosin (H&E) for morphological evaluation to aid in scoring.

[0402] ROR2 membrane expression in tumors is scored semiquantitatively for membrane staining (complete or partial) and cytoplasmic staining (diffuse or granular). Membrane and cytoplasmic reactivity are scored separately. For ROR2 membrane staining, the percentage of differential intensity, H score, and percent score ≥ 1+ are the main components of scoring, as shown below. For ROR2 cytoplasmic staining, the main components of scoring are the percentage of differential intensity and H score.

[0403] When scoring tumor tissue for ROR2, surrounding staining in the stroma, non-tumor areas, and adjacent normal tissue is excluded and scored numerically. Areas of ischemia or necrosis are not scored in any cancer indication. Percentage scores are assigned to represent the penetration of cell membrane staining per specimen. Percentages are estimated and reported in increments including, but not limited to, any of the following increments: 0, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, 99, or 100%. In certain embodiments of the invention, ROR2-expressing tumors have a TmPS of at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, or at least 95%.

[0404] To fully understand ROR2 expression on the cell membrane in non-macrophage tumors across cancer indications, we use both standard percent score and H-score approaches to capture the patterns of reactivity observed.

[0405] Both approaches require recording the percentage of tumor cells with ROR2 staining at the corresponding differential intensity using a 4-point semiquantitative scale (0, 1+, 2+, 3+), where 0=null, negative, or nonspecific staining, 1+=low or weak staining, 2+=moderate or moderate staining, and 3+=high or strong staining.

[0406] Percentage score method A percentile score is calculated by summing the percentage of intensities ≥1+, ≥2+, or ≥3+, and thus the score ranges from 0 to 100. Percent score 1+ or greater = (% of 1+) + (% of 2+) + (% of 3+) Percent score 2+ or greater = (% of 2+) + (% of 3+) Percentage score 3+ or greater = (% at 3+).

[0407] H-score method The H score is calculated by summing the percentage of cells with expression intensity (brown staining) multiplied by the corresponding differential intensity on a 4-point semiquantitative scale (0, 1+, 2+, 3+), thus the score ranges from 0 to 300. H-Score = [(% below 1) x 0] + [(% 1+) x 1] + [(% 2+) x 2] + [(% 3+) x 3]

[0408] Analysis of ROR2 expression in human cancer tissues is semiquantitative, with numerical scoring data providing a measure of reactivity: a specimen is considered "positive" for ROR2 if 10% or more of the tumor cells show plasma membrane staining of intensity 1+ or higher.

[0409] The following examples are illustrative of the soft gelatin capsules of the present disclosure, but are not limiting. Other suitable modifications and adaptations of the variety of conditions and parameters normally found in the art and obvious to those skilled in the art are within the scope of the present disclosure. EXAMPLES

[0410] Example 1: Conditionally Active Biological (CAB) Antibodies Against ROR2

[0411] In this example, an antibody against human ROR2 was produced. A humanized antibody against ROR2 was used as a wild-type antibody to generate a CAB antibody against ROR2. DNA encoding the wild-type antibody (heavy and light chain variable regions) was evolved to generate a mutant antibody heavy and light chain variable region library. The mutant heavy and light chain variable regions in the library were screened for selective binding affinity to human ROR2 at pH 6.0 compared to pH 7.4 by ELISA (Figures 2A-2B and 3A-3B). At the same time, the expression level of the mutant antibody was also optimized for the purpose of providing a higher yield in the subsequent manufacturing process. Screening was performed in serum using a FLAG tag because of the presence of human antibodies in serum that may cause false positives for screening. The generated conditionally active antibodies were found to have a higher binding affinity to ROR2 at pH 6.0 than that at pH 7.4 (Tables 1 and 2).

[0412] The CAB antibody did not show aggregation in buffer as demonstrated in FIG. 4. The CAB antibody was analyzed by size exclusion chromatography. As shown in FIG. 4, only one peak was detected, demonstrating that the antibody showed little or no aggregation. FIG. 5 shows that some of the selected mutant antibodies (scFv) demonstrated higher binding affinity to ROR2 at pH 6.0 than at pH 7.4. Furthermore, FIG. 5 also shows that increasing the temperature from room temperature to 60° C. did not significantly alter the selectivity of the antibody.

[0413] All of the conditionally active antibodies exhibited high expression levels as shown in Table 4 below, where the column "Clone" indicates the antibody and the expression level in mg / ml is shown in the second column.

[0414] These antibody clones were sent to the service provider at requested expression levels ("order amounts") that represent the expected expression levels. However, the actual expression levels ("delivered amounts") of some of these antibodies were extremely high and exceeded the expected expression levels. At least three clones had expression levels that exceeded the expected expression levels (BAP048.7.067-HC-FLAG, BAP048.7-C02D09-FLAG, and BAP048.7-A03F01-FLAG). [Table 4]

[0415] Surface plasmon resonance (SPR) was also used to assay the conditionally active antibodies to measure the on and off rates for binding to ROR2. SPR assays can be used to measure the on and off rates for protein binding. The in vivo on and off rates (in animals and humans) of conditionally active antibodies are important characteristics.

[0416] It was observed that the conditionally active antibodies had good binding affinity at pH 6.0 and little or no binding affinity at pH 7.4 (FIGS. 6A-6B). SPR assays showed that those same conditionally active antibodies were highly selective at pH 6.0 compared to pH 7.4 (FIGS. 6A-6B).

[0417] Example 2: Anti-ROR2 antibodies conjugated to model toxins

[0418] The anti-ROR2 antibodies of the invention were conjugated to a model toxin (eg, paclitaxel) to generate a conditionally active antibody-drug conjugate (ROR2-CAB-ADC).

[0419] Tumors were induced in mice by injection of MDA-MB-436 tumor cells to generate xenograft mice. ROR2-CAB-ADC was then injected into the xenograft mice at a dose of 0.3 or 1 mg / kg once a week for 2 weeks. Controls used in this study included PBS buffer as vehicle and toxin alone (paclitaxel). The study showed that ROR2-CAB-ADC provided a significantly greater reduction in tumor size compared to the control (Figures 7A-7C). Results for the 0.3 mg / kg dose group are presented in Figure 7B, and results for the 1 mg / kg dose group are presented in Figure 7C. This study showed that anti-ROR2 antibodies conjugated to toxins are effective in reducing tumor size.

[0420] Example 3. pH-dependent binding affinity of conditionally active antibody BAP048

[0421] The binding affinity of one of the conditionally active antibodies identified by the present invention, BAP048 (or BAP048.7 as shown in some of the figures), was tested by pH titration. The wild-type antibody was used as a control. As shown in Figure 8, the conditionally active antibody BAP048 is more active at pH lower than pH 6.5, but less active at pH 7.0. The wild-type antibody does not show a pH dependency for its binding affinity.

[0422] Example 4. Cross-species binding affinity of conditionally active antibody BAP048

[0423] The conditionally active antibody BAP048 was selected for its conditional binding affinity to human ROR2. The conditionally active antibody was tested for its binding affinity to three targets: human ROR2 (hROR2), cynomolgus monkey ROR2 (cynoROR2), and mouse ROR2 (mROR2) using ELISA (Figure 9). The conditionally active antibody BAP048 showed nearly identical binding affinity t...

Claims

1. A pharmaceutical composition for use as a medicament, comprising a pharmaceutically acceptable carrier and an immunoconjugate for treating cancer at a dose of 0.3 mg / kg to 3.3 mg / kg of a subject's body weight administered every two or three weeks, the immunoconjugate comprises a ROR2-binding antibody or ROR2-binding antibody fragment thereof having a heavy chain variable region comprising three complementarity-determining regions having H1, H2, and H3 amino acid sequences; (a) the H1 sequence is GYTX １ TEX ２ X ３ X ４ H (SEQ ID NO: 1); (b) the H2 sequence is X ５ X ６ X ７ X ８ NNGGTGYNQKFKG (SEQ ID NO: 2); (c) The H3 sequence is ９ X １０ X １１ SX １２ YX １３ YX １４ X １５ SYFX １６ X １７ X １８ (SEQ ID NO: 3); X １ is F or E, X ２ is Y or D, X ３ is T or C, X ４ is M or D or E or Y, X ５ is G or S, X ６ is I or E, X ７ is N or C or L or V, X ８ is T, D or E, X ９ is A, M or T, X １０ is R or H, X １１ is G or E, X １２ is L or F, X １３ is S or G, X １４ is G or D, X １５ is N or E, X １６ is D or L, X １７ is Y or C or T, X １８ is W or L, and the ROR2-binding antibody or ROR2-binding antibody fragment thereof comprises a light chain variable region comprising three complementarity-determining regions having L1, L2, and L3 amino acid sequences; (d) the L1 sequence is SATSSX １９ X ２０ X ２１ MX ２２ (SEQ ID NO: 7); (e) the L2 sequence is X ２３ TSNLAS (SEQ ID NO: 8); (f) the L3 sequence is QX ２４ X ２５ SX ２６ YPFX ２７ X ２８ (SEQ ID NO: 9); X １９ is V or E, X ２０ is S or D, X ２１ is Y or C or D, X ２２ is H or G or L, X ２３ is G or C or H or P, X ２４ is Q or E, X ２５ is R or H, X ２６ is S or D or G or I or Q or V, X ２７ is T or D, X ２８ is F or D or E, However, X １ From X ２８ and cannot simultaneously be F, Y, T, M, G, I, N, T, A, R, G, L, S, G, N, D, Y, W, V, S, Y, H, G, Q, R, S, T, and F, respectively.

2. The pharmaceutical composition of claim 1, comprising an immunoconjugate at a dose of 0.3 mg / kg to 1.8 mg / kg of subject body weight.

3. The pharmaceutical composition of claim 1, comprising an immunoconjugate at a dose of 1.8 mg / kg of the subject's body weight.

4. 2. The pharmaceutical composition of claim 1, wherein the heavy chain variable region has an amino acid sequence selected from the sequences of SEQ ID NOs: 18-26, and the light chain variable region has an amino acid sequence selected from SEQ ID NOs: 13-17 and 27.

5. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding antibody fragment has a higher binding affinity for ROR2 protein in a tumor microenvironment having a pH in the range of 5.8 to 6.8 compared to the binding affinity in a non-tumor microenvironment having a pH in the range of 7.0 to 7.

6.

6. The pharmaceutical composition of claim 5, wherein the antibody or antigen-binding antibody fragment has a ratio of binding affinity for ROR2 protein at a pH of 7.0 to 7.6 in a tumor microenvironment to binding affinity for ROR2 protein at a pH of 5.8 to 6.8 in a non-tumor microenvironment of at least 1.5:1, at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, or at least 10:

1.

7. The pharmaceutical composition of claim 1, wherein the antibody or antigen-binding antibody fragment is a chimeric antibody, a multispecific antibody, a humanized antibody, or an antigen-binding fragment thereof.

8. The pharmaceutical composition of claim 1, wherein the immunoconjugate comprises at least one agent selected from a chemotherapeutic agent, a radioactive atom, a cytostatic agent, and a cytotoxic agent.

9. The pharmaceutical composition described in claim 8, wherein the antibody or its antigen-binding antibody fragment and at least one agent of the immunoconjugate are covalently bound to a linker molecule.

10. The pharmaceutical composition of claim 8, wherein the at least one drug is selected from maytansinoids, auristatins, dolastatins, calicheamicins, pyrrolobenzodiazepines, and anthracyclines.

11. The pharmaceutical composition of claim 1, wherein the immunoconjugate comprises BA3021-cleavable linker-MMAE n , where BA3021 is an antibody or antigen-binding antibody fragment having a heavy chain variable region comprising SEQ ID NO:16; and a light chain variable region comprising SEQ ID NO:21; and n is an integer from 1 to 4, inclusive.

12. A pharmaceutical composition for treating cancer, comprising a pharmaceutically acceptable carrier and an immunoconjugate at a dose of 0.3 mg / kg to 3.3 mg / kg of a subject's body weight, the immunoconjugate comprises an antibody or antigen-binding antibody fragment thereof having a heavy chain variable region comprising three complementarity determining regions having H1, H2, and H3 amino acid sequences; (a) the H1 sequence is GYTX 1 TEX 2 X 3 X 4 H (SEQ ID NO: 1); (b) the H2 sequence is X5X6X7X8NNGGTGYNQKFKG (SEQ ID NO: 2); (c) the H3 sequence is X9X10X11SX12YX13YX14X15SYFX16X17X18 (SEQ ID NO: 3); X 1 is F or E; X 2 is Y or D; X 3 is T or C; X 4 is M, D, E, or Y; X 5 is G or S; X 6 is I or E; X 7 is N, C, L or V; X 8 is T, D or E; X 9 is A, M or T; X 10 is R or H; X 11 is G or E; X 12 is L or F; X 13 is S or G; X 14 is G or D; X 15 is N or E; X 16 is D or L; X 17 is Y, C or T; X 18 is W or L, and the antibody or antigen-binding antibody fragment thereof comprises a light chain variable region comprising three complementarity determining regions having L1, L2, and L3 amino acid sequences; (d) the L1 sequence is SATSSX 19 X 20 X 21 MX 22 (SEQ ID NO: 7); (e) the L2 sequence is X 23 TSNLAS (SEQ ID NO: 8); (f) the L3 sequence is QX24X25SX26YPFX27X28 (SEQ ID NO: 9); X 19 is V or E; X 20 is S or D; X 21 is Y, C or D; X 22 is H, G or L; X 23 is G, C, H or P; X 24 is Q or E; X 25 is R or H; X 26 is S or D or G or I or Q or V; X 27 is T or D; X 28 is F, D or E; The pharmaceutical composition, wherein X 1 to X 28 cannot simultaneously be F, Y, T, M, G, I, N, T, A, R, G, L, S, G, N, D, Y, W, V, S, Y, H, G, Q, R, S, T, and F, respectively.

13. The pharmaceutical composition of claim 12, comprising an immunoconjugate at a dose of 0.3 mg / kg to 1.8 mg / kg of subject body weight.

14. The pharmaceutical composition of claim 12, comprising an immunoconjugate at a dose of 1.8 mg / kg of subject body weight.

15. The pharmaceutical composition described in any one of claims 12 to 14, wherein the cancer is a ROR2-expressing cancer.

16. The pharmaceutical composition described in claim 15, wherein the ROR2-expressing cancer is a ROR2-expressing tumor having a tumor membrane P score of at least 1.

17. The pharmaceutical composition described in any one of claims 12 to 14, wherein the cancer is selected from sarcoma, ovarian cancer, melanoma, non-small cell lung cancer, breast cancer, and head and neck cancer.

18. The pharmaceutical composition of any one of claims 12 to 14, wherein the antibody or antigen-binding antibody fragment has a higher binding affinity for ROR2 protein in a tumor microenvironment having a pH in the range of 5.8 to 6.8 compared to the binding affinity in a non-tumor microenvironment having a pH in the range of 7.0 to 7.

6.

19. The immunoconjugate is BA3021-cleavable linker-MMAE ｎ 19. The pharmaceutical composition of claim 18, wherein BA3021 is an antibody or antigen-binding antibody fragment having a heavy chain variable region comprising SEQ ID NO: 16; and a light chain variable region comprising SEQ ID NO: 21; and n is an integer from 1 to 4, inclusive.

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