Humanized antibodies to mucin-16 and methods of use thereof

Abstract

This article provides compositions, methods, and uses of anti-mucin-16 (MUC16) agents involving epitopes of immune-specific binding mucin-16 (MUC16). This article also provides uses and methods for the administration, treatment, or prevention of disorders and diseases such as cancer associated with positive MUC16 expression.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application No. 62 / 845,065, filed May 8, 2019, the entire contents of which are incorporated herein by reference.

[0003] Government Support Statement

[0004] This invention was developed with government support under licenses P01 CA190174-01, P01 CA190174-02, and P01 CA190174-03 granted by the National Institutes of Health in the United States. The government owns certain rights to this invention. Background Technology

[0005] Mucins are important biomolecules for cell homeostasis and epithelial surface protection. Alterations in mucin expression in cancers such as ovarian cancer can be used as biomarkers for diagnosis, prognosis and treatment (Singh AP et al., Lancet Oncol 2008; 9(11):1076-85). MUC16 is a mucin overexpressed on most ovarian cancer cells and is an established alternative serum marker for the detection and progression of ovarian cancer (CA-125) (Badgwell D et al., Dis Markers 23(5-6):397410(2007); Bast RC, Jr et al., Int J Gynecol Cancer 15 Suppl 3:274-81(2005); Fritsche HA et al., Clin Chem 44(7):1379-80(1998); and Krivak TC et al., Gynecol Oncol 115(1):81-5(2009)).

[0006] MUC16 is a highly glycosylated mucin composed of a cleaved and released large extracellular domain (CA-125) and a preserved domain (MUC-CD) (Figure 1). The MUC-CD contains a non-repeating extracellular domain (MUC16 extracellular domain) near the cleavage site, a transmembrane domain, and a cytoplasmic tail region with potential phosphorylation sites. Distal to the cleavage site, the released extracellular domain (CA-125) contains 16–20 tandem repeats of 156 amino acids each, each with numerous potential glycosylation sites (O'Brien TJ et al., Tumor Biol 22(6):348–66(2001)). Because the MUC16 antigen is normally expressed at low levels only in normal tissues such as the uterus, endometrium, fallopian tubes, ovaries, and serosa of the peritoneum and thoracic cavity, MUC16 is a potentially attractive target for immunotherapy, including targeted therapy for cancer.

[0007] The majority of the extracellular domain of MUC16 is cleaved and secreted (i.e., CA-125), which limits the practicality of this portion of MUC16 as a target antigen for ovarian cancer. Many reported MUC16 monoclonal antibodies bind to epitopes on the large secretory CA-125 portion of the glycoprotein and not to the retained extracellular domain of MUC16 (Bellone S Am J Obstet Gynecol 200(1):75el-10(2009); Berek JS. Expert Opin Biol Ther.4(7):1159-65(2004); O'Brien TJ et al., Int J Biol Markers 13(4):188-95(1998)). Therefore, for diagnostic and therapeutic purposes, there is a need to generate novel antibodies targeting the unshed region of MUC16. Summary of the Invention

[0008] This article provides compositions, methods, and uses of anti-mucin 16 (MUC16) constructs comprising an antibody moiety that specifically binds to mucin 16 (MUC16) and modulates the expression and / or activity of MUC16 for the management or treatment of MUC16-mediated disorders, such as cancer.

[0009] In some embodiments, this document provides an anti-mucin 16 (MUC16) construct comprising an antibody moiety that specifically recognizes the mucin 16 (MUC16) polypeptide, wherein the antibody moiety comprises a humanized heavy chain variable domain and a humanized light chain variable domain of a 4H11 or 18C6 mouse monoclonal antibody. In some embodiments, the antibody portion comprises (a)(i) a variable heavy (VH) chain comprising heavy chain complementarity-determining regions 1 (HC-CDR1), HC-CDR2, and HC-CDR3 of SEQ ID NO:17, 18, and 19, respectively, and heavy chain framework regions 1 (HC-FW1), HC-FW2, and HC-FW3 of SEQ ID NO:136, 137, and 138, respectively, wherein one or more amino acids selected from amino acid positions 1, 3, 5, 11, and 19 of SEQ ID NO:136, amino acid positions 5, 7, 8, and 9 of SEQ ID NO:137, and amino acid positions 12, 14, 18, 22, and 23 of SEQ ID NO:138 are humanized relative to mouse HC-FW1, HC-FW2, and HC-FW3 of SEQ ID NO:124, 125, and 126, respectively; and (ii) a variable light (VL) chain comprising SEQ ID NO:124, 125, and 126, respectively. The light chain complementarity-determining regions 1 (LC-CDR1), LC-CDR2, and LC-CDR3 of NO:14, 15, and 16, and the light chain framework regions 1 (LC-FW1), LC-FW2, LC-FW3, and LC-FW4 of SEQ ID NO:120, 121, 122, and 123, respectively, wherein one or more amino acids selected from positions 3, 9, 15, 18, and 22 of SEQ ID NO:120, amino acid positions 7 and 27 of SEQ ID NO:122, and amino acid positions 3 and 9 of SEQ ID NO:123 are humanized relative to mouse LC-FW1, LC-FW2, LC-FW3, and LC-FW4 of SEQ ID NO:104, 105, 106, and 107, respectively; or (b)(i) comprising a variable heavy (VH) chain of SEQ ID NO:4 or 5; and (ii) comprising a variable light (VL) chain of SEQ ID NO:2 or 3;Or (c)(i) a variable heavy (VH) chain comprising heavy chain complementarity-determining regions 1 (HC-CDR1), HC-CDR2, and HC-CDR3 of SEQ ID NO:35, 36, and 37, respectively, and heavy chain framework regions 1 (HC-FW1), HC-FW2, HC-FW3, and HC-FW4 of SEQ ID NO:175, 176, 177, and 178, respectively, wherein one or more amino acids selected from amino acid positions 10, 11, 12, 13, 15, 19, and 23 of SEQ ID NO:175, amino acid positions 5, 14, 16, 18, 22, and 23 of SEQ ID NO:177, and amino acid position 6 of SEQ ID NO:178, respectively, are relative to SEQ ID NO:35, 36, and 37, respectively. (ii) humanized mouse HC-FW1, HC-FW2, HC-FW3, and HC-FW4 of SEQ ID NO:159, 160, 161, and 162; and (ii) a variable light (VL) chain comprising light chain complementarity-determining regions 1 (LC-CDR1), LC-CDR2, and LC-CDR3 of SEQ ID NO:32, 33, and 34, respectively, and light chain framework regions 1 (LC-FW1), LC-FW2, LC-FW3, and LC-FW4 of SEQ ID NO:155, 156, 157, and 158, respectively, wherein one or more amino acids selected from positions 7, 9, 11, and 18 of SEQ ID NO:155, amino acid position 5 of SEQ ID NO:156, and amino acid positions 9 and 18 of SEQ ID NO:157 are relative to SEQ ID NO:159, 160, 161, and 162, respectively. Humanization of mouse LC-FW1, LC-FW2, LC-FW3, and LC-FW4 of NO:139, 140, 141, and 142; or (d)(i) comprising a variable heavy (VH) chain of SEQ ID NO:22 or 23; and (ii) comprising a variable light (VL) chain of SEQ ID NO:20 or 21.

[0010] In some embodiments, the HC-FW1 of (a)(i) contains SEQ ID NO: 130; the HC-FW2 of (a)(i) contains SEQ ID NO: 131; the HC-FW3 of (a)(i) contains SEQ ID NO: 132; the LC-FW1 of (a)(ii) contains SEQ ID NO: 112; the LC-FW2 of (a)(ii) contains SEQ ID NO: 113; the LC-FW3 of (a)(ii) contains SEQ ID NO: 114; and / or the LC-FW4 of (a)(ii) contains SEQ ID NO: 115. In some embodiments, the HC-FW1 of (a)(i) contains SEQ ID NO: 133; the HC-FW2 of (a)(i) contains SEQ ID NO: 134; the HC-FW3 of (a)(i) contains SEQ ID NO: 135; the LC-FW1 of (a)(ii) contains SEQ ID NO: 116; the LC-FW2 of (a)(ii) contains SEQ ID NO: 117; the LC-FW3 of (a)(ii) contains SEQ ID NO: 118; and / or the LC-FW4 of (a)(ii) contains SEQ ID NO: 119.

[0011] In some embodiments, the HC-FW1 of (c)(i) contains SEQ ID NO: 167; the HC-FW2 of (c)(i) contains SEQ ID NO: 168; the HC-FW3 of (c)(i) contains SEQ ID NO: 169; the HC-FW4 of (c)(i) contains SEQ ID NO: 170; the LC-FW1 of (c)(ii) contains SEQ ID NO: 147; the LC-FW2 of (c)(ii) contains SEQ ID NO: 148; the LC-FW3 of (c)(ii) contains SEQ ID NO: 149; and / or the LC-FW4 of (c)(ii) contains SEQ ID NO: 150. In some embodiments, the HC-FW1 of (c)(i) contains SEQ ID NO:171; the HC-FW2 of (c)(i) contains SEQ ID NO:172; the HC-FW3 of (c)(i) contains SEQ ID NO:173; the HC-FW4 of (c)(i) contains SEQ ID NO:174; the LC-FW1 of (c)(ii) contains SEQ ID NO:151; the LC-FW2 of (c)(ii) contains SEQ ID NO:152; the LC-FW3 of (c)(ii) contains SEQ ID NO:153; and / or the LC-FW4 of (c)(ii) contains SEQ ID NO:154.

[0012] In some embodiments, the antibody portion immune-specifically recognizes human MUC16. In some embodiments, the antibody portion immune-specifically recognizes human MUC16 of SEQ ID NO:53. In some embodiments, the antibody portion immune-specifically binds to the MUC16 c114 polypeptide comprising the amino acid sequence of SEQ ID NO:44. In some embodiments, the MUC16 is glycosylated. In some embodiments, the MUC16 is N-glycosylated at Asn1800 or Asn1806. In some embodiments, the antibody portion of the anti-adhesion protein 16 (MUC16) construct provided herein comprises (a)(i) a heavy chain containing SEQ ID NO:12 or 13 and (ii) a light chain containing SEQ ID NO:10 or 11; or (b)(i) a heavy chain containing SEQ ID NO:30 or 31 and (ii) a light chain containing SEQ ID NO:28 or 29.

[0013] In some embodiments, the antibody moiety of the anti-mucoprotein 16 (MUC16) construct provided herein binds immunospecifically to the extracellular domain of MUC16. In some embodiments, the antibody moiety is a full-length antibody, Fab, Fab', F(ab')2, Fv, or a single-chain Fv (scFv). In some embodiments, the antibody moiety is a single-chain Fv (scFv), and the scFv comprises any one of SEQ ID NO: 53-68. In some embodiments, the VH chain and the VL chain are human VH and VL chains. In some embodiments, the antibody moiety is a monoclonal antibody.

[0014] In some embodiments, the anti-MUC16 construct provided herein inhibits the in vitro invasion of MUC16-expressing tumor cells in a Matrigel invasion assay. In some embodiments, the tumor cells are ovarian tumor cells.

[0015] In some embodiments, the antibody moiety comprises human-derived heavy and light chain constant regions. In some embodiments, the heavy chain constant region has isotypes selected from γ1, γ2, γ3, and γ4. In some embodiments, the light chain constant region has isotypes selected from κ and λ. In some embodiments, the antibody moiety is an immunoglobulin comprising two identical heavy chains and two identical light chains. In some embodiments, the immunoglobulin is IgG.

[0016] In some embodiments, the anti-MUC16 construct provided herein is single-specific. In some embodiments, the anti-MUC16 construct provided herein is multi-specific. In some embodiments, the anti-MUC16 construct provided herein is bispecific. In some embodiments, the anti-MUC16 construct provided herein is a tandem scFv, a bispecific antibody (Db), a single-chain bispecific antibody (scDb), a dual-affinity retargeting (DART) antibody, F(ab')2, a dual variable domain (DVD) antibody, a club-and-mortar structure (KiH) antibody, a docking-locked (DNL) antibody, a chemically cross-linked antibody, a heteropolypeptide, or a heteroconjugate antibody. In some embodiments, the anti-MUC16 construct provided herein is a tandem scFv comprising two scFvs linked by a peptide linker. In some embodiments, the antibody portion that specifically recognizes MUC16 is a first antibody portion, and wherein the anti-MUC16 construct further comprises a second antibody portion that specifically recognizes a second antigen. In some embodiments, the second antigen is an antigen on the surface of T cells. In some embodiments, the second antigen is CD3. In some embodiments, the second antigen is selected from CD3γ, CD3δ, CD3ε, and CD3ζ. In some embodiments, the second antigen is CD3ε. In some embodiments, the multispecific or bispecific anti-MUC16 construct comprises an anti-CD3 antibody moiety. In some embodiments, the multispecific or bispecific anti-MUC16 construct comprises any one of SEQ ID NO:42, 69-75, and 88-95.

[0017] In some implementations, the anti-MUC16 construct provided herein is a chimeric antigen receptor (CAR).

[0018] In some embodiments, the CAR includes a co-stimulatory domain. In some embodiments, the CAR includes a CD3zeta(ζ) chain cytoplasmic signaling domain. In some embodiments, the CAR includes the scFv of any one of SEQ ID NO:53-68. In some embodiments, the CAR includes any one of SEQ ID NO:80-87 and 97-103.

[0019] In some implementations, the anti-MUC16 constructs provided herein are further conjugated to peptides, detection agents, imaging agents, therapeutic agents, or cytotoxic agents.

[0020] In some embodiments, this document also provides polypeptides comprising one or more of the amino acid sequences of SEQ ID NO:2-5, 10-13, 20-23 and 28-31 or amino acids of the anti-MUC16 construct provided herein.

[0021] In some embodiments, this document also provides a polynucleotide comprising a nucleic acid sequence encoding one or more polypeptides comprising an amino acid sequence of one or more of SEQ ID NO: 2-5, 10-13, 20-23, and 28-31 or amino acids of the anti-MUC16 construct provided herein. In some embodiments, this document provides a vector comprising the polynucleotide provided herein operatively linked to a promoter.

[0022] In some embodiments, this document also provides cells comprising the anti-MUC16 construct, peptide, polynucleotide, or vector provided herein. In some embodiments, the cells are mammalian cells. In some embodiments, the cells are immune cells. In some embodiments, the cells are lymphocytes. In some embodiments, the cells are T cells or B cells.

[0023] In some embodiments, this document also provides pharmaceutical compositions comprising: a therapeutically effective amount of the presently provided anti-MUC16 construct, the presently provided peptide, the presently provided polynucleotide, or the presently provided vector; and a pharmaceutically acceptable vector.

[0024] In some embodiments, this document also provides a method of treating a patient in need of MUC16-related disease or disorder, comprising administering to the patient a pharmaceutical composition comprising a therapeutically effective amount of the anti-MUC16 construct, peptide, polynucleotide, or carrier provided herein. In some embodiments, the MUC16-related disease or disorder is cancer. In some embodiments, the cancer is ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer. In some embodiments, the cancer is metastatic cancer. In some embodiments, the pharmaceutical composition inhibits or reduces metastasis in the patient. In some embodiments, the patient is a human patient.

[0025] In some embodiments, this document also provides methods for generating effector cells, which include genetically modifying cells with one or more nucleic acids encoding the anti-MUC16 construct provided herein.

[0026] In some embodiments, this document also provides a method comprising: introducing one or more nucleic acids encoding an anti-MUC16 construct provided herein into one or more primary cells isolated from a patient, and administering the cells containing said one or more nucleic acids to said patient. In some embodiments, the method further comprises amplifying the cells prior to administering the cells to the patient. In some embodiments, the primary cells are lymphocytes. In some embodiments, the primary cells are T cells.

[0027] In some embodiments, the treatment methods provided herein further include administering a therapeutically effective amount of another therapeutic agent to the patient. In some embodiments, the therapeutic agent is an anticancer agent. In some embodiments, the therapeutic agent is a chemotherapy agent.

[0028] In some embodiments, this document also provides a method for detecting MUC16 in a sample, comprising: (a) contacting the sample with an anti-MUC16 construct provided herein; and (b) directly or indirectly detecting the binding between the anti-MUC16 construct and MUC16 present in the sample. In some embodiments, the anti-MUC16 construct is conjugated to a detectable marker. In some embodiments, the detectable marker is a chromogenic agent, an enzyme, a radioisotope, an isotope, a fluorescent agent, a toxic agent, a chemiluminescent agent, or a nuclear magnetic resonance contrast agent. In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in the sample is detected directly by detecting the detectable marker. In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in the sample is detected indirectly using a secondary antibody.

[0029] In some embodiments, this document also provides a method for diagnosing an individual suspected of having a MUC16-related disease or disorder, comprising: a) administering an effective amount of the anti-MUC16 construct provided herein to the individual; and b) directly or indirectly determining the binding level between the anti-MUC16 construct and any MUC16 in the individual, wherein a binding level above a threshold level indicates that the individual has the MUC16-related disease or disorder. In some embodiments, the anti-MUC16 construct is conjugated to a detectable marker. In some embodiments, the detectable marker is a chromogenic agent, an enzyme, a radioisotope agent, an isotope agent, a fluorescent agent, a toxic agent, a chemiluminescent agent, or a magnetic resonance imaging agent. In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in the sample is detected directly by detecting the detectable marker. In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in the sample is detected indirectly using a secondary antibody.

[0030] A method for diagnosing an individual suspected of having a MUC16-related disease or disorder includes a) contacting a sample containing cells derived from the individual with an anti-MUC16 construct provided herein; and b) determining the number of cells in the sample that bind to the anti-MUC16 construct, wherein a value for the number of cells binding to the anti-MUC16 construct above a threshold level indicates that the individual has the MUC16-related disease or disorder. In some embodiments, the anti-MUC16 construct is conjugated to a detectable marker. In some embodiments, the detectable marker is a chromophore, enzyme, radioisotope, isotope, fluorescent agent, toxic agent, chemiluminescent agent, or magnetic resonance imaging (MRI) contrast agent.

[0031] In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in the sample is detected directly by detecting the detectable marker. In some embodiments, the binding between the anti-MUC16 construct and any MUC16 in the sample is detected indirectly using a secondary antibody.

[0032] In some embodiments, this document also provides the use of the anti-MUC16 construct, anti-MUC16 peptide, polynucleotide encoding the anti-MUC16 construct or anti-MUC16 peptide, vector comprising said polynucleotide, or cells comprising any said peptide and its polynucleotide for the treatment of diseases or disorders associated with positive MUC16 expression. In some embodiments, said disease or disorder associated with positive MUC16 expression is cancer.

[0033] In some embodiments, this document also provides the use of the anti-MUC16 construct, anti-MUC16 peptide, polynucleotide encoding the anti-MUC16 construct or anti-MUC16 peptide, vector comprising said polynucleotide, or cell comprising any of said peptide and its polynucleotide in the manufacture of a medicament for treating a disease or disorder associated with positive MUC16 expression. In some embodiments, said disease or disorder associated with positive MUC16 expression is cancer.

[0034] In some embodiments, this document also provides the use of the anti-MUC16 construct, anti-MUC16 peptide, polynucleotide encoding the anti-MUC16 construct or anti-MUC16 peptide, vector comprising said polynucleotide, or cell comprising any said peptide and its polynucleotide for the diagnosis of diseases or disorders associated with positive MUC16 expression. In some embodiments, said disease or disorder associated with positive MUC16 expression is cancer. Attached Figure Description

[0035] Figure 1A shows a schematic diagram of the structure of MUC16. Figure 1B shows a schematic diagram and amino acid sequence of a truncated form of MUC16 (referred to as MUC16c114, SEQ ID NO:44), which contains an extracellular domain of 58 amino acids, a transmembrane domain of 25 amino acids, and a cytoplasmic tail region of 31 amino acids. The numbering in the figures is based on the original publication identifying Muc16, namely Yin and Lloyd (2001) J Biol Chem 276:27371-27375.

[0036] Figure 2 shows the amino acid alignment between the extracellular domains of wild-type MUC16-C114 (SEQ ID NO:44) and N30 mutant MUC16-C114 (SEQ ID NO:50).

[0037] Figures 3A-3E illustrate the in vitro characterization of antibodies binding to the carboxyl terminus of MUC16. Figure 3A shows a cartoon diagram of the molecular layout of the MUC16 superstructure, highlighting four distinct regions: the N-terminal domain, the tandem repeat [TR] region, the SEA (spermin, enterokinase, and aggregate protein) domain, and the carboxyl-terminal domain, which includes a juxtamembrane [JM] region or an extracellular domain and a transmembrane [TM] region. The sequence of MUC16 peptide-2 is shown; this peptide-2 is found within the extracellular domain and is the target binding site of the antibody tested in this study. Figure 3B provides a graph showing the saturation binding assay results of radiolabeled variants of two lead antibody candidates. 89 Zr]Zr-DFO-9C9 (left image) and [ 89 The binding affinity curve of Zr-DFO-4H11 (right figure) (solid line) compared to the control antibody (dashed line). Figure 3C shows [ 89 Zr]Zr-DFO-4H11 and [ 89 The intracellular characteristics of Zr-DFO-9C9 are shown in SKOV3. c114 The minimum uptake of any antibody in cells at 4°C, but compared with [ 89 Compared to the slow uptake of Zr-DFO-9C9, [ 89 The relatively rapid uptake of Zr-DFO-4H11 at 37°C; Figure 3D provides a graphical representation showing [ 89 Zr]Zr-DFO-4H11 and [ 89 Comparable in vitro serum stability of Zr-DFO-9C9. Figure 3E provides a graphical representation showing the stability in the presence of excess unlabeled 9C9 antibody. 89The blocking of binding of Zr-DFO-4H11 to biotinylated MUC16 peptide-2 captured on streptavidin-functionalized magnetic beads (compare unblocked (middle bar) with blocked (right bar); also showing the control sample without MUC16 peptide-2 (left bar)).

[0038] Figures 4A-4D Explanation [ 89 Zr]Zr-DFO-9C9 and [ 89 In vivo characterization of the radiopharmacological properties of Zr-DFO-4H11. Figure 4A provides […]. 89 Zr]Zr-DFO-9C9 (170-200 μCi; 6.29-7.4 MBq, suspended in 200 μL of Chelex-treated PBS via lateral tail vein injection) in SKOV3 c114 Representative PET images of xenografts [top: coronal sections; bottom: maximum intensity projection (MIP)] showing the tumor (T) profile at 24 h post-injection (pi) and the gradually increasing active uptake within the tumor up to 96 h pi. High active concentrations were observed in the liver (L) and kidney (K) at early time points, but gradually decreased at subsequent time points. Figure 4B is provided. 89 Zr]Zr-DFO-4H11 (170-200 μCi; 6.29-7.4 MBq, suspended in 200 μL of Chelex-treated PBS via lateral tail vein injection) in SKOV3 c114 A representative series of PET images from xenografts [top: coronal sections; bottom: maximum intensity projection (MIP)], depicting the contours of tumors (T) and lymph nodes (LN) at 24 h post-injection (pi), where the PET signal intensity in the tumor gradually increases up to 96 h pi. [With] 89 Zr-DFO-4H11 yielded high-contrast PET images, and the liver (L) and lymph nodes (LN) were the only non-tumor tissues exhibiting background activity at subsequent time points. Figure 4C provides [ 89 Zr]Zr-DFO-9C9 and [ 89 Zr]Zr-DFO-4H11 in SKOV3 c114 A graphical representation of in vivo biodistribution in xenografts, showing high and comparable tumor uptake associated with the activities of two radioimmunoconjugates. 89 Zr]Zr-DFO-9C9 and [ 89 Zr]Zr-DFO-4H11 in SKOV3 c114Tumor uptake may be blocked in the presence of an excess of unlabeled antibody co-injected with the corresponding radiolabeled variant of the antibody, and is significantly higher than that in isotype controls. The difference in in vivo active concentrations in non-tumor tissues was most significant between the kidney and axillary lymph nodes (LNs). 89 Zr]Zr-DFO-9C9 exhibits significantly higher performance than [ 89 The renal activity concentrations of Zr-DFO-4H11 and isotype control, while [ 89 Zr]Zr-DFO-4H11 is shown in the injection [ 89 Significantly higher activity concentrations of LN were observed in Zr-DFO-9C9 mice or isotype control mice. **Indicates p-value ≤ 0.005; ***Indicates p-value ≤ 0.0005; ***Indicates p-value ≤ 0.00005. Figure 4D provides a bar chart illustrating [ 89 Zr]Zr-DFO-9C9 and [ 89 Comparison of in vivo radiopharmacological characteristics of Zr-DFO-4H11, such as evaluation based on the tumor to background (T:B) active concentration ratio in the target vital organ.

[0039] Figures 5A-5E illustrate the in vitro characterization of the humanized 4H11 antibody. Figure 5A provides a cartoon illustration of the DFO-conjugated humanized 4H11 antibody (DFO-hu4H11). Figure 5B provides a histogram from flow cytometry analysis, showing the interaction between DFO-hu4H11 and SKOV3. c114 Binding to cells (or SKOV3+ cells, solid lines) compared to the lack of binding to SKOV3 cells (dashed lines); Figure 5C provides... 89 Zr-labeled hu4H11 antibody ([ 89 Cartoon image of Zr-DFO-hu4H11. Figure 5D provides [ 89 Quality control of Zr-DFO-hu4H11 shows high radiochemical purity in transient thin-layer chromatography compared to size-exclusion purified radioimmunoconjugates; Figure 5E provides […]. 89 Graphical representation of low nonspecific binding and high (>90%) immunoreactivity fraction of Zr-DFO-hu4H11 to biotinylated MUC16 peptide-2 captured on streptavidin-functionalized DynaBeads. This is achieved by means of a significant excess of unlabeled DFO-hu4H11. 89 The specificity of target binding was established by blocking the binding of Zr]Zr-DFO-hu4H11 to MUC16 peptide-2 on magnetic beads.

[0040] Figures 6A-6C illustrate the in vivo characterization of the radiopharmacological features of the humanized 4H11 antibody. Figure 6A provides […].89 Zr]Zr-DFO-hu4H11 (200 μCi; 7.4 MBq, suspended in 200 μL of Chelex-treated PBS via lateral tail vein injection) in SKOV3 c114 Representative PET images of xenografts [top: coronal section; bottom: maximum intensity projection (MIP)] showing a clear outline of the tumor (T) at 36 h, followed by gradual accumulation of most of the injected activity within the tumor at 96 h pi; Figure 6B illustrates [ 89 The in vivo biodistribution of Zr-DFO-hu4H11 was shown, exhibiting high active concentrations in tumors and ≤8% ID / g in most non-tumor background organs except for bone and axillary lymph nodes. Active tumor uptake could be blocked by co-injection of a 40-fold overdose of unlabeled DFO-hu4H11. **Indicates p-value ≤0.005; Figure 6C provides a bar chart showing [ 89 In vivo radiopharmacological characteristics of Zr]Zr-DFO-hu4H11, as evaluated based on the tumor to background (T:B) ratio of active concentration in the target vital organ.

[0041] Figures 7A-7D illustrate the […] 89 In vivo and in vitro analysis of the biodistribution of Zr-DFO-hu4H11. Figure 7A provides […]. 89 Zr]Zr-DFO-hu4H11 (250 μCi; 9.25 MBq, suspended in 200 μL of Chelex-treated PBS via lateral caudal vein injection) in bilateral xenografts (left shoulder: SKOV3) c114 Representative series of PET images in SKOV3 tumor (top: coronal section; middle: transverse section; bottom: PET-CT overlay with maximum intensity projection (MIP)) showing SKOV3 tumor; right shoulder: right shoulder tumor. c114 Preferred uptake in tumors. Figure 7B provides representative autoradiographic images from ex vivo analysis of harvested bilateral tumors (from mice shown in 7A), revealing that SKOV3 tumors showed almost no signal compared to those exposed to autoradiography in the same cassette. c114 Higher and heterogeneous signal intensity distribution within the tumor (dashed circles: highly active hot spots; dashed triangles: low to inactive cold spots). Figure 7C shows hematoxylin and eosin (H&E) staining of a tumor section (shown in 7B), revealing necrotic areas (dashed triangles) and areas with nests of actively dividing tumor cells (dashed circles). Figure 7D shows formalin-fixed paraffin-embedded SKOV3 cells. c114 Comparative H&E staining of tumors (left) and SKOV3 tumors (right) reveals significant differences in tumor structure and the morphology of the cells constituting the tumor.

[0042] Figures 8A-8B Explanation [ 89 PET imaging of Zr-DFO-hu4H11 in MUC16-expressing cell lines and HGSOC patient-derived xenograft models. Figure 8A provides […]. 89 Zr-DFO-hu4H11 (150 μCi; 5.55 MBq, suspended in 200 μL of Chelex-treated PBS via lateral tail vein injection) at 72 hpi in nude mice [left: coronal section; right: maximum intensity projection (MIP)], the nude mice carrying a subcutaneous xenografted MUC16-positive OVCAR3 tumor on the right shoulder. Figure 8B is provided. 89 Zr-DFO-hu4H11 (150 μCi; 5.55 MBq, suspended in 200 μL of Chelex-treated PBS via lateral tail vein injection) at 72 hpi in two mice carrying PDX of HGSOC tumors in the right shoulder, and the representative PET images show high activity concentrations in the tumor (T) and some persistent activity in the blood pool (BP) (including the heart and descending aorta).

[0043] Figures 9A-9D illustrate the in vitro binding of 4H11 and 18C6 mouse mAbs and humanized antibodies to the MUC16+OVCAR3 cell line and transfected cell lines expressing MUC16 c344 and c114 peptides, as well as the binding of these antibodies to the control MUC16 cell line, using FACS analysis. - A2780 and SKOV3 cell lines showed no in vitro binding. Figure 9A shows the mean fluorescence of binding between 4H11 and 18C6 mouse mAb antibodies and the tested cell lines. Figure 9B shows the percentage of fluorescently positive cells binding between 4H11 and 18C6 mouse mAb antibodies and the tested cell lines. Figure 9C shows the mean fluorescence of binding between 4H11 and 18C6 humanized antibodies and the tested cell lines. Figure 9D shows the percentage of fluorescently positive cells binding between 4H11 and 18C6 humanized antibodies and the tested cell lines.

[0044] Figures 10A and 10B demonstrate, through FACS analysis, the in vitro binding of the 4H11 humanized antibody to the MUC16+OVCAR3 cell line and the SKOV3 transfectant cell line expressing MUC16 c344 and c114 peptides, while binding to the control MUC16... - SKOV3 cell line was not bound in vitro.

[0045] Figure 11 illustrates that, in the Matrigel invasion assay, the 4H11 humanized antibody inhibited the invasion of MUC16+OVCAR3, OVCA-433, and CAOV3 cell lines compared to untreated cells.

[0046] Figure 12 illustrates that, in the Matrigel invasion assay, the 4H11 humanized antibody inhibited the invasion of SKOV3 cell lines transfected with MUC16 c344 and c114 peptides compared to untreated cells. SKOV3 cell lines expressing the mutant MUC16 peptide N123mutc114 were used as a negative control for invasion. Detailed Implementation

[0047] This application provides, in one aspect, an anti-MUC16 antibody agent, such as an anti-MUC16 construct, which includes an antibody portion that specifically recognizes epitopes of MUC16, such as epitopes of the retained extracellular domain of MUC16 (MUC16 extracellular domain).

[0048] Using phage display technology, scFvs specific to the preserved extracellular domain of human MUC16 were identified. Flow cytometry assays confirmed that these antibodies recognize cancer cell lines expressing MUC16. This application thereby provides anti-MUC16 antibody agents, such as anti-MUC16 constructs, comprising an antibody moiety that specifically binds to MUC16. The anti-MUC16 antibody agents include, for example, anti-MUC16 antibodies (e.g., full-length anti-MUC16 antibodies and their antigen-binding fragments), anti-MUC16 scFvs, anti-MUC16 antibody fusion proteins (e.g., anti-MUC16 Fc fusion proteins and chimeric antigen receptors (CARs)), multispecific antibodies (e.g., bispecific antibodies), and their anti-MUC16 antibody conjugates (i.e., anti-MUC16 immunoconjugates).

[0049] In another aspect, nucleic acids encoding anti-MUC16 antibody agents are provided, such as anti-MUC16 antibodies (e.g., full-length anti-MUC16 antibodies and their antigen-binding fragments), anti-MUC16 scFv, anti-MUC16 antibody fusion proteins (e.g., anti-MUC16 Fc fusion proteins and chimeric antigen receptors (CARs)), multispecific antibodies (e.g., bispecific antibodies), and their anti-MUC16 antibody conjugates (i.e., anti-MUC16 immunoconjugates).

[0050] On the other hand, compositions comprising anti-MUC16 antibody agents, such as pharmaceutical compositions, are provided, said anti-MUC16 antibody agents such as full-length anti-MUC16 antibody and its antigen-binding fragment, anti-MUC16 scFv, anti-MUC16 antibody fusion protein (e.g., anti-MUC16 Fc fusion protein and chimeric antigen receptor (CAR)), multispecific antibody (e.g., bispecific antibody) and its anti-MUC16 antibody conjugate (i.e., anti-MUC16 immunoconjugate).

[0051] This article also provides methods for preparing and using the aforementioned anti-MUC16 antibody agents and antibodies (e.g., for the treatment of cancer), as well as kits and articles that can be used in such methods.

[0052] This document also discloses kits for detecting and / or treating MUC16-related symptoms, comprising at least one anti-MUC16 antibody of the present technology or a functional variant thereof (e.g., a substitutional variant) and instructions for use. In some embodiments, the anti-MUC16 antibody is conjugated to one or more detectable markers. In one embodiment, the one or more detectable markers include radiolabels, fluorescent markers, or chromophores.

[0053] Alternatively or additionally, in some embodiments, the kit further comprises a secondary antibody that specifically binds to the anti-MUC16 antibody agent described herein. In some embodiments, the secondary antibody is conjugated to at least one detectable label selected from radiolabeled, fluorescently labeled, or chromogenicly labeled labels.

[0054] definition

[0055] Unless otherwise defined, all technical and scientific terms used herein shall have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. The following references provide general definitions for many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition, 1994); The Cambridge Dictionary of Science and Technology (Walker, ed., 1988); The Glossary of Genetics, 5th edition, R. Rieger et al. (eds.), Springer Verlag (1991); and Hale and Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise stated, the following terms shall have the meanings assigned to them as follows. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure.

[0056] As used herein, the terms “MUC16” or “MUC16 polypeptide” or “MUC16 peptide” refer to MUC16-linked mucins, as described in Yin BW and Lloyd KO, 2001, J Biol Chem. 276(29):27371-5. GenBank TM Accession number NP_078966.2 (SEQ ID NO:1) provides an exemplary human MUC16 nucleic acid sequence. GenBank TM Accession number NP 078966.2 (SEQ ID NO:1) provides an exemplary human MUC16 amino acid sequence. Native MUC16 comprises an intracellular domain, a transmembrane domain, an extracellular domain near the presumed cleavage site, and a large, highly glycosylated region of 12–20 repeats (each repeat being 156 amino acids long) (Figure 1A). “Immature” MUC16 refers to SEQ ID NO:1, which contains the MUC16 signal sequence (amino acid residues 1–60 of SEQ ID NO:1). “Mature MUC16” refers to native MUC16 expressed on the cell surface, i.e., where the signal sequence has been removed through cellular processing, such as SEQ ID NO:51, where the first 60 amino acid residues of SEQ ID NO:1 have been removed (i.e., SEQ ID NO:1 is the “immature” form of MUC16).

[0057] The polypeptide represented by the amino acid sequence of SEQ ID NO:44 is referred to herein as MUC16 C114 and consists of 114 amino acid residues at the C-terminus of mature MUC16 (SEQ ID NO:51 is the sequence of mature MUC16). MUC16 C114 contains an extracellular domain of 58 amino acids, a transmembrane domain of 25 amino acids, and a cytoplasmic tail of 31 amino acids (Fig. 1B). MUC16c114 is capable of N-glycosylation at asparagine amino acid residues at positions 1, 24, and 30 of SEQ ID NO:44 (also referred to as amino acid positions Asn1777, Asn1800, and Asn1806, according to the original MUC16 publication Yin BW and Lloyd KO, 2001, J Biol Chem. 276(29):27371-5).

[0058] As used herein, the singular forms “a”, “an”, and “the” are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0059] As used herein, the term “about” when used to modify a numerical value or range indicates that a deviation of 5% to 10% above or below the value or range is still within the intended meaning of the value or range.

[0060] As used herein, the term “administering” a drug to a subject includes any route by which a drug is introduced into or delivered to a subject to perform its intended function. Administration can be performed via any suitable route, including but not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, and other suitable routes as described herein. Administration includes self-administration and administration by another person.

[0061] The term "amino acid" refers to naturally occurring amino acids and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally encoded amino acids include 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrolysine and selenocysteine. Amino acid analogs are reagents having the same basic chemical structure as naturally occurring amino acids (i.e., α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (such as ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. In some embodiments, the amino acid forming the polypeptide is in the D form. In some embodiments, the amino acid forming the polypeptide is in the L form. In some embodiments, the first plurality of amino acids forming the polypeptide are in the form of D, and the second plurality of amino acids forming the polypeptide are in the form of L.

[0062] Amino acids are represented in this article by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides are represented by their commonly accepted single-letter codes.

[0063] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs). The terms cover amino acid chains of any length, including full-length proteins, where the amino acid residues are linked by covalent peptide bonds.

[0064] As used herein, the term "antibody" refers not only to the complete antibody molecule but also to a fragment of an antibody molecule that retains the ability to bind to immunogens. Such fragments are well known in the art and are commonly used both in vitro and in vivo. Therefore, as used herein, the term "antibody" refers not only to the complete immunoglobulin molecule but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments lacking the Fc fragment of the complete antibody are cleared from circulation more quickly and can bind less nonspecifically to tissues than the complete antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Antibodies of this technology include complete natural antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, multispecific antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single-chain V region fragments (scFv), single-domain antibodies (e.g., nanobodies and single-domain camelid antibodies), V NAR Antibodies include fragments, bispecific T-cell adaptor antibodies, microantibodies, disulfide-linked Fv (sdFv), and anti-idiotype (anti-Id) antibodies, intracellular antibodies, fusion peptides, unconventional antibodies, and antigen-binding fragments of any of the above antibodies. Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0065] In some embodiments, an antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V). H ) and heavy chain constant region (C H The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as V in this paper). L ) and light chain constant region C L Composition. The constant region of the light chain consists of a structural domain C. L Composition. V H and V L The region can be further subdivided into highly variable regions, called complementary determinant regions (CDRs), and more conservative regions, called framing regions (FRs). Each V H and V LComposed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. As used herein, interchangeably, the terms “antigen-binding portion,” “antigen-binding fragment,” or “antigen-binding region” of an antibody refer to the region or portion of the antibody that binds to the antigen and confers antigen-specificity to the antibody; fragments of antigen-binding proteins (e.g., antibodies) include one or more fragments of the antibody that retain the ability to specifically bind antigens (e.g., peptide / HLA complexes). It has been shown that the antigen-binding function of an antibody can be performed by fragments of the full-length antibody. Examples of antigen-binding portions covered within the term “antibody fragment” of an antibody include: Fab fragments, i.e., those composed of V… L V H C L The monovalent segment composed of the CHI structural domain; the F(ab)2 segment, which is a divalent segment containing two Fab segments connected by a disulfide bridge in the hinge region; and the segment composed of V H The Fd fragment composed of the CHI domain; the V-arm of the antibody. L and V H Fv segments composed of structural domains; composed of V H dAb fragments composed of structural domains (Ward et al., Nature 341:544-546, 1989); and separated complementarity-determining regions (CDRs).

[0066] Antibodies and antibody fragments may be derived wholly or partially from mammals (e.g., humans, non-human primates, goats, guinea pigs, hamsters, horses, mice, rats, rabbits, and sheep) or from non-mammal animals that produce antibodies (e.g., chickens, ducks, geese, snakes, and tailed amphibians). Antibodies and antibody fragments may be produced in vivo or in vitro, such as from yeast or bacteriophages (e.g., as monoclonal antibodies or antibody fragments or as part of an antibody library).

[0067] Furthermore, although the two structural domains V of the Fv fragment L and V H Encoded by individual genes, but linked together using recombination methods via synthetic adapters, which allow them to be made into a single protein chain, in which V L and V HRegions pair to form monovalent molecules. These are called single-chain Fvs (scFvs); see, for example, Bird et al., Science 242:423-426 (1988); and Huston et al., Proc. Natl. Acad. Sci. 85:5879-5883 (1988). These antibody fragments are obtained using conventional techniques known to those skilled in the art and are screened for utility in the same manner as intact antibodies.

[0068] "Isolated antibody" or "isolated antigen-binding protein" is an antibody or antigen-binding protein that has been identified and isolated and / or recovered from components of its natural environment. "Synthetic antibody" or "recombinant antibody" is typically produced using recombinant techniques or peptide synthesis techniques known to those skilled in the art.

[0069] As used herein, the term "single-chain variable fragment" or "scFv" refers to the variable region (V) of the heavy chain of an immunoglobulin (e.g., mouse or human). H ) and light chain variable region (V L (covalently linked to form V) H :V L A fusion protein of heterodimers. Heavy chain (V H ) and light chains (V L It can bind directly or via a peptide-encoded linker (e.g., about 10, 15, 20, or 25 amino acids), which will bind V H N-terminus and V L Connect the C end, or connect the V end. H C-terminus and V L The N-terminal linker is used. Linkers are typically enriched with glycine for flexibility and with serine or threonine for solubility. Linkers can connect the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain.

[0070] Despite the removal of the constant region and the introduction of a linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be derived from proteins containing V... H and V LNucleic acid expression of the coding sequence, as described in Huston et al., Proc. Nat. Acad. Sci. USA, 85:5879-5883 (1988). See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778; and U.S. Patent Publications Nos. 20050196754 and 20050196754. Antagonistic scFvs with inhibitory activity have been described (see, for example, Zhao et al., Hybridoma (Larchmt) 27(6):455-51 (2008); Peter et al., J Cachexia Sarcopenia Muscle (2012); Shieh et al., J Imunol 183(4):2277-85 (2009); Giomallelli et al., Thromb Haemost 97(6):955-63 (2007); Fife et al., J Clin Invst 116(8):2252-61 (2006); Brocks et al., Immunotechnology 3(3):173-84 (1997); Moosmayer et al., Ther Immunol 2(10):31-40 (1995). Agonistic scFvs with stimulatory activity have been described (see, for example, Peter et al., J Biol Chem 25278(38):36740-7(2003); Xie et al., Nat Biotech 15(8):768-71(1997); Ledbetter et al., Crit Rev Immunol 17(5-6):427-55(1997); Ho et al., BioChim Biophys Acta 1638(3):257-66(2003)).

[0071] As used herein, "antigen" refers to a molecule that an antibody (or an antigen-binding fragment thereof) can selectively bind to. Target antigens can be proteins, carbohydrates, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds. In some embodiments, the target antigen can be a peptide (e.g., the MUC16 peptide). Antigens can also be administered to animals to elicit an immune response.

[0072] The term "antigen-binding fragment" refers to a fragment of a complete immunoglobulin structure having a polypeptide moiety responsible for binding to an antigen. Examples of antigen-binding fragments that can be used in this art include, but are not limited to, scFv, (scFv)2, scFvFc, Fab, Fab', and F(ab')2.

[0073] As used herein, the term "biological sample" or "sample" means sample material derived from living cells. Biological samples can include tissues, cells, cellular protein or membrane extracts, and biological fluids (e.g., ascites or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells, and fluids present within the subject's body. Biological samples of this technology include, but are not limited to, samples taken from: breast tissue, kidney tissue, cervix, endometrium, head or neck, gallbladder, parotid gland tissue, prostate, brain, pituitary gland, kidney tissue, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid tissue, heart tissue, lung tissue, bladder, adipose tissue, lymph node tissue, uterus, ovarian tissue, adrenal gland tissue, testicular tissue, tonsils, thymus, blood, hair, cheek, skin, serum, plasma, CSF, sperm, prostatic fluid, semen, urine, feces, sweat, saliva, sputum, mucus, bone marrow, lymph, and tears. Biological samples can also be obtained from visceral biopsies or from cancer. Biological samples can be obtained from subjects for diagnosis or research; or they can be obtained from disease-free individuals as controls or for basic research. Samples can be obtained using standard methods, including, for example, venipuncture and surgical biopsy. In some embodiments, biological samples are tissue samples obtained via needle aspiration biopsy.

[0074] As used herein, a “bispecific antibody” or “BsAb” refers to an antibody capable of simultaneously binding to two targets with different structures (e.g., two different target antigens or two different epitopes on the same target antigen). Various bispecific antibody structures are known in the art. In some embodiments, each antigen-binding moiety in the bispecific antibody includes V… H and / or V L District; in some such implementation schemes, V H and / or V L The regions are those found in specific monoclonal antibodies. In some embodiments, bispecific antibodies contain two antigen-binding moieties, each including a V from a different monoclonal antibody. H and / or V L In some embodiments, the bispecific antibody contains two antigen-binding moieties, one of which includes a V... H and / or V L immunoglobulin molecules in the region, the V H and / or V L The region contains a CDR from the first monoclonal antibody; and another antigen-binding region includes a V... H and / or V L Antibody fragments in the region (e.g., Fab, F(ab'), F(ab')2, Fd, Fv, dAB, scFv, etc.), the V Hand / or V L The region contains a CDR derived from the second monoclonal antibody.

[0075] As used herein, the term "associative" refers to the association of two molecules by any method known to those skilled in the art. Suitable types of association include chemical bonds and physical bonds. Chemical bonds include, for example, covalent bonds and coordinate bonds. Physical bonds include, for example, hydrogen bonds, dipole interactions, van der Waals forces, electrostatic interactions, hydrophobic interactions, and aromatic ring stacking.

[0076] As used herein, a “control” is an alternative sample used in an experiment for comparative purposes. A control can be “positive” or “negative.” For example, in cases where the purpose of the experiment is to determine the relevance of a therapeutic agent to the treatment of a specific type of disease, a positive control (a composition known to exhibit the desired therapeutic effect) and a negative control (a subject or sample that does not receive the therapy or receives a placebo) are typically used.

[0077] As used herein, the term "shared FR" refers to a shared frame (FR) antibody region within an immunoglobulin sequence. The FR region of an antibody does not come into contact with the antigen.

[0078] As used herein, the term "effective amount" means an amount sufficient to achieve the desired therapeutic and / or preventive effect, such as an amount resulting in the prevention or reduction of one or more signs or symptoms associated with the disease or condition described herein. In the case of therapeutic or preventive application, the amount of composition administered to a subject will vary depending on the composition, the degree, type, and severity of the disease, and individual characteristics such as general health status, age, sex, weight, and drug tolerance. A technician will be able to determine the appropriate dosage based on these and other factors. The composition may also be administered in combination with one or more other therapeutic compounds. In the methods described herein, a therapeutic composition may be administered to a subject having one or more signs or symptoms of the disease or condition described herein. As used herein, a "therapeutic effective amount" of a composition refers to the level of composition in which the physiological effects of the disease or condition are improved or eliminated. A therapeutic effective amount may be given in one or more administrations.

[0079] As used herein, the term "expression" refers to the process of polynucleotide transcription into mRNA and / or the subsequent translation of transcribed mRNA into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells may include mRNA splicing. Gene expression levels can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. In one aspect, the expression level of a gene from a sample can be directly compared to the expression level of said gene from a control or reference sample. In another aspect, the expression level of a gene from a sample can be directly compared to the expression level of said gene from the same sample after administration of the compositions disclosed herein. The term “expression” also refers to one or more of the following events: (1) generating an RNA template from a DNA sequence within a cell (e.g., by transcription); (2) processing RNA transcripts within a cell (e.g., by splicing, editing, 5' cap formation and / or 3' end formation); (3) translating an RNA sequence into a polypeptide or protein within a cell; (4) post-translational modification of a polypeptide or protein within a cell; (5) presenting a polypeptide or protein on the cell surface; and (6) secreting, presenting or releasing a polypeptide or protein from a cell.

[0080] The term "linker" refers to a synthetic sequence (e.g., an amino acid sequence) that connects or links two sequences (e.g., two polypeptide domains). In some embodiments, the linker contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid sequences.

[0081] As used herein, the “humanized” form of a nonhuman (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a nonhuman immunoglobulin. For most, humanized antibodies are human immunoglobulins in which hypervariable residues of the receptor are replaced by hypervariable residues (donor antibodies) from a nonhuman species (such as mice, rats, rabbits, or nonhuman primates) possessing the desired specificity, affinity, and capability. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies may include residues not found in the receptor or donor antibody. These modifications are made to further enhance antibody properties such as binding affinity. Typically, humanized antibodies will contain substantially all of at least one, usually two, variable domains (e.g., Fab, Fab', F(ab')2, or Fv), wherein all or substantially all of the hypervariable loops correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are those of the common FR sequences of human immunoglobulins, but said FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR is typically no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody may also optionally contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region. For further details, see Jones et al., Nature 321:522-525 (1986); Reichmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, for example, Ahmed and Cheung, FEBS Letters 588(2):288-297 (2014).

[0082] As used herein, the term "hypervariate region" refers to the amino acid residues in an antibody responsible for antigen binding. Hypervariate regions typically contain amino acid residues from the "complementarity-determining region" or "CDR" (e.g., V...). L Before and after residues 24-34 (L1), 50-56 (L2), and 89-97 (L3), and V H The residues before and after 31-35B (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Maryland. (1991)) and / or those residues from the “hypervariate ring” (e.g., V) LResidues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the group, as well as V H 26-32(H1), 52A-55(H2) and 96-101(H3) (Chothia and Lesk J.Mol.Biol.196:901-917(1987)).

[0083] As used herein, “F(ab)” refers to an antibody structural fragment that binds to an antigen but is monovalent and does not have an Fc portion. For example, an antibody digested by papain produces two F(ab) fragments and one Fc fragment (e.g., the heavy (H) chain constant region; the Fc region that does not bind to the antigen).

[0084] As used in this article, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of intact IgG antibodies, wherein this fragment has two antigen-binding (ab) groups. 1 (bivalent) region, where each (ab) 1 The F(ab') region contains two separate amino acid chains (a portion of the H chain linked by an SS bond for binding the antigen and a light (L) chain) with the remaining H chain portions linked together. The "F(ab')2" fragment can be divided into two separate Fab' fragments.

[0085] As used herein, “CDR” is defined as the complementarity-determining region amino acid sequence of an antibody, which serves as a hypervariable region of the immunoglobulin heavy and light chains. See, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 4th USDepartment of Health and Human Services, National Institutes of Health (1987). Typically, an antibody contains three heavy chain and three light chain CDRs, or CDR regions, within the variable region. The CDR provides most of the contact residues for the antibody to bind to an antigen or epitope. In some embodiments, the Kabat system is used to characterize the CDR region (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., USDepartment of Health and Human Services, NIH Publication No. 91-3242 (1991)).

[0086] As used herein, the terms “constant region” or “constant domain” are interchangeable and have the meanings commonly found in the art. A constant region is an antibody portion that does not directly participate in antibody-antigen binding but can exhibit various effector functions (such as interaction with Fc receptors), such as the carboxyl-terminal portion of the light chain and / or heavy chain. The constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence than the variable domains of immunoglobulins.

[0087] As used herein, “epitope” is a term in the art and can refer to a local region of an antigen that an antibody can specifically bind to. An epitope can be, for example, a series of amino acids of a polypeptide (linear or continuous epitope), or an epitope can be, for example, formed together by two or more discontinuous regions of one or more polypeptides (conformal, nonlinear, discontinuous, or non-continuous epitope).

[0088] As used herein, the term "ligand" refers to a molecule that binds to a receptor. Specifically, a ligand binds to a receptor on another cell, thereby allowing cell-to-cell recognition and / or interaction.

[0089] As used herein, the term "affinity" refers to a measure of binding strength. Unbound by theory, affinity depends on the tightness of the stereochemical coordination between the antibody's binding site and the antigenic determinant, the size of the contact area between them, and the distribution of charged and hydrophobic groups. Affinity also includes the term "affinity," which refers to the strength of the antigen-antibody bond after the formation of a reversible complex (e.g., monovalent or polyvalent). Methods for calculating the affinity of an antibody for an antigen are known in the art, including the use of binding assays to calculate affinity. Antibody activity in functional assays (e.g., flow cytometry assays) also reflects antibody affinity. Antibodies and their affinity can be phenotypically characterized and compared using functional assays (e.g., flow cytometry assays). Nucleic acid molecules useful in the currently disclosed subject matter include any nucleic acid molecule encoding a polypeptide or a fragment thereof. In some embodiments, nucleic acid molecules useful in the currently disclosed subject matter include nucleic acid molecules encoding an antibody or an antigen-binding moiety thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but typically exhibit significant identity. Polynucleotides that have “significant homology” or “significant identity” with an endogenous sequence are generally capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. “Hybridization” means pairing between complementary polynucleotide sequences (e.g., the genes described herein) or portions thereof under various stringent conditions to form a double-stranded molecule. (See, for example, Wahl, GM and SLBerger, Methods Enzymol. 152:399 (1987); Kimmel, AR, Methods Enzymol. 152:507 (1987)).

[0090] As used herein, the terms “immune-specific binding,” “immune-specific recognition,” “specific binding,” and “specific recognition” are similar terms in the context of antibodies and refer to antibodies and their antigen-binding fragments that bind to antigens (e.g., epitopes or immune complexes) via antigen-binding sites, as understood by those skilled in the art, and do not exclude cross-reactivity of antibodies or antigen-binding fragments with other antigens.

[0091] The terms “substantially homologous” or “substantially identical” mean a polypeptide or nucleic acid molecule exhibiting at least 50% or greater homology or identity with a reference amino acid sequence (e.g., any amino acid sequence described herein) or nucleic acid sequence (e.g., any nucleic acid sequence described herein). For example, such sequences are at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% homologous or identical at the amino acid level or nucleic acid level to a sequence used for comparison (e.g., wild-type or native sequences). In some embodiments, substantially homologous or substantially identical polypeptides contain one or more amino acid substitutions, insertions, or deletions relative to a sequence used for comparison. In some embodiments, substantially homologous or substantially identical polypeptides contain one or more non-natural amino acids or amino acid analogs (including D-amino acids and trans amino acids) to replace the homologous sequence.

[0092] Sequence analysis software, such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, 53705), BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs, is typically used to measure sequence homology or sequence identity. Such software matches similar sequences by assigning degrees of homology to different substitutions, deletions, and / or other modifications. In an exemplary method for determining the degree of identity, the BLAST program can be used, where e -3 With e -100 The probability scores between them indicate closely related sequences.

[0093] As used herein, the term "analyte" refers to a structure-related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0094] As used herein, the term "conserved sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the currently disclosed anti-MUC16 antibody or its antigen-binding fragment containing an amino acid sequence. Conserved modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into human scFvs of currently disclosed anti-MUC16 antibodies or their antigen-binding fragments using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be grouped according to their physicochemical properties, such as charge and polarity. Conserved amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid residue having the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be categorized by polarity: polar amino acids include arginine (basic polar), asparagine and aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Therefore, one or more amino acid residues in the CDR region can be substituted by other amino acid residues in the same group, and the retained function of the altered antibody (i.e., the functions listed in (c) through (1) above) can be tested using the functional assays described herein. In some embodiments, no more than one, two, three, four, or five residues in the designated sequence or CDR region are altered.

[0095] As used herein, the term "heterologous nucleic acid molecule or polypeptide" refers to a nucleic acid molecule (e.g., cDNA, DNA, or RNA molecule) or polypeptide that is not normally present in cells or samples obtained from cells. This nucleic acid may originate from another organism, or it may be, for example, an mRNA molecule that is not normally expressed in cells or samples.

[0096] As used herein, the term “adjustment” refers to a positive or negative change. Exemplary adjustments include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0097] As used herein, the term “increase” means a positive change of at least about 5%, including but not limited to positive changes of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0098] As used herein, the term “reduction” means a negative change of at least about 5%, including but not limited to negative changes of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or about 100%.

[0099] As used herein, an "isolated" polynucleotide or nucleic acid molecule is a polynucleotide or nucleic acid molecule isolated from other nucleic acid molecules present in a natural source of nucleic acid molecules (e.g., in mice or humans). Furthermore, "isolated" nucleic acid molecules (such as cDNA molecules) may be substantially free of other cell material or culture medium when produced by recombinant technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, the language "substantially free" includes formulations of polynucleotide or nucleic acid molecules having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% of other materials (e.g., cell material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals).

[0100] As used herein, the term "isolated cell" refers to a cell that is separated from the molecular and / or cellular components of the natural accompanying cell.

[0101] As used herein, the term "tumor formation" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion to other tissues or organs. Tumor formation is typically uncontrolled and progressive, and occurs without inducing or causing the cessation of normal cell proliferation. Tumor formation can affect a wide variety of cell types, tissues, or organs, including but not limited to those selected from: bladder, colon, bone, brain, breast, cartilage, glial tissue, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lungs, lymph nodes, nerve tissue, ovary, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid gland, trachea, genitourinary tract, ureter, urethra, uterus, and vagina, or their tissues or cell types. Tumor formation includes cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).

[0102] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic compounds, and absorption-delaying compounds that are compatible with drug administration. Pharmaceutically acceptable carriers and formulations thereof are known to those skilled in the art and are described, for example, in the following literature: Remington's Pharmaceutical Sciences (20th edition, A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA).

[0103] As used herein, the term "recombinant" when used with respect to, for example, cells or nucleic acids, proteins or vectors, indicates that said cells, nucleic acids, proteins or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or indicates that said material is derived from cells that have been so modified. Thus, for example, recombinant cells express genes not found in the native (non-recombinant) form of said cells, or express native genes that would otherwise be abnormally expressed, underexpressed, or not expressed at all.

[0104] As used in this article, the term “separate” therapeutic use refers to the simultaneous or substantially simultaneous administration of at least two active ingredients via different routes.

[0105] As used herein, the term "sequential" therapeutic use refers to the administration of at least two active ingredients at different times, via the same or different routes of administration. More specifically, sequential use means initiating the administration of one or more other active ingredients after the complete administration of one active ingredient. Therefore, an active ingredient may be administered minutes, hours, or days before the administration of one or more other active ingredients. In this case, there is no concurrent treatment.

[0106] As used in this article, the term “simultaneous” therapeutic use refers to the application of at least two active ingredients via the same route and simultaneously or substantially simultaneously.

[0107] As used herein, the terms “subject,” “individual,” or “patient” can refer to a single organism, a vertebrate, a mammal, or a human. In some embodiments, the individual, patient, or subject is a human.

[0108] As used herein, “treating” encompasses treating a subject, such as a person, for a disease or disorder as described herein, and includes: (i) suppressing the disease or disorder, i.e., preventing its development; (ii) alleviating the disease or disorder, i.e., causing the remission of said disorder; (iii) slowing the progression of said disorder; and / or (iv) suppressing, alleviating, or slowing the progression of one or more symptoms of said disease or disorder. In some embodiments, treatment means, for example, that symptoms associated with the disease are alleviated, reduced, cured, or in a state of remission.

[0109] It should also be understood that the various treatments for the disorders described herein are intended to be “fundamental,” encompassing both complete and less complete treatment, and in which some biological or medical outcome is achieved. The treatment may be a continuous, extended course of treatment for a chronic condition, or a single or several applications of treatment for an acute condition.

[0110] Anti-MUC16 antibody agent

[0111] This document provides anti-MUC16 antibody agents that specifically bind to the immune system of MUC16. In some embodiments, the anti-MUC16 antibody agent specifically binds to the retained extracellular domain of MUC16. In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 construct comprising an antibody moiety that specifically binds to the immune system of MUC16. In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 antibody (e.g., a full-length anti-MUC16 antibody or its antigen-binding fragment). In some embodiments, the anti-MUC16 antibody agent binds to cells expressing MUC16 (e.g., cancer cells expressing MUC16).

[0112] Anti-MUC16 antibody agents (such as anti-MUC16 antibodies or their antigen-binding fragments) may include, for example, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies (BsAb)), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetramers comprising two heavy chains and two light chains, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain variable fragments (scFv), camel-derived antibodies, affybody and disulfide-linked Fv (dsFv), Fc fusion proteins, immunoconjugates, or fragments thereof. Such antibodies and antigen-binding fragments can be prepared by methods known in the art.

[0113] In some implementations, the anti-MUC16 antibody agent is a full-length antibody (e.g., full-length IgG) that specifically binds to MUC16 or its antigen-binding fragment.

[0114] In some implementations, referring to an antibody that specifically binds to MUC16 means that the antibody's affinity for MUC16 is at least about 10 times (including, for example, at least about 10, 10) greater than its affinity for binding to non-target targets. 2 10 3 10 4 10 5 10 6 Or 10 7 (Any of the two). In some embodiments, the non-target is an antigen that is not MUC16. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence-activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). K d It can be determined by methods known in the art, such as surface plasmon resonance (SPR) measurements using instruments such as Biacore or dynamic repulsion measurements (KinExA) using instruments such as Sapidyne.

[0115] While this document discusses in detail anti-MUC16 antibody agents containing human sequences (e.g., human heavy chain and light chain variable domain sequences containing human CDR sequences), non-human anti-MUC16 antibody agents are also considered. In some embodiments, a non-human anti-MUC16 antibody agent comprises a human CDR sequence and a non-human frame sequence derived from an anti-MUC16 antibody agent as described herein. In some embodiments, the non-human frame sequence includes any sequence that can be used to generate synthetic heavy chain and / or light chain variable domains using one or more human CDR sequences as described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, macaques), etc. In some embodiments, a non-human anti-MUC16 antibody agent comprises an anti-MUC16 antibody agent generated by grafting one or more human CDR sequences as described herein onto a non-human frame sequence (e.g., mouse or chicken frame sequences).

[0116] The complete amino acid sequence of an exemplary human MUC16 includes or is composed of the amino acid sequence of SEQ ID NO:1. In some embodiments, the anti-MUC16 antibody agent described herein specifically recognizes an epitope within human MUC16. In some embodiments, the anti-MUC16 antibody agent described herein specifically recognizes an epitope within a reserved extracellular domain of human MUC16. In some embodiments, the anti-MUC16 antibody agent described herein immune-specifically binds to the extracellular domain of MUC16 (Figure 1). In some embodiments, the anti-MUC16 antibody agent described herein immune-specifically binds to cells expressing human MUC16. In some embodiments, the anti-MUC16 antibody agent described herein immune-specifically binds to cells expressing a recombinant MUC16 peptide. In some embodiments, the MUC16 peptide is MUC16-c344 having the amino acid sequence shown in SEQ ID NO:43. In some embodiments, the MUC16 peptide is MUC16-c114 having the amino acid sequence shown in SEQ ID NO:44.

[0117] In some embodiments, the anti-MUC16 antibody agent cross-reacts with MUC16 peptides from species other than humans. In some embodiments, the anti-MUC16 antibody agent is completely specific to human MUC16 and does not exhibit species-specific or other types of non-human cross-reactivity.

[0118] In some embodiments, the anti-MUC16 antibody agent specifically recognizes MUC16 expressed on the cell surface of cancer cells (such as solid tumors). In some embodiments, the anti-MUC16 antibody agent specifically recognizes MUC16 expressed on the cell surface of one or more of the following: ovarian cancer cells, breast cancer cells, prostate cancer cells, colon cancer cells, lung cancer cells, brain cancer cells, pancreatic cancer cells, kidney cancer cells, fallopian tube cancer cells, uterine (e.g., endometrial) cancer cells, primary peritoneal cancer cells, or cancer cells of any other tissue expressing MUC16. In some embodiments, the anti-MUC16 antibody agent specifically recognizes MUC16 expressed on the cell surface of cancer cell lines (e.g., ovarian cancer cell lines such as OVCAR3, OVCA-432, OVCA-433, and CAOV3).

[0119] In some embodiments, the anti-MUC16 antibody agent cross-reacts with at least one allelic variant of the MUC16 protein or a fragment thereof. In some embodiments, the allelic variant has up to about 30 amino acid substitutions (such as any one of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30) when compared to naturally occurring MUC16 or a fragment thereof, such as conserved amino acid substitutions. In some embodiments, the anti-MUC16 antibody agent does not cross-react with any allelic variant of the MUC16 protein or a fragment thereof.

[0120] In some embodiments, the anti-MUC16 antibody agent cross-reacts with at least one interspecies variant of the MUC16 protein. In some embodiments, for example, the MUC16 protein or a fragment thereof is human MUC16, and the interspecies variant of the MUC16 protein or a fragment thereof is its mouse or rat variant. In some embodiments, the anti-MUC16 antibody agent does not cross-react with any interspecies variant of the MUC16 protein.

[0121] In some embodiments, any anti-MUC16 antibody agent described herein comprises an anti-MUC16 antibody portion that specifically binds to MUC16.

[0122] In some embodiments, the anti-MUC16 antibody portion includes an antibody heavy chain variable domain and an antibody light chain variable domain. In some embodiments, the anti-MUC16 antibody portion includes the antibody heavy chain variable domain and the antibody light chain variable domain of the humanized 18C6 anti-MUC16 antibody.

[0123] Humanized 4H11 anti-MUC16 antibody

[0124] In some embodiments, the anti-MUC16 antibody agent described herein comprises the antibody heavy chain variable domain and / or antibody light chain variable domain of the 4H11 anti-MUC16 antibody (PCT Publication No. WO2011 / 119979), wherein one or more amino acid residues of one or more framework regions of the 4H11 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain are modified with corresponding amino acids in the human antibody heavy chain framework region (HC-FW) or light chain framework region (LC-FW).

[0125] In some embodiments, amino acid residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more of the frame region of the mouse 4H11 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain are modified to the corresponding amino acids in human antibody HC-FW and / or LC-FW. In some embodiments, the human LC-FW is derived from the immunoglobulin κ variable 4-1 (IGKV4-1) gene or the immunoglobulin κ conjugating 2 (IGKJ2) gene. In some embodiments, the human HC-FW is derived from the immunoglobulin heavy chain variable 3-21 (IGHV3-21) gene. In some embodiments, the anti-MUC16 antibody agents described herein are more mouse-like, meaning that about 10 or fewer amino acid residues of the frame region of the mouse 4H11 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain are modified with corresponding amino acids in the human antibody HC-FW or LC-FW. In some embodiments, the more human amino acid substitutions are made relative to the mouse 4H11 anti-MUC16 antibody sequence, the lower the expected immunogenicity of the anti-MUC16 antibody agent when administered to humans. In some embodiments, one or more amino acids may be unmodified relative to the mouse sequence to maintain the antibody's structure and / or activity.

[0126] In some embodiments, the anti-MUC16 antibody moiety comprises a heavy chain variable domain containing one, two, or three HC-CDRs of SEQ ID NO:4 or 5. In some embodiments, the anti-MUC16 antibody moiety comprises heavy chain variable domains of HC-CDR1, HC-CDR2, and HC-CDR3 containing a heavy chain variable domain of SEQ ID NO:4 or 5. In some embodiments, the anti-MUC16 antibody moiety comprises heavy chain variable domains containing HC-CDR1, HC-CDR2, and HC-CDR3 as shown in SEQ ID NO:17, 18, and 19, respectively.

[0127] In some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains containing heavy chain framework regions 1 (HC-FW1), HC-FW2, and HC-FW3 as shown in SEQ ID NO:124, 125, and 126, respectively, wherein one or more amino acid residues in HC-FW1, HC-FW2, and / or HC-FW3 are modified to the corresponding amino acids in human HC-FW1, HC-FW2, and / or HC-FW3, respectively. In some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains containing heavy chain framework regions 1 (HC-FW1), HC-FW2, and HC-FW3 as shown in SEQ ID NO:124, 125, and 126, respectively, wherein one or more amino acid residues in HC-FW1, HC-FW2, and / or HC-FW3 are modified to the corresponding amino acids in human HC-FW1, HC-FW2, and / or HC-FW3 as shown in SEQ ID NO:127, 128, and 129.

[0128] Alternatively or concurrently, in some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains containing HC-FW1, HC-FW2, and HC-FW3 as shown in SEQ ID NO: 136, 137, and 138, wherein X at position 1 of SEQ ID NO: 136 is S or E, X at position 3 of SEQ ID NO: 136 is K or Q, X at position 5 of SEQ ID NO: 136 is Q or V, X at position 11 of SEQ ID NO: 136 is F or L, and X at position 19 of SEQ ID NO: 136 is K or R; X at position 5 of SEQ ID NO: 137 is S or A, X at position 7 of SEQ ID NO: 137 is E or G, X at position 8 of SEQ ID NO: 137 is M or K, and X at position 9 of SEQ ID NO: 137 is R or G; X at position 12 of SEQ ID NO: 138 is T or S, and X at position 138 is Q or V, respectively. X at position 14 of SEQ ID NO:138 is H or Y, X at position 18 of SEQ ID NO:138 is G or N, X at position 22 of SEQ ID NO:138 is S or A, and / or X at position 23 of SEQ ID NO:138 is G or E.

[0129] In some embodiments, the anti-MUC16 antibody moiety comprises heavy chain variable domains containing HC-FW1, HC-FW2, and HC-FW3, respectively, of SEQ ID NO:130, 131, and 132. In some embodiments, the anti-MUC16 antibody moiety comprises heavy chain variable domains containing HC-FW1, HC-FW2, and HC-FW3, respectively, of SEQ ID NO:130, 131, and 132, or variants thereof comprising up to about 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:130, 131, and 132. In some embodiments, the anti-MUC16 antibody moiety comprises heavy chain variable domains containing HC-FW1, HC-FW2, and HC-FW3, respectively, of SEQ ID NO:133, 134, and 135. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing HC-FW1, HC-FW2, and HC-FW3 of SEQ ID NO:133, 134, and 135, respectively, or a variant thereof containing up to about 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:133, 134, and 135.

[0130] In some embodiments, the anti-MUC16 antibody portion includes a heavy chain variable domain containing SEQ ID NO:4 or 5.

[0131] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing one, two, or three LC-CDRs of SEQ ID NO: 2 or 3. In some embodiments, the anti-MUC16 antibody portion comprises light chain variable domains of LC-CDR1, LC-CDR2, and LC-CDR3 containing a light chain variable domain of SEQ ID NO: 2 or 3. In some embodiments, the anti-MUC16 antibody portion comprises light chain variable domains containing LC-CDR1, LC-CDR2, and LC-CDR3 as shown in SEQ ID NO: 14, 15, and 16, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing SEQ ID NO: 2 or 3. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain as shown in SEQ ID NO: 2 or 3.

[0132] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing light chain framework region 1 (LC-FW1), LC-FW2, LC-FW3, and LC-FW4 as shown in SEQ ID NO: 104, 105, 106, and 107, respectively, wherein one or more amino acid residues in LC-FW1, LC-FW2, LC-FW3, and / or LC-FW4 are modified with the corresponding amino acid in human LC-FW1, LC-FW2, LC-FW3, and / or LC-FW4. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing LC-FW1, LC-FW2, LC-FW3, and LC-FW4, as shown in SEQ ID NO:104, 105, 106, and 107, respectively, wherein one or more amino acid residues of LC-FW1, LC-FW2, LC-FW3, and / or LC-FW4 are modified to the corresponding amino acids of human LC-FW1, LC-FW2, LC-FW3, and / or LC-FW4 as shown in SEQ ID NO:108, 109, 110, and 111.

[0133] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing LC-FW1, LC-FW2, LC-FW3, and LC-FW4 as shown in SEQ ID NO:120, 121, 122, and 123, wherein X at position 3 of SEQ ID NO:120 is E or V, X at position 9 of SEQ ID NO:120 is S or D, X at position 15 of SEQ ID NO:120 is A or L, X at position 18 of SEQ ID NO:120 is K or R, X at position 22 of SEQ ID NO:120 is S or N; X at position 7 of SEQ ID NO:122 is T or S, X at position 27 of SEQ ID NO:122 is L or V, X at position 3 of SEQ ID NO:123 is P or Q, and / or X at position 9 of SEQ ID NO:123 is V or I.

[0134] In some embodiments, the anti-MUC16 antibody moiety comprises a light chain variable domain containing LC-FW1, LC-FW2, LC-FW3, and LC-FW4, respectively, of SEQ ID NO:112, 113, 114, and 115. In some embodiments, the anti-MUC16 antibody moiety comprises a light chain variable domain containing LC-FW1, LC-FW2, LC-FW3, and LC-FW4, respectively, of SEQ ID NO:112, 113, 114, and 115, or a variant comprising up to about 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:112, 113, 114, and 115. In some embodiments, the anti-MUC16 antibody moiety comprises a light chain variable domain containing LC-FW1, LC-FW2, LC-FW3, and LC-FW4 of SEQ ID NO: 116, 117, 118, and 119, respectively. In some embodiments, the anti-MUC16 antibody moiety comprises a light chain variable domain containing LC-FW1, LC-FW2, LC-FW3, and LC-FW4 of SEQ ID NO: 116, 117, 118, and 119, respectively, or a variant comprising up to about 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 116, 117, 118, and 119.

[0135] In some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains of HC-CDR1, HC-CDR2, and HC-CDR3 containing the heavy chain variable domain of SEQ ID NO:4 or 5, and light chain variable domains of LC-CDR1, LC-CDR2, and LC-CDR3 containing the light chain variable domain of SEQ ID NO:2 or 3. In some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains of HC-CDR1, HC-CDR2, and HC-CDR3 as shown in SEQ ID NO:17, 18, and 19, respectively, and light chain variable domains of LC-CDR1, LC-CDR2, and LC-CDR3 as shown in SEQ ID NO:14, 15, and 16, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing SEQ ID NO:4 or 5, and a light chain variable domain containing SEQ ID NO:2 or 3. In some embodiments, the anti-MUC16 antibody portion includes the heavy chain variable domain shown in SEQ ID NO:4 or 5, and the light chain variable domain shown in SEQ ID NO:2 or 3.

[0136] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing the following: HC-FW1 shown in SEQ ID NO:130, HC-FW2 shown in SEQ ID NO:131, HC-FW3 shown in SEQ ID NO:132, LC-FW1 shown in SEQ ID NO:112, LC-FW2 shown in SEQ ID NO:113, LC-FW3 shown in SEQ ID NO:114, and / or LC-FW4 shown in SEQ ID NO:115.

[0137] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing the following: HC-FW1 shown in SEQ ID NO:133, HC-FW2 shown in SEQ ID NO:134, HC-FW3 shown in SEQ ID NO:135, LC-FW1 shown in SEQ ID NO:116, LC-FW2 shown in SEQ ID NO:117, LC-FW3 shown in SEQ ID NO:118, and / or LC-FW4 shown in SEQ ID NO:119.

[0138] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing HC-CDR1, HC-CDR2, and HC-CDR3 as shown in SEQ ID NO:17, 18, and 19, and HC-FW1, HC-FW2, and HC-FW3 as shown in SEQ ID NO:130, 131, and 132, respectively; and a light chain variable domain containing LC-CDR1, LC-CDR2, and LC-CDR3 as shown in SEQ ID NO:14, 15, and 16, and LC-FW1, LC-FW2, LC-FW3, and LC-FW4 as shown in SEQ ID NO:112, 113, 114, and 115, respectively.

[0139] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing HC-CDR1, HC-CDR2, and HC-CDR3 as shown in SEQ ID NO:17, 18, and 19, and HC-FW1, HC-FW2, and HC-FW3 as shown in SEQ ID NO:133, 134, and 135, respectively; and a light chain variable domain containing LC-CDR1, LC-CDR2, and LC-CDR3 as shown in SEQ ID NO:14, 15, and 16, and LC-FW1, LC-FW2, LC-FW3, and LC-FW4 as shown in SEQ ID NO:116, 117, 118, and 119, respectively.

[0140] In some embodiments, the antibody heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:4 or 5, or a variant thereof comprising up to about 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:4 or 5. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO:2 or 3, or a variant thereof comprising up to about 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:2 or 3.

[0141] Humanized 18C6 anti-MUC16 antibody agent

[0142] In some embodiments, the anti-MUC16 antibody agent described herein comprises the antibody heavy chain variable domain and / or antibody light chain variable domain of the 18C6 anti-MUC16 antibody (PCT Publication No. WO2016 / 149368), wherein one or more amino acid residues of one or more framework regions of the 18C6 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain are modified with corresponding amino acids in the human antibody heavy chain framework region (HC-FW) or light chain framework region (LC-FW).

[0143] In some embodiments, amino acid residues 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more of the frame region of the mouse 18C6 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain are modified to the corresponding amino acids in human antibody HC-FW and / or LC-FW. In some embodiments, human LC-FW is derived from the immunoglobulin κ variable 2-28 (IGKV2-28) gene or the immunoglobulin κ conjugating 4 (IGKJ4) gene. In some embodiments, human HC-FW is derived from the immunoglobulin heavy chain variable 2-5 (IGHV2-5) gene. In some embodiments, the anti-MUC16 antibody agents described herein are more mouse-like, meaning that about 10 or fewer amino acid residues of the frame region of the mouse 18C6 anti-MUC16 heavy chain variable domain and / or antibody light chain variable domain are modified with corresponding amino acids in the human antibody HC-FW or LC-FW. In some embodiments, the more human amino acid substitutions relative to the mouse 18C6 anti-MUC16 antibody sequence, the lower the expected immunogenicity of the anti-MUC16 antibody agent when administered to humans. In some embodiments, one or more amino acids may be unmodified relative to the mouse sequence to maintain the antibody's structure and / or activity.

[0144] In some embodiments, the anti-MUC16 antibody moiety comprises a heavy chain variable domain containing one, two, or three HC-CDRs of SEQ ID NO: 22 or 23. In some embodiments, the anti-MUC16 antibody moiety comprises heavy chain variable domains of HC-CDR1, HC-CDR2, and HC-CDR3 containing the heavy chain variable domain of SEQ ID NO: 22 or 23. In some embodiments, the anti-MUC16 antibody moiety comprises heavy chain variable domains containing HC-CDR1, HC-CDR2, and HC-CDR3 as shown in SEQ ID NO: 35, 36, and 37, respectively.

[0145] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing heavy chain framework region 1 (HC-FW1), HC-FW2, HC-FW3, and HC-FW4 as shown in SEQ ID NO:159, 160, 161, and 162, respectively, wherein one or more amino acid residues in HC-FW1, HC-FW2, HC-FW3, and / or HC-FW4 are modified with the corresponding amino acid in human HC-FW1, HC-FW2, HC-FW3, and / or HC-FW4. In some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains containing heavy chain framework region 1 (HC-FW1), HC-FW2, HC-FW3, and HC-FW4, as shown in SEQ ID NO:159, 160, 161, and 162, respectively, wherein one or more amino acid residues in HC-FW1, HC-FW2, HC-FW3, and / or HC-FW4 are modified to the corresponding amino acids in human HC-FW1, HC-FW2, HC-FW3, and / or HC-FW4 as shown in SEQ ID NO:163, 164, 165, and 166, respectively.

[0146] In some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains containing heavy chain framework regions 1 (HC-FW1), HC-FW2, HC-FW3, and HC-FW4, respectively, as shown in SEQ ID NO:175, 176, 177, and 178, wherein X at position 10 of SEQ ID NO:175 is G or T, X at position 11 of SEQ ID NO:175 is I or L, X at position 12 of SEQ ID NO:175 is L or V, X at position 13 of SEQ ID NO:175 is Q or K, X at position 15 of SEQ ID NO:175 is S or T, X at position 19 of SEQ ID NO:175 is S or T, X at position 23 of SEQ ID NO:175 is S or T; X at position 5 of SEQ ID NO:177 is S or T, X at position 14 of SEQ ID NO:177 is F or V, and X at position 15 of SEQ ID NO:175 is S or T. X at position 16 of SEQ ID NO:177 is K or T, X at position 18 of SEQ ID NO:177 is A or T, X at position 22 of SEQ ID NO:177 is T or P, X at position 23 of SEQ ID NO:177 is A or V; and / or X at position 6 of SEQ ID NO:178 is S or L.

[0147] In some embodiments, the anti-MUC16 antibody moiety comprises a heavy chain variable domain containing HC-FW1, HC-FW2, HC-FW3, and HC-FW4, respectively, of SEQ ID NO:167, 168, 169, and 170. In some embodiments, the anti-MUC16 antibody moiety comprises a heavy chain variable domain containing HC-FW1, HC-FW2, HC-FW3, and HC-FW4, respectively, of SEQ ID NO:167, 168, 169, and 170, or a variant comprising up to about 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:167, 168, 169, and 170. In some embodiments, the anti-MUC16 antibody moiety comprises a heavy chain variable domain containing HC-FW1, HC-FW2, HC-FW3, and HC-FW4, respectively, of SEQ ID NO:171, 172, 173, and 174. In some embodiments, the anti-MUC16 antibody moiety comprises a heavy chain variable domain containing HC-FW1, HC-FW2, HC-FW3, and HC-FW4, respectively, of SEQ ID NO:171, 172, 173, and 174, or a variant comprising up to about 5 amino acid substitutions (such as about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:171, 172, 173, and 174.

[0148] In some embodiments, the anti-MUC16 antibody portion includes a heavy chain variable domain containing SEQ ID NO:22 or 23.

[0149] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing one, two, or three LC-CDRs of SEQ ID NO: 20 or 21. In some embodiments, the anti-MUC16 antibody portion comprises LC-CDR1, LC-CDR2, and LC-CDR3 light chain variable domains containing the light chain variable domain of SEQ ID NO: 20 or 21. In some embodiments, the anti-MUC16 antibody portion comprises light chain variable domains containing LC-CDR1, LC-CDR2, and LC-CDR3 as shown in SEQ ID NO: 32, 33, and 34, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing SEQ ID NO: 20 or 21. In some embodiments, the anti-MUC16 antibody portion comprises the light chain variable domain shown in SEQ ID NO: 20 or 21.

[0150] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing light chain framework region 1 (LC-FW1), LC-FW2, LC-FW3, and LC-FW4 as shown in SEQ ID NO: 139, 140, 141, and 142, respectively, wherein one or more amino acid residues in LC-FW1, LC-FW2, LC-FW3, and / or LC-FW4 are modified with the corresponding amino acid in human LC-FW1, LC-FW2, LC-FW3, and / or LC-FW4. In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing LC-FW1, LC-FW2, LC-FW3, and LC-FW4 as shown in SEQ ID NO:139, 140, 141, and 142, respectively, wherein one or more amino acid residues in LC-FW1, LC-FW2, LC-FW3, and / or LC-FW4 are modified to the corresponding amino acids in human LC-FW1, LC-FW2, LC-FW3, and / or LC-FW4 as shown in SEQ ID NO:143, 144, 145, and 146, respectively.

[0151] In some embodiments, the anti-MUC16 antibody portion comprises a light chain variable domain containing LC-FW1, LC-FW2, LC-FW3, and LC-FW4 as shown in SEQ ID NO:155, 156, 157, and 158, respectively, wherein X at position 7 of SEQ ID NO:155 is A or S, X at position 9 of SEQ ID NO:155 is P or L, X at position 11 of SEQ ID NO:155 is V or L, X at position 18 of SEQ ID NO:155 is S or P, X at position 5 of SEQ ID NO:156 is R or K, X at position 9 of SEQ ID NO:157 is R or S, and / or X at position 18 of SEQ ID NO:157 is R or K.

[0152] In some embodiments, the anti-MUC16 antibody moiety comprises a light chain variable domain containing LC-FW1, LC-FW2, LC-FW3, and LC-FW4, respectively, of SEQ ID NO:147, 148, 149, and 150. In some embodiments, the anti-MUC16 antibody moiety comprises a light chain variable domain containing LC-FW1, LC-FW2, LC-FW3, and LC-FW4, respectively, of SEQ ID NO:147, 148, 149, and 150, or a variant comprising up to about 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:147, 148, 149, and 150. In some embodiments, the anti-MUC16 antibody moiety comprises a light chain variable domain containing LC-FW1, LC-FW2, LC-FW3, and LC-FW4 of SEQ ID NO: 151, 152, 153, and 154, respectively. In some embodiments, the anti-MUC16 antibody moiety comprises a light chain variable domain containing LC-FW1, LC-FW2, LC-FW3, and LC-FW4 of SEQ ID NO: 151, 152, 153, and 154, or a variant thereof comprising up to about 5 amino acid substitutions (such as about 1, 2, 3, 4, or any 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 151, 152, 153, and 154.

[0153] In some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains of HC-CDR1, HC-CDR2, and HC-CDR3 containing the heavy chain variable domain of SEQ ID NO: 22 or 23, and light chain variable domains of LC-CDR1, LC-CDR2, and LC-CDR3 containing the light chain variable domain of SEQ ID NO: 20 or 21. In some embodiments, the anti-MUC16 antibody portion comprises heavy chain variable domains of HC-CDR1, HC-CDR2, and HC-CDR3 as shown in SEQ ID NO: 35, 36, and 37, and light chain variable domains of LC-CDR1, LC-CDR2, and LC-CDR3 as shown in SEQ ID NO: 32, 33, and 34, respectively. In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing SEQ ID NO: 22 or 23, and a light chain variable domain containing SEQ ID NO: 20 or 21. In some embodiments, the anti-MUC16 antibody portion includes the heavy chain variable domain shown in SEQ ID NO:22 or 23, and the light chain variable domain shown in SEQ ID NO:20 or 21.

[0154] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing the following: HC-FW1 shown in SEQ ID NO:167, HC-FW2 shown in SEQ ID NO:168, HC-FW3 shown in SEQ ID NO:169, HC-FW4 shown in SEQ ID NO:170, LC-FW1 shown in SEQ ID NO:147, LC-FW2 shown in SEQ ID NO:148, LC-FW3 shown in SEQ ID NO:149, and / or LC-FW4 shown in SEQ ID NO:150.

[0155] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing the following: HC-FW1 shown in SEQ ID NO:171, HC-FW2 shown in SEQ ID NO:172, HC-FW3 shown in SEQ ID NO:173, HC-FW4 shown in SEQ ID NO:174, LC-FW1 shown in SEQ ID NO:151, LC-FW2 shown in SEQ ID NO:152, LC-FW3 shown in SEQ ID NO:153, and / or LC-FW4 shown in SEQ ID NO:154.

[0156] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing HC-CDR1, HC-CDR2, and HC-CDR3 as shown in SEQ ID NO:35, 36, and 37, and HC-FW1, HC-FW2, HC-FW3, and HC-FW4 as shown in SEQ ID NO:167, 168, 169, and 170, respectively; and a light chain variable domain containing LC-CDR1, LC-CDR2, and LC-CDR3 as shown in SEQ ID NO:32, 33, and 34, and LC-FW1, LC-FW2, LC-FW3, and LC-FW4 as shown in SEQ ID NO:147, 148, 149, and 150, respectively.

[0157] In some embodiments, the anti-MUC16 antibody portion comprises a heavy chain variable domain containing HC-CDR1, HC-CDR2, and HC-CDR3 as shown in SEQ ID NO:35, 36, and 37, and HC-FW1, HC-FW2, HC-FW3, and HC-FW4 as shown in SEQ ID NO:171, 172, 173, and 174, respectively; and a light chain variable domain containing LC-CDR1, LC-CDR2, and LC-CDR3 as shown in SEQ ID NO:32, 33, and 34, and LC-FW1, LC-FW2, LC-FW3, and LC-FW4 as shown in SEQ ID NO:151, 152, 153, and 154, respectively.

[0158] In some embodiments, the antibody heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:22 or 23, or a variant thereof comprising up to about 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:22 or 23. In some embodiments, the light chain variable domain comprises the amino acid sequence of SEQ ID NO:20 or 21, or a variant thereof comprising up to about 5 amino acid substitutions (such as any one of about 1, 2, 3, 4, or 5) or having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO:20 or 21.

[0159] Exemplary antibody sequences of the humanized 4H11 and 18C6 antibody agents provided herein are shown in the table below. The exemplary CDR sequences in Table 1 were predicted using the IgBLAST algorithm. See, for example, Ye J. et al., Nucleic Acids Research 41:W34-W40 (2013), the disclosure of which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that many algorithms are known for predicting CDR positions in the variable regions of the antibody heavy and light chains, and antibody agents containing CDRs from the antibodies described herein but based on prediction algorithms other than IgBLAST are within the scope of this art.

[0160] Exemplary antibody heavy and light chain variable region sequences are based on INTERNATIONAL IMMUNOGENETICS INFORMATION (IMGT) is used to define this. See, for example, Lefranc, M.-P. et al., Nucleic Acids Res., 43:D413-422 (2015), the disclosure of which is incorporated herein by reference in its entirety. Those skilled in the art will recognize that V contains antibodies from the antibodies described herein. H or V L Antibody agents based on sequences but algorithms other than IMGT are within the scope of this technology.

[0161] Table 1. Exemplary anti-MUC16 antibody CDR sequences.

[0162]

[0163] Table 2. Exemplary anti-MUC16 antibody VH and VL domain sequences.

[0164]

[0165] In some implementations, the anti-MUC16 antibody portion includes an antibody heavy chain constant region and an antibody light chain constant region.

[0166] In some embodiments, the anti-MUC16 antibody portion includes an IgG1 heavy chain constant region. In some embodiments, the anti-MUC16 antibody portion includes an IgG2 heavy chain constant region. In some embodiments, the anti-MUC16 antibody portion includes an IgG3 heavy chain constant region.

[0167] In some embodiments, the anti-MUC16 antibody portion comprises the IgG1 heavy chain constant region. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 8, 9, 26, 27, or 47.

[0168] In some embodiments, the anti-MUC16 antibody portion comprises an IgG4 heavy chain constant region. In some embodiments, the IgG4 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:48.

[0169] In some embodiments, the anti-MUC16 antibody portion includes a λ light chain constant region. In some embodiments, the light chain constant region includes or consists of the amino acid sequence of SEQ ID NO: 6, 7, 24, 25, or 49.

[0170] In some implementations, the anti-MUC16 antibody portion includes the κ light chain constant region.

[0171] Full-length anti-MUC16 antibody

[0172] In some embodiments, the anti-MUC16 antibody is a full-length anti-MUC16 antibody. In some embodiments, the full-length anti-MUC16 antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the full-length anti-MUC16 antibody includes an IgG constant domain, such as the constant domain of any of IgG1, IgG2, IgG3, and IgG4, including variants thereof. In some embodiments, the full-length anti-MUC16 antibody includes a λ light chain constant region. In some embodiments, the full-length anti-MUC16 antibody includes a κ light chain constant region. In some embodiments, the full-length anti-MUC16 antibody is a full-length human anti-MUC16 antibody. In some embodiments, the full-length anti-MUC16 antibody includes an Fc sequence of a mouse immunoglobulin. In some embodiments, the full-length anti-MUC16 antibody includes an Fc sequence that has been altered or otherwise modified to give it enhanced antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) effector function.

[0173] Therefore, for example, in some embodiments, a full-length anti-MUC16 antibody comprising a constant domain of IgG1 or IgG4 is provided, wherein the anti-MUC16 antibody specifically binds to MUC16 on tumor cells. In some embodiments, IgG1 is human IgG1. In some embodiments, IgG1 is human IgG4. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 8, 9, 26, or 27. In some embodiments, the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 6, 7, 24, or 25. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 8, 9, 26, or 27, and the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 6, 7, 24, or 25. In some embodiments, the binding of an anti-MUC16 antibody to cells expressing MUC16 (e.g., cancer cells expressing MUC16) inhibits tumor growth or metastasis or induces tumor regression. In some embodiments, the binding of an anti-MUC16 antibody to cells expressing MUC16 (e.g., cancer cells expressing MUC16) inhibits in vitro Matrigel invasion of MUC16-expressing cells.

[0174] In some embodiments, a full-length anti-MUC16 antibody comprising a constant domain of IgG1 or IgG4 is provided, wherein the anti-MUC16 antibody comprises a) a heavy chain variable domain containing SEQ ID NO: 4 or 5; and b) a light chain variable domain containing SEQ ID NO: 2 or 3. In some embodiments, IgG1 is human IgG1. In some embodiments, IgG4 is human IgG4. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 8 or 9. In some embodiments, the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 6 or 7. In some embodiments, the anti-MUC16 antibody comprises a heavy chain containing SEQ ID NO: 12 or 13 and a light chain containing SEQ ID NO: 10 or 11.

[0175] In some embodiments, a full-length anti-MUC16 antibody comprising a constant domain of IgG1 or IgG4 is provided, wherein the anti-MUC16 antibody comprises a) a heavy chain variable domain containing SEQ ID NO: 22 or 23; and b) a light chain variable domain containing SEQ ID NO: 20 or 21. In some embodiments, IgG1 is human IgG1. In some embodiments, IgG4 is human IgG4. In some embodiments, the anti-MUC16 heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 26 or 27. In some embodiments, the anti-MUC16 light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 24 or 25. In some embodiments, the anti-MUC16 antibody comprises a heavy chain containing SEQ ID NO: 30 or 31 and a light chain containing SEQ ID NO: 28 or 29.

[0176] Chimeric anti-MUC16 construct

[0177] In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 chimeric antigen receptor (CAR) or a variant thereof that specifically binds to the MUC16 immune response. In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 CAR. CARs are well known in the art, and the anti-MUC16 antibody agent can be any CAR known in the art, such as that described in Sadelain et al., Nature 545:423-431 (2017), the disclosure of which is expressly incorporated herein by reference.

[0178] As used herein, the term "chimeric antigen receptor (CAR)" refers to an artificially constructed hybrid single-chain protein or single-chain polypeptide containing a single-chain variable fragment (scFv) as part of an extracellular antigen-binding domain that is directly or indirectly connected to a transmembrane domain (e.g., a transmembrane domain of an immune cell co-stimulatory signaling molecule) that is in turn directly or indirectly connected to an intracellular immune cell (e.g., a T cell or NK cell) signaling domain. The intracellular signaling domain (ISD) contains a primary signaling sequence from an antigen-dependent TCR-associated T cell activation molecule or a primary immune cell signaling sequence, such as a portion of the intracellular domain of CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. ISDs may also contain co-stimulatory signaling sequences; for example, a portion of the intracellular domain of non-antigen-dependent co-stimulatory molecules such as CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, etc. CARs are characterized by their ability to redirect the specificity and reactivity of immune cells (e.g., T cells or NK cells) to selected targets in an MHC-restricted (in the case of TCR-mimicking antibodies) or non-MHC-restricted (in the case of antibodies against cell surface proteins) manner, thereby utilizing the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition gives CAR-expressing immune cells (e.g., T cells or NK cells) the ability to recognize antigens independently of antigen processing, thereby bypassing major mechanisms of tumor escape.

[0179] In some embodiments, the anti-MUC16 CAR comprises an anti-MUC16 antibody portion of any of the anti-MUC16 antibody portions described herein. For example, in some embodiments, an anti-MUC16 CAR comprising an anti-MUC16 antibody portion is provided. In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 CAR comprises a) a heavy chain variable domain containing SEQ ID NO:4 or 5; and b) a light chain variable domain containing SEQ ID NO:2 or 3. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:4 or 5, or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:2 or 3, or a variant thereof having at least about 95% sequence identity. In some embodiments, the anti-MUC16 CAR comprises a sequence selected from SEQ ID NO:80-83 or 97-99.

[0180] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 CAR comprises a) a heavy chain variable domain containing SEQ ID NO: 22 or 23; and b) a light chain variable domain containing SEQ ID NO: 20 or 21. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 22 or 23, or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 20 or 21, or a variant thereof having at least about 95% sequence identity. In some embodiments, the anti-MUC16 CAR comprises a sequence selected from SEQ ID NO: 84-87 or 100-103.

[0181] In some embodiments, the anti-MUC16 antibody agent is an anti-MUC16 chimeric receptor comprising a transmembrane domain of a T-cell receptor (TCR). For example, in some embodiments, the anti-MUC16 antibody agent is an antibody-T-cell receptor (abTCR) as described in PCT patent application publication number WO2017070608, the disclosure of which is expressly incorporated herein by reference and may be included in one or more claims herein. In some embodiments, the anti-MUC16 abTCR comprises any anti-MUC16 antibody portion according to any anti-MUC16 antibody portion described herein. For example, in some embodiments, an anti-MUC16 abTCR comprising an anti-MUC16 antibody portion is provided.

[0182] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 abTCR comprises a) a heavy chain variable domain containing SEQ ID NO:4 or 5; and b) a light chain variable domain containing SEQ ID NO:2 or 3. In some embodiments, the heavy chain variable domain of the anti-MUC16 abTCR comprises the amino acid sequence of SEQ ID NO:4 or 5, or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:2 or 3, or a variant thereof having at least about 95% sequence identity.

[0183] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 abTCR comprises a) a heavy chain variable domain containing SEQ ID NO: 22 or 23; and b) a light chain variable domain containing SEQ ID NO: 20 or 21. In some embodiments, the heavy chain variable domain of the anti-MUC16 abTCR comprises the amino acid sequence of SEQ ID NO: 22 or 23, or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 20 or 21, or a variant thereof having at least about 95% sequence identity.

[0184] In some embodiments, the anti-MUC16 antibody agent is a chimeric co-stimulatory receptor comprising an anti-MUC16 antibody moiety that specifically binds to MUC16 and a co-stimulatory signaling domain. In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor is capable of stimulating immune cells, which are functionally expressed on the surface of said immune cells upon binding to MUC16. In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor lacks a functional primary immune cell signaling sequence. In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor lacks any primary immune cell signaling sequence. In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor comprises a single polypeptide chain comprising an anti-MUC16 antibody moiety, a transmembrane domain, and a co-stimulatory signaling domain. In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor comprises a first polypeptide chain and a second polypeptide chain, wherein the first and second polypeptide chains together form the anti-MUC16 antibody moiety, the transmembrane module, and the co-stimulatory signaling module comprising the co-stimulatory signaling domain. In some embodiments, the first and second polypeptide chains are separate polypeptide chains, and the anti-MUC16 chimeric co-stimulatory receptor is a multimer, such as a dimer. In some embodiments, the first and second polypeptide chains are covalently linked, such as by peptide bonds or by another chemical bond (such as a disulfide bond). In some embodiments, the first and second polypeptide chains are linked by at least one disulfide bond. In some embodiments, the anti-MUC16 antibody moiety is Fab, Fab', (Fab')2, Fv, or a single-chain Fv (scFv). In some embodiments, the anti-MUC16 scFv comprises a sequence selected from any one of SEQ ID NO:53-68.

[0185] Examples of costimulatory immune cell signaling domains in anti-MUC16 chimeric costimulatory receptors used in this technology include cytoplasmic sequences of co-receptors of T cell receptors (TCRs) that can coordinate with chimeric receptors (e.g., CAR or abTCRs) to initiate signal transduction upon chimeric receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capabilities.

[0186] It is known that the signals generated by the TCR alone are insufficient to fully activate T cells, and secondary or co-stimulatory signals are also required. Therefore, T cell activation can be described as being mediated by two different classes of intracellular signaling sequences: those that initiate antigen-dependent primary activation via the TCR (referred to herein as “primary immune cell signaling sequences”) and those that function in a non-antigen-dependent manner to provide secondary or co-stimulatory signals (referred herein as “co-stimulatory immune cell signaling sequences”).

[0187] Primary immune cell signaling sequences that function in a stimulatory manner may contain signaling motifs called immune receptor tyrosine activation motifs or ITAMs. Examples of primary immune cell signaling sequences containing ITAMs include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. A “functional” primary immune cell signaling sequence is a sequence capable of transducing immune cell activation signals when operatively coupled to an appropriate receptor. A “non-functional” primary immune cell signaling sequence may contain fragments or variants of a primary immune cell signaling sequence that do not transduce immune cell activation signals. The anti-MUC16 chimeric costimulatory receptor described herein lacks a functional primary immune cell signaling sequence, such as a functional signaling sequence containing an ITAM. In some embodiments, the anti-MUC16 chimeric costimulatory receptor lacks any primary immune cell signaling sequence.

[0188] Co-stimulatory immune cell signaling sequences can be part of the intracellular domain of co-stimulatory molecules, including, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0189] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 chimeric co-stimulatory receptor comprises a) a heavy chain variable domain containing SEQ ID NO:4 or 5; and b) a light chain variable domain containing SEQ ID NO:2 or 3. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:4 or 5, or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO:2 or 3, or a variant thereof having at least about 95% sequence identity.

[0190] In some embodiments, the anti-MUC16 antibody portion of the anti-MUC16 chimeric co-stimulatory receptor comprises a) a heavy chain variable domain containing SEQ ID NO: 22 or 23; and b) a light chain variable domain containing SEQ ID NO: 20 or 21. In some embodiments, the heavy chain variable domain comprises the amino acid sequence of SEQ ID NO: 22 or 23, or a variant thereof having at least about 95% (e.g., at least about 96%, 97%, 98%, or 99%) sequence identity, and the light chain variable domain comprises the amino acid sequence of SEQ ID NO: 20 or 21, or a variant thereof having at least about 95% sequence identity.

[0191] In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor is expressed in immune cells. In some embodiments, the anti-MUC16 chimeric co-stimulatory receptor is expressed in immune cells expressing another chimeric receptor. In some embodiments, the other chimeric receptor is a CAR or abTCR. In some embodiments, the other chimeric receptor binds to MUC16. In some embodiments, the other chimeric receptor does not bind to MUC16. In some embodiments, the other chimeric receptor binds to a cancer-associated antigen, said cancer being characterized by high MUC16 expression and / or high aerobic glycolysis. In some embodiments, the other chimeric receptor binds to an antigen associated with any of the cancers described herein (such as kidney cancer, cervical cancer, prostate cancer, breast cancer, colon cancer, brain cancer, or prostate cancer). In some embodiments, the other chimeric receptor binds to an antigen associated with kidney cancer. In some embodiments, said kidney cancer is renal cell carcinoma (RCC).

[0192] In some embodiments, the RCC is a metastatic RCC. In some embodiments, the immune cell is a T cell. In some embodiments, the expression of the anti-MUC16 chimeric co-stimulatory receptor in the immune cell is inducible. In some embodiments, the expression of the anti-MUC16 chimeric co-stimulatory receptor in the immune cell is induced after signal transduction via other chimeric receptors.

[0193] Combining affinity

[0194] Binding affinity can be derived from K d K off K on or K a Indicated. As used in this article, the term "K" is used to refer to... off "K" refers to the dissociation rate constant of the antibody agent from the antibody / antigen complex, as determined by the kinetic selection settings. As used herein, the term "K" is... on "K" refers to the binding rate constant of an antibody agent associating with an antigen to form an antibody / antigen complex. As used herein, the term "equilibrium dissociation constant" is... d"" refers to the dissociation constant of a specific antibody-antigen interaction, and describes the concentration required for the antigen to occupy half of all antibody-binding domains present in the antibody molecule solution at equilibrium, and is equal to K. off / K on K d The measurements presuppose that all binding agents are in solution. In cases where the antibody binds to the cell wall (e.g., in a yeast expression system), the corresponding equilibrium rate constant is expressed as EC50, which gives K d A good approximation of the affinity constant K. a It is the dissociation constant K d The reciprocal of.

[0195] Using the dissociation constant (K) d This serves as an indicator of the affinity of the antibody moiety for the antigen. For example, it can be easily analyzed using the Scatchard method with antibodies labeled with various markers, or by using Biacore (manufactured by Amersham Biosciences) (i.e., analyzing biomolecular interactions via surface plasmon resonance) according to the user manual and accompanying kit. K can be derived using these methods. d The value is expressed in units M (moles). The K value of an antibody that specifically binds to the target. d For example, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤10 -13 M.

[0196] The binding specificity of an antibody agent can be determined empirically using methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA, RIA, ECL, IRMA, EIA, BIAcore, and peptide scanning. In some embodiments, the binding affinity of an anti-MUC16 antibody agent is measured by testing the binding affinity of the anti-MUC16 antibody agent to cells expressing MUC16 on their surface (e.g., HepG2 cells).

[0197] In some implementations, the anti-MUC16 antibody agent specifically binds to the K+ target MUC16 (e.g., nMUC16). d For about 10 -7 M to approximately 10 -13 M (as per approximately 10) -7 M to approximately 10 -13 M, approximately 10 -9 M to approximately 10 -13M or approximately 10 -10 M to approximately 10 -12 M). Therefore, in some embodiments, the binding K between the anti-nMUC16 antibody and nMUC16. d The binding K between anti-sMUC16 antibody and sMUC16 d Or the binding of K between anti-MUC16 antibody and MUC16 (in any form). d It is about 10 -7 M to approximately 10 - 13 M, approximately 1×10 -7 M to approximately 5 × 10 -13 M, approximately 10 -7 M to approximately 10 -12 M, approximately 10 -7 M to approximately 10 -11 M, approximately 10 -7 M to approximately 10 -10 M, approximately 10 -7 M to approximately 10 -9 M, approximately 10 -8 M to approximately 10 -13 M, approximately 1×10 -8 M to approximately 5 × 10 -13 M, approximately 10 -8 M to approximately 10 -12 M, approximately 10 -8 M to approximately 10 -11 M, approximately 10 -8 M to approximately 10 -10 M, approximately 10 -8 M to approximately 10 -9 M, approximately 5×10 -9 M to approximately 1×10 -13 M, approximately 5×10 -9 M to approximately 1×10 -12 M, approximately 5×10 -9 M to approximately 1×10 -11 M, approximately 5×10 -9 M to approximately 1×10 -10 M, approximately 10 -9 M to approximately 10 -13 M, approximately 10 -9 M to approximately 10 -12 M, approximately 10 -9 M to approximately 10 -11 M, approximately 10 -9 M to approximately 10 -10 M, approximately 5×10 -10 M to approximately 1×10 -13 M, approximately 5×10 -10 M to approximately 1×10-12 M, approximately 5×10 -10 M to approximately 1×10 -11 M, approximately 10 -10 M to approximately 10 -13 M, approximately 1×10 -10 M to approximately 5 × 10 -13 M to approximately 1×10 -10 M to approximately 1×10 -12 M, approximately 1×10 -10 M to approximately 5 × 10 -12 M, approximately 1×10 -10 M to approximately 1×10 -11 M, approximately 10 -11 M to approximately 10 -13 M, approximately 1×10 -11 M to approximately 5 × 10 -13 M, approximately 10 -11 To about 10 -12 M or approximately 10 -12 M to approximately 10 -13 M. In some implementations, the binding between the anti-nMUC16 antibody and nMUC16 is K. d It is about 10 -7 M to approximately 10 -13 M.

[0198] In some implementations, the binding of the anti-MUC16 antibody to the non-target K d The K value is greater than the binding between the anti-MUC16 antibody and the target. d Furthermore, in some embodiments herein, the binding affinity of the anti-MUC16 antibody to a target (e.g., MUC16 bound to the cell surface) is higher than its binding affinity to a non-target. In some embodiments, the non-target is an antigen that is not MUC16. In some embodiments, the K-value of the binding between the anti-MUC16 antibody (targeting nMUC16) and a non-MUC16 target is... d It could be the K binding between the anti-MUC16 antibody and the target MUC16. d At least about 10 times, such as about 10-100 times, about 100-1000 times, about 10 3 -10 4 times, approximately 10 4 -10 5 times, approximately 10 5 -10 6 times, approximately 10 6 -10 7 times, approximately 10 7 -10 8 times, approximately 10 8 -10 9times, approximately 10 9 -10 10 times, approximately 10 10 -10 11倍 Or about 10 11 -10 12 times.

[0199] In some implementations, the anti-MUC16 antibody agent binds to a non-target K d For about 10 -1 M to approximately 10 -6 M (as per approximately 10) -1 M to approximately 10 -6 M, approximately 10 -1 M to approximately 10 -5 M or approximately 10 -2 M to approximately 10 -4 M). In some embodiments, the non-target is an antigen that is not MUC16. Therefore, in some embodiments, the binding K between the anti-MUC16 antibody and the non-MUC16 target. d It is about 10 -1 M to approximately 10 -6 M, approximately 1×10 -1 M to approximately 5 × 10 -6 M, approximately 10 -1 M to approximately 10 -5 M, approximately 1×10 -1 M to approximately 5 × 10 - 5 M, approximately 10 -1 M to approximately 10 -4 M, approximately 1×10 -1 M to approximately 5 × 10 -4 M, approximately 10 -1 M to approximately 10 -3 M, approximately 1×10 -1 M to approximately 5 × 10 -3 M, approximately 10 -1 M to approximately 10 -2 M, approximately 10 -2 M to approximately 10 -6 M, approximately 1×10 -2 M to approximately 5 × 10 -6 M, approximately 10 -2 M to approximately 10 -5 M, approximately 1×10 - 2 M to approximately 5 × 10 -5 M, approximately 10 -2 M to approximately 10 -4 M, approximately 1×10 -2 M to approximately 5 × 10 -4 M, approximately 10 -2M to approximately 10 -3 M, approximately 10 -3 M to approximately 10 -6 M, approximately 1×10 -3 M to approximately 5 × 10 -6 M, approximately 10 -3 M to approximately 10 -5 M, approximately 1×10 -3 M to approximately 5 × 10 -5 M, approximately 10 -3 M to approximately 10 -4 M, approximately 10 -4 M to approximately 10 -6 M, approximately 1×10 -4 M to approximately 5 × 10 -6 M, approximately 10 -4 M to approximately 10 -5 M or approximately 10 -5 M to approximately 10 -6 M.

[0200] In some implementations, when referring to the anti-MUC16 antibody agent specifically recognizing the target MUC16 (e.g., cell surface-bound MUC16) with high binding affinity and binding non-targets with low binding affinity, the anti-MUC16 antibody agent will have approximately 10 -7 M to approximately 10 -13 M (as per approximately 10) -7 M to approximately 10 -13 M, approximately 10 -9 M to approximately 10 -13 M or approximately 10 -10 M to approximately 10 -12 M) of K d It binds to the target MUC16 (e.g., cell surface-bound MUC16) and will be at approximately 10 -1 M to approximately 10 -6 M (as per approximately 10) -1 M to approximately 10 -6 M, approximately 10 -1 M to approximately 10 -5 M or approximately 10 -2 M to approximately 10 -4 M) of K d Binds to non-targets.

[0201] In some embodiments, when referring to the specific recognition of cell surface-bound MUC16 by an anti-MUC16 antibody agent, the binding affinity of the anti-MUC16 antibody agent is compared to that of a control anti-MUC16 antibody agent. In some embodiments, the K-value of the binding between the control anti-MUC16 antibody agent and cell surface-bound MUC16 is specified. dIt could be the K-linked binding between the anti-nMUC16 antibody agent described in this article and MUC16 bound to the cell surface. d At least approximately 2 times, such as approximately 2 times, approximately 3 times, approximately 4 times, approximately 5 times, approximately 6 times, approximately 7 times, approximately 8 times, approximately 9 times, approximately 10 times, approximately 10-100 times, approximately 100-1000 times, approximately 10 3 -10 4 times, approximately 10 4 -10 5 times, approximately 10 5 -10 6 times, approximately 10 6 -10 7 times, approximately 10 7 -10 8 times, approximately 10 8 -10 9 times, approximately 10 9 -10 10 times, approximately 10 10 -10 11 times or about 10 11 -10 12 times.

[0202] Functional activity of anti-Muc16 antibody

[0203] In some embodiments, the anti-MUC16 antibody agent described herein, or its antigen-binding fragment, inhibits in vitro Matrigel invasion of cells recombinantly expressing the MUC16 peptide. In some embodiments, MUC16 comprises SEQ ID NO:44 (MUC16 c114). In some embodiments, the cells recombinantly expressing glycosylated MUC16 c114 are SKOV3 cells. In some embodiments, the MUC16 peptide is glycosylated. In some embodiments, the glycosylated form of the MUC16 peptide is N-glycosylated at amino acid residue Asn30 (corresponding to Asn1806 of mature MUC16 (SEQ ID NO:1)). In some embodiments, the MUC16 peptide is N-glycosylated at amino acid residues Asn24 and Asn30 (corresponding to Asn1800 and Asn1806 of mature MUC16 (SEQ ID NO:1), respectively). In some embodiments, the MUC16 peptide undergoes N-glycosylation at amino acid residues Asn1, Asn24, and Asn30 (also referred to as Asn1777, Asn1800, and Asn1806, respectively, in Yin and Lloyd (2001) J Biol Chem 276:27371-27375). In some embodiments, the glycosylation comprises an N-linked chitobiose. In some embodiments, the glycosylation consists of an N-linked chitobiose. In some embodiments, Matrigel invasion is inhibited by at least 1.25, 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold compared to in vitro Matrigel invasion of cells treated with a control antibody (e.g., an antibody not targeting MUC16). In some embodiments, such as when cells are treated with a control antibody (e.g., an antibody that does not target MUC16), Matrigel invasion is inhibited by approximately 1.25, 1.5, 1.75, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold in vitro compared to Matrigel invasion of the cells in vitro.

[0204] Assays used to determine the inhibition of Matrigel invasion mediated by MUC16 anti-MUC16 antibody agents or antigen-binding fragments are known to those skilled in the art. For example, BD BioCoat... TM Matrigel TMInvasive inserts or chambers (catalog number 354480, in 24-well plates) and control inserts (catalog number 354578, in 24-well plates) were available from BD Biosciences, Massachusetts. The Matrigel invasion assay was performed according to the manufacturer's protocol. In short, the Matrigel chambers in the 24-well plates (stored at -20°C) and the control inserts (stored at 4°C) were allowed to reach room temperature. Both inserts were rehydrated for 2 hours in a 37°C, 5% CO2 humidified incubator with 0.5 mL of serum-free medium in the inserts and in the outer wells of the 24-well plate. The cultured SKOV3 cells were treated with trypsin and washed with medium. One million cells were aliquoted into another centrifuge tube and washed three times with serum-free medium. These cells were then conditioned to yield 5,000 cells per 0.5 mL serum-free medium. The culture medium was removed from the rehydrated inserts, and the inserts were transferred to new 24-well plates containing 0.75 mL of medium containing 10% fetal bovine serum (FBS) in each well as a chemical attractant. Cells (5,000 cells) in 0.5 mL of serum-free medium were immediately added to the inserts. Proper care was taken to ensure no air bubbles were trapped in the inserts or outer wells. The 24-well plates were incubated at 37°C in a 5% CO2 humidified incubator for 48 h. After incubation, non-invasive cells were removed from the upper surface of the membrane by “wiping” with a cotton swab inserted into either the Matrigel or control insert, applying gentle pressure as the swab tip moved across the membrane surface. This wiping was repeated using a second swab moistened with culture medium. The inserts were then stained for 30 min in new 24-well plates containing 0.5 mL of 0.5% crystal violet dye in distilled water. After staining, the inserts were rinsed in three beakers of distilled water to remove excess dye. The insert was air-dried in a new 24-well plate. Invading cells were manually counted under an inverted microscope at 200x magnification. Counts were taken from three fields of view of the membrane and recorded in the figure.

[0205] In some embodiments, in mouse model studies, the anti-MUC16 antibody agent or its antigen-binding fragment described herein can inhibit or reduce metastasis, suppress tumor growth, or induce tumor regression. For example, tumor cell lines can be introduced into athymic nude mice, and the anti-MUC16 antibody agent or its antigen-binding fragment described herein can be administered to athymic mice once or multiple times, with tumor progression of the injected tumor cells monitored over periods of weeks and / or months. In some cases, administration of the anti-MUC16 antibody agent or its antigen-binding fragment to athymic nude mice can be performed prior to the introduction of tumor cell lines. In one embodiment, for the mouse xenograft model described herein, SKOV3 cells expressing MUC16 c114 are used.

[0206] In some embodiments, in mouse models, the anti-MUC16 antibody agent or its antigen-binding fragment described herein inhibits tumor growth or induces tumor regression by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% compared to mice subjected to mimicry treatment, as assessed by methods described herein or known to those skilled in the art. In some embodiments, in mouse models, the anti-MUC16 antibody agent or its antigen-binding fragment described herein inhibits tumor growth or induces tumor regression by at least about 25% or 35%, optionally about 75%, compared to mice subjected to mimicry treatment, as assessed by methods described herein or known to those skilled in the art. In some embodiments, in mouse models, the anti-MUC16 antibody agent or its antigen-binding fragment described herein inhibits tumor growth or induces tumor regression by at least about 1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times, as assessed by methods described herein or known to those skilled in the art, compared to mice subjected to simulant treatment. Simulant-treated mice may be treated, for example, with phosphate-buffered saline or a control (e.g., an anti-IgG antibody).

[0207] Tumor growth inhibition or regression can be determined, for example, by monitoring tumor size over a period of time (e.g., by physical measurement of palpable tumors) or other visual detection methods. For instance, tumor cell lines can be generated to recombinantly express visualization agents such as green fluorescent protein (GFP) or luciferase, and GFP can then be visualized in vivo using a microscope. In vivo visualization of luciferase can be performed by administering a luciferase substrate to xenograft mice and detecting the luminescence produced due to luciferase processing of the substrate. The extent or level of detection of GFP or luciferase is correlated with tumor size in xenograft mice.

[0208] In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein may increase animal survival in tumor xenograft models, as compared to mice subjected to simulant treatment. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein increases mouse survival in tumor xenograft models by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methods described herein or known to those skilled in the art, as evaluated by methods described herein or known to those skilled in the art. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein increases mouse survival in tumor xenograft models by at least about 25% or 35%, optionally about 75%, as assessed by methods described herein or known to those skilled in the art, as compared to mice subjected to simulant treatment in tumor xenograft models. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein increases mouse survival in tumor xenograft models by at least about 1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, as assessed by methods described herein or known to those skilled in the art. Survival can be determined, for example, by plotting a survival curve of the number of surviving mice relative to time (e.g., days or weeks) after injection of the tumor cell line. Simulated-treatment mice can be treated, for example, with phosphate-buffered saline or a control (e.g., an anti-IgG antibody).

[0209] In some embodiments, after contacting cells expressing the MUC16 peptide with the anti-MUC16 antibody agent or its antigen-binding fragment described herein, the anti-MUC16 antibody agent or its antigen-binding fragment is internalized into the cells. When referring to molecules internalized by cells, "internalized" or "internalization" means that a molecule in contact with the extracellular surface of the cell membrane crosses the cell membrane to the intracellular surface of the cell membrane and / or to the cytoplasm. In some embodiments, the cells recombinantly expressing glycosylated MUC16 c114 are SKOV3 cells. In some embodiments, the glycosylated form of MUC16 c114 is, for example, N-glycosylation at Asn1, Asn24, and Asn30 of SEQ ID NO:44 (also referred to as Asn1777, Asn1800, and Asn1806, respectively, in Yin and Lloyd (2001) J Biol Chem 276:27371-27375). In some embodiments, the glycosylation comprises an N-linked chitobiose. In some embodiments, the glycosylation consists of an N-linked chitobiose.

[0210] Assays, such as those using radiolabeled antibodies to determine the internalization of the anti-MUC16 antibody agent or its antigen-binding fragment described herein into cells, are known to those skilled in the art. For example, studies can be conducted on SKOV3 cells expressing MUC16 c114. 89 Internalization of Zr-labeled antibodies. In short, approximately 1x10... 5Cells were seeded in 12-well plates and incubated overnight at 37°C in a 5% CO2 incubator. A volume of radiolabeled protein was added to each well, and the plates were incubated at 37°C and 4°C for 1, 5, 12, and 24 hours. After each incubation period, the culture medium was collected, and the cells were washed with 1 mL of phosphate-buffered saline (PBS). Surface binding activity was collected by washing the cells at 4°C with 1 mL of 100 mM acetic acid and 100 mM glycine (1:1, pH 3.5). Adhering cells were then lysed with 1 mL of 1 M NaOH. Each wash was collected, and the activity was counted. The percentage of internalization was determined using the ratio of the activity of the final wash to the total activity of all washes. In some embodiments, the assay was performed at 37°C. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment undergoes internalization in at least 1%, 2%, 3%, 5%, 6%, 7%, 8%, 9%, or 10% of cells incubated with the anti-MUC16 antibody or its antigen-binding fragment. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment undergoes internalization in about 1%, 2%, 3%, 5%, 6%, 7%, 8%, 9%, or 10% of cells incubated with the anti-MUC16 antibody or its antigen-binding fragment. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment undergoes internalization within 1, 2, 3, 4, 8, 12, 16, 20, or 24 hours after cells are contacted with the anti-MUC16 antibody or its antigen-binding fragment.

[0211] Nucleic acid

[0212] Also considered are nucleic acid molecules encoding anti-MUC16 antibody agents or antigen-binding fragments thereof (such as anti-MUC16 antibodies, for example, full-length anti-MUC16 antibodies). In some embodiments, a nucleic acid (or set of nucleic acids) encoding a full-length anti-MUC16 antibody (including any full-length anti-MUC16 antibody described herein) or an antigen-binding fragment thereof is provided. In some embodiments, the nucleic acid (or set of nucleic acids) encoding the anti-MUC16 antibody agent described herein may also contain a nucleic acid sequence encoding a peptide tag (such as a protein purification tag, e.g., a His-tag, HA tag).

[0213] Also considered here are isolated host cells containing an anti-MUC16 antibody, an isolated nucleic acid encoding a polypeptide component of the anti-MUC16 antibody, or a vector containing a nucleic acid encoding a polypeptide component of the anti-MUC16 antibody described herein.

[0214] This application also includes variants of these nucleic acid sequences. For example, the variants include nucleotide sequences that hybridize under at least moderately stringent hybridization conditions to a nucleic acid sequence encoding an anti-MUC16 antibody agent of this application (such as an anti-MUC16 antibody, for example, a full-length anti-MUC16 antibody), its antigen-binding fragment, or an anti-MUC16 antibody portion thereof.

[0215] The present invention also provides a vector in which the nucleic acid of the present invention is inserted.

[0216] In summary, the expression of an anti-MUC16 antibody (e.g., a full-length anti-MUC16 antibody) or its antigen-binding fragment via a natural or synthetic nucleic acid encoding an anti-MUC16 antibody can be achieved by inserting the nucleic acid into a suitable expression vector, such that the nucleic acid is operatively linked to 5' and 3' regulatory elements (including, for example, promoters (e.g., lymphocyte-specific promoters)) and a 3' untranslated region (UTR). The vector is suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, a start sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0217] The nucleic acids of this technology can also be used for nucleic acid immunotherapy and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, this technology provides gene therapy vectors.

[0218] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors, including but not limited to plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0219] Alternatively, expression vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (see, for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0220] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the cells of a recipient, either in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses (such as lentiviruses) are validating tools for achieving long-term gene transfer because they allow for the long-term stable integration of transgenes and their spread in daughter cells. Lentiviral vectors offer the added advantage of being able to transduce non-proliferating cells, such as hepatocytes, compared to vectors derived from oncogenic retroviruses (such as murine leukemia virus). They also have the added advantage of lower immunogenicity.

[0221] Additional promoter elements (such as enhancers) regulate the frequency of transcription initiation. Typically, these elements are located within a 30-110 bp region upstream of the start site, but recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, allowing promoter function to be preserved when elements are reversed or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can increase to 50 bp before activity begins to decline.

[0222] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide operatively linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rouss' sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, this technique should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of this technique. Inducible promoters provide molecular switches that enable expression when expression of a polynucleotide sequence operatively linked to the promoter is required, or shut down expression when it is not required. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0223] In some embodiments, the expression of the anti-MUC16 antibody is inducible. In some embodiments, the nucleic acid encoding the anti-MUC16 antibody is operatively linked to an inducible promoter (including any inducible promoter described herein).

[0224] Inducible promoters

[0225] Inducible promoters provide molecular switches that enable expression when expression of a polynucleotide sequence operatively linked to the promoter is required, or shut down expression when it is not required. Exemplary inducible promoter systems for eukaryotic cells include, but are not limited to, hormone-regulating elements (e.g., see Mader, S. and White, JH Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)), synthetic ligand-regulating elements (e.g., Spencer, DM et al. 1993 Science 262:1019-1024), and ionizing radiation-regulating elements (e.g., see Manome, Y. et al., Biochemistry 32:10607-10613 (1993); Datta, R. et al., Proc. Natl. Acad. Sci. USA 89:1014-10153 (1992)). Other exemplary inducible promoter systems for use in in vitro or in vivo mammalian systems are reviewed in Gingrich et al., Annual Rev. Neurosci 21:377-405 (1998). In some embodiments, the inducible promoter system for expressing an anti-MUC16 antibody is the Tet system. In some embodiments, the inducible promoter system for expressing an anti-MUC16 antibody is a lac repressor system derived from Escherichia coli (E. coli).

[0226] An exemplary inducible promoter system used in this technology is the Tet system. Such systems are based on the Tet system described by Gossen et al., (1993). In one exemplary embodiment, the target polynucleotide is under the control of a promoter containing one or more Tet operator (TetO) sites. In the inactive state, the Tet repressor (TetR) binds to the TetO site and represses transcription from the promoter. In the active state, for example, in the presence of an inducer (such as tetracycline (Tc), anhydrous tetracycline, doxycycline (Dox), or an active analog thereof), the inducer causes the release of TetR from TetO, thereby allowing transcription to occur. Doxycycline is a member of the tetracycline antibiotic family and has the chemical name 1-dimethylamino-2,4a,5,7,12-pentahydroxy-11-methyl-4,6-dioxo-1,4a,11,11a,12,12a-hexahydrotetraphenyl-3-carboxamide.

[0227] In one implementation, TetR performs codon optimization for expression in mammalian cells (e.g., mouse or human cells). Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, allowing for a large number of variations in the nucleotide sequence of a given nucleic acid without any alteration to the amino acid sequence encoded by the nucleic acid. However, many organisms exhibit differences in codon usage, a phenomenon known as "codon bias" (i.e., biased use of one or more specific codons for a given amino acid). Codon bias is often associated with the presence of a predominant tRNA species for a particular codon, which in turn increases the efficiency of mRNA translation. Therefore, coding sequences derived from a specific organism (e.g., prokaryotes) can be customized through codon optimization to improve expression in different organisms (e.g., eukaryotes).

[0228] Other specific variations of the Tet system include the following “Tet-Off” and “Tet-On” systems. In the Tet-Off system, transcription is inactive in the presence of Tc or Dox. In this system, the expression of target nucleic acids under the transcriptional control of tetracycline-controlled transactivator proteins (tTA), consisting of TetR fused to a strong transactivation domain from VP16 of herpes simplex virus, regulates the expression of target nucleic acids under the transcriptional control of tetracycline-responsive promoter elements (TREs). TREs consist of TetO sequence polymers fused to a promoter (typically a small promoter sequence derived from the immediate early promoter of human cytomegalovirus (hCMV)). In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and expression of the target gene remains inactive.

[0229] Conversely, in the Tet-On system, transcription is active in the presence of either Tc or Dox. The Tet-On system is based on the inverse tetracycline-controlled transactivator rtTA. Like tTA, rtTA is a fusion protein composed of a TetR repressor and a VP16 transactivation domain. However, a four-amino acid change in the TetR DNA-binding moiety alters the binding signature of rtTA, allowing it to recognize the tetO sequence in the TRE of the target transgene only in the presence of Dox. Therefore, in the Tet-On system, transcription of the TRE-regulated target gene is stimulated by rtTA only in the presence of Dox.

[0230] Another inducible promoter system is the lac repressor system from *E. coli* (see Brown et al., Cell 49:603-612 (1987)). The lac repressor system functions by regulating the transcription of a target polynucleotide operatively linked to a promoter containing the lac operator gene (lacO). The lac repressor (lacR) binds to LacO, thereby preventing the transcription of the target polynucleotide. Expression of the target polynucleotide is induced by a suitable inducer such as isopropyl-β-D-thiogalactopyranoside (IPTG).

[0231] To assess the expression of a polypeptide or its fraction, the expression vector to be introduced into cells may also contain a selective marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector. In other respects, the selective marker may be carried on a separate DNA segment and used in co-transfection procedures. Both the selective marker and the reporter gene may be side-linked with appropriate regulatory sequences to enable expression in host cells. Useful selective markers include, for example, antibiotic resistance genes, such as neo.

[0232] Reporter genes are used to identify potentially infected cells and evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is absent or not expressed in the recipient organism or tissue and encodes a polypeptide whose expression exhibits easily detectable properties, such as enzymatic activity. Reporter gene expression is measured at an appropriate time after DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tel et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available. Typically, constructs with a minimum 5' flanking region that exhibit the highest expression level of the reporter gene are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate the ability of drugs to regulate promoter-driven transcription.

[0233] In some embodiments, a nucleic acid encoding a full-length anti-MUC16 antibody according to any full-length anti-MUC16 antibody described herein is provided. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding the heavy and light chains of a full-length anti-MUC16 antibody. In some embodiments, one or more nucleic acid sequences are each contained in a separate vector. In some embodiments, at least some nucleic acid sequences are contained in the same vector. In some embodiments, all nucleic acid sequences are contained in the same vector. The vector may be selected, for example, from mammalian expression vectors and viral vectors (such as those derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses).

[0234] Methods for introducing genes into cells and expressing them within those cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, using any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0235] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York). In some embodiments, the introduction of polynucleotides into host cells is performed via calcium phosphate transfection.

[0236] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian (e.g., human) cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0237] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as a delivery medium in vitro and in vivo is the liposome (e.g., an artificial membrane capsule).

[0238] In the case of using non-viral delivery systems, an exemplary delivery medium is liposomes. Consider using lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, nucleic acids can be associated with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linker molecules associated with both liposomes and oligonucleotides, embedded in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in micelles, or complexed with micelles, or otherwise associated with lipids. Combinations of lipids, lipid / DNA, or lipid / expression vector associations are not limited to any particular structure in solution. For example, they can exist as bilayers, micelles, or “collapsed” structures. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic. For example, lipids include fat droplets that are naturally present in the cytoplasm, as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives (such as fatty acids, alcohols, amines, amino alcohols, and aldehydes).

[0239] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to inhibitors of this technology, various assays can be performed to confirm the presence of recombinant DNA sequences in host cells. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as DNA blotting and RNA blotting, RT-PCR and PCR; and "biochemical" assays, such as detecting the presence or absence of specific peptides, for example by immunological means (ELISA and Western blotting) or by assays described herein to identify agents within the scope of this technology.

[0240] Preparation of anti-MUC16 antibody agent and anti-MUC16 antibody fraction

[0241] In some embodiments, the anti-MUC16 antibody agent is a monoclonal antibody or derived from a monoclonal antibody. In some embodiments, the anti-MUC16 antibody agent contains V derived from a monoclonal antibody. H and V L A domain or a variant thereof. In some embodiments, the anti-MUC16 antibody agent also comprises a C domain derived from a monoclonal antibody. H 1 and C L Domains or variants thereof. Monoclonal antibodies can be prepared, for example, using methods known in the art, including hybridoma methods, phage display methods, or methods using recombinant DNA. Additionally, exemplary phage display methods are described herein and in the following examples.

[0242] In the hybridoma approach, hamsters, mice, or other suitable host animals are typically immunized with an immunomodulator to induce the production or ability to produce antibodies that specifically bind to the immunomodulator. Alternatively, lymphocytes can be immunized in vitro. The immunomodulator may comprise a peptide or fusion protein of the target protein. Generally, peripheral blood lymphocytes (“PBLs”) are used if human-derived cells are desired, or spleen cells or lymph node cells are used if non-human mammalian-derived cells are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent (such as polyethylene glycol) to form hybridoma cells. The immortalized cell line is typically transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Rat or mouse myeloma cell lines are commonly used. The hybridoma cells can be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma culture medium will typically include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which prevents the growth of HGPRT-deficient cells.

[0243] In some embodiments, the immortalized cell lines are efficiently fused, supporting stable, high-level expression of the selected antibody in the producing cells, and are sensitive to culture media such as HAT medium. In some embodiments, the immortalized cell lines are mouse myeloma cell lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center in San Diego, California, and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human xenogeneic myeloma cell lines have also been described for the production of human monoclonal antibodies.

[0244] The presence of monoclonal antibodies against the polypeptide in the culture medium for hybridoma cells can then be determined. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). Such techniques and assays are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0245] After identifying the desired hybridoma cells, the clones can be subcloned using a limiting dilution procedure and grown using standard methods. Goding, same as above. Suitable media for this purpose include, for example, Dalberg modified Eagle medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo as ascites in mammals.

[0246] Subclonal secreted monoclonal antibodies can be separated or purified from culture medium or ascites fluid using routine immunoglobulin purification procedures, such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0247] In some embodiments, according to any of the anti-MUC16 antibody agents described herein, the anti-MUC16 antibody agent comprises a sequence from a clone selected from an antibody library (such as a phage library displaying an scFv or Fab fragment). The clone can be identified by screening a combination library against antibody fragments having one or more desired activities. For example, various methods for generating phage display libraries and screening such libraries against antibodies having the desired binding characteristics are known in the art. Such methods are reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totow, NJ, 2001), and further described in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (edited by Lo, Human Press, NJ, 2001). Press, Totowar, New Jersey, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0248] In some phage display methods, VH and VL gene libraries are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phages, as described by Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as scFv fragments or as Fab fragments. Libraries derived from immunogenic sources provide high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, natural libraries (e.g., from humans) can be cloned to provide antibodies from a single source against a variety of non-self and self antigens without any immunization, as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be synthesized by cloning an unrearranged V gene segment from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and performing rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Publications Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0249] Anti-MUC16 antibody agents can be prepared using a phage display library of partially screened anti-MUC16 antibodies specific to the target MUC16 (e.g., nMUC16). The library can be a human scFv phage display library with at least 1 x 10⁻⁶ cells / mL. 9 (e.g., at least about 1×10) 9 2.5×10 9 5×10 9 7.5×10 9 1×10 10 2.5×10 10 5×10 10 7.5×10 10 Or 1×10 11(any one of) a unique human antibody fragment. In some embodiments, the library is a primordial human library constructed from DNA extracted from human PMBCs and spleens from healthy donors, covering all heavy and light chain subfamilies. In some embodiments, the library is a primordial human library constructed from DNA extracted from PBMCs isolated from patients with various diseases such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semi-synthetic human library in which the heavy chain CDR3 is completely randomized, wherein all amino acids (except cysteine) are equally likely to be present at any given position (see, for example, Hoet, RM et al., Nat. Biotechnol. 23(3):344-348, 2005). In some embodiments, the heavy chain CDR3 of the semi-synthetic human library is about 5 to about 24 amino acids (e.g., any one of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0250] Phage clones that bind to the target MUC16 (e.g., nMUC16) with high affinity can be selected by iteratively binding the phage to the target MUC16 (which binds to a solid support, such as beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phages and elution of specifically bound phages. The bound phage clones are then eluted and used to infect suitable host cells, such as *E. coli* XL1-Blue, for expression and purification. In the example of cell panning, HEK293 cells overexpressing MUC16 on their cell surface are mixed with a phage library, the cells are then collected, and the bound clones are eluted and used to infect suitable host cells for expression and purification (see all examples). Multiple rounds (such as about 2, 3, 4, 5, 6, or more rounds) of panning can be performed using solution panning, cell panning, or a combination of both to enrich phage clones that specifically bind to the target MUC16. The specific binding of enriched phage clones to the target MUC16 can be tested using any method known in the art, including, for example, ELISA and FACS.

[0251] Monoclonal antibodies can also be prepared using recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of this technology can be readily isolated and sequenced using conventional procedures, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies. Hybridoma cells as described above or MUC16-specific phage clones of this technology can be used as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells that do not normally produce immunoglobulins (such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to obtain monoclonal antibody synthesis in recombinant host cells. The DNA can also be modified, for example, by replacing homologous non-human sequences with coding sequences for human heavy and light chain constant domains and / or frame regions (U.S. Patent No. 4,816,567; Morrison et al., ibid.) or by covalently attaching all or part of the coding sequence for a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. This non-immunoglobulin polypeptide can replace the constant domain of the antibody agent of this technology, or it can replace the variable domain of an antigen combination site of the antibody agent of this technology, to produce a chimeric bivalent antibody agent.

[0252] The antibody may be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, one method involves recombinantly expressing an immunoglobulin light chain and a modified heavy chain. The heavy chain is typically truncated at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine ​​residue may be substituted with or deleted from another amino acid residue to prevent cross-linking.

[0253] In vitro methods are also suitable for preparing monovalent antibodies. Antibody digestion to produce its fragments, particularly Fab fragments, can be performed using any method known in the art.

[0254] An antibody variable domain having the desired binding specificity (antibody-antigen combination site) can be fused to an immunoglobulin constant domain sequence. The fusion preferably has an immunoglobulin heavy chain constant domain comprising at least a portion of a hinge, CH2, and CH3 regions. In some embodiments, a first heavy chain constant region (CH1) containing the site required for light chain binding is present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism.

[0255] Human and humanized antibodies

[0256] Anti-MUC16 antibody agents (e.g., full-length anti-MUC16 antibodies) or their antigen-binding fragments can be humanized antibody agents or human antibody agents. Humanized forms of non-human (e.g., mouse) antibody moieties are typically chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding sequences of the antibody) containing minimal sequences derived from non-human immunoglobulins. Humanized antibody moieties comprise human immunoglobulins, immunoglobulin chains, or fragments thereof (recipient antibodies), wherein residues of the recipient's CDR are replaced by residues of a CDR from a non-human species such as mouse, rat, or rabbit (donor antibody) having the desired specificity, affinity, and capability. In some cases, Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibody moieties may also contain residues not found in either the recipient antibody or the input CDR or framework sequence. Typically, humanized antibodies can substantially contain all of at least one (and usually two) variable domains, wherein all or substantially all CDR regions correspond to those of non-human immunoglobulins, and all or substantially all FR regions are those of human immunoglobulin common sequences.

[0257] In general, humanized antibody agents have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as “input” residues, which are typically derived from the “input” variable domain. According to some embodiments, humanization can be performed essentially by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence, following the methods of Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). Thus, such “humanized” antibody moieties are antibody moieties (US Patent No. 4,816,567) in which a significantly smaller than complete human variable domain has been replaced by a corresponding sequence from a non-human species. In practice, humanized antibody moieties are typically human antibody moieties in which some CDR residues and possibly some FR residues are replaced with residues from similar sites in rodent antibodies.

[0258] As an alternative to humanization, human antibody portions can be generated. For example, it is now possible to generate transgenic animals (e.g., mice) capable of producing a complete library of human antibodies after immunization in the absence of endogenous immunoglobulin production. For example, homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice has been described as resulting in complete suppression of endogenous antibody production. Transferring human germline immunoglobulin gene arrays into such germline mutant mice results in the production of human antibodies after antigen challenge. See, for example, Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807; and WO 97 / 17852. Alternatively, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice) in which endogenous immunoglobulin genes have been partially or completely inactivated. Upon stimulation, human antibody production is observed, which is very similar to that observed in humans in all aspects, including gene rearrangement, assembly, and antibody library. This method is described in, for example, the following publications: U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; and Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0259] Human antibody agents can also be generated from in vitro activated B cells (see U.S. Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Techniques by Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies. Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner et al., J. Immunol., 147(1):86-95 (1991).

[0260] Anti-MUC16 antibody variant

[0261] In some embodiments, consider amino acid sequence variants of the anti-MUC16 antibody agents (e.g., full-length anti-MUC16 antibodies) or their antigen-binding fragments provided herein. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody agent. Amino acid sequence variants of the antibody agent can be prepared by introducing appropriate modifications into the nucleotide sequence encoding said antibody agent or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody agent. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen binding).

[0262] In some embodiments, an anti-MUC16 antibody agent with one or more amino acid substitutions is provided. Target sites for substitutional mutagenesis include HVR and FR. Amino acid substitutions can be introduced into the target antibody agent, and products can be screened for desired activities, such as preserved / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0263] Conservative substitutions are shown in Table 3 below.

[0264] Table 3: Conservative Substitution

[0265]

[0266]

[0267] Amino acids can be classified into different categories based on common side-chain characteristics: hydrophobic: leucine, Met, Ala, Val, Leu, Ile; neutral-hydrophilic: Cys, Ser, Thr, Asn, Gln; acidic: Asp, Glu; basic: His, Lys, Arg; residues affecting chain orientation: Gly, Pro; and aromatic: Trp, Tyr, Phe. Non-conservative substitution involves exchanging members of one of these categories for another.

[0268] Exemplary substitution variants are affinity-matured antibody agents that can be conveniently generated, for example, using phage display-based affinity maturation techniques. In short, one or more CDR residues are mutated and the variant antibody moiety is displayed on a phage, and screening is performed for specific biological activities, such as binding affinity. Modifications (e.g., substitutions) can be made in the HVR to, for example, improve antibody affinity. Such modifications can be made in HVR “hotspots” (i.e., residues encoded by codons that undergo mutations at a high frequency during somatic maturation) (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or specificity-determining residues (SDRs), and the resulting variant V is tested. H or V L The binding affinity. For example, affinity maturation through construction and reselection from secondary libraries has been described in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totova, NJ, (2001)).

[0269] In some implementations of affinity maturation, diversity is introduced into the selectable variant gene to be matured via any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-guided mutagenesis). A secondary library is then constructed. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-guided approach, in which several HVR residues are randomized (e.g., 4-6 residues at a time). The HVR residues involved in antigen binding can be specifically identified, for example, using alanine scan mutagenesis or modeling. Specifically, CDR-H3 and CDR-L3 are typically targeted.

[0270] In some embodiments, substitution, insertion, or deletion can occur within one or more HVRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes (e.g., conserved substitutions as described herein) that do not substantially reduce binding affinity can be made in the HVR. Such changes can be located outside the HVR "hotspot" or SDR. In some embodiments of the variant VH and VL sequences provided above, each HVR is unchanged or contains no more than one, two, or three amino acid substitutions.

[0271] A useful method for identifying antibody agent residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, residues or target residue groups (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and substituted with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be determined to identify contact points between the antibody and antigen. Such contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine if they contain the desired properties.

[0272] Amino acid sequence insertions include fusion of amino and / or carboxyl termini, ranging in length from one residue to polypeptides containing one hundred or more residues, and intra-sequence insertions of single or multiple amino acid residues. Examples of terminator insertions include antibody agents having an N-terminal methionyl residue. Other insertion variants of the antibody agent molecule include fusion of the N-terminus or C-terminus of the antibody agent with an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody agent.

[0273] Fc region variants

[0274] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of the antibody agent provided herein (e.g., a full-length anti-MUC16 antibody or an anti-MUC16 Fc fusion), thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC effector function, typically associated with binding to the Fc receptor (FcR). In some embodiments, the Fc region variant has reduced ADCC effector function. There are numerous examples of Fc sequence variations or mutations that can alter effector function. For example, WO 00 / 42072 and Shields et al., J Biol. Chem. 9(2):6591-6604 (2001) describe antibody variants with enhanced or reduced binding to the FcR. The contents of these publications are expressly incorporated herein by reference.

[0275] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (e.g., cancer cells) whose membrane surface antigens have been bound by a specific antibody (e.g., an anti-MUC16 antibody). Typical ADCC involves the activation of NK cells via antibody. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of an antibody bound to the surface of the target cell. The most common Fc receptor on the surface of NK cells is called CD16 or FcγRIII. The binding of the Fc receptor to the Fc region of the antibody leads to NK cell activation, cytolysis, granule release, and subsequent apoptosis of the target cell. The contribution of ADCC to tumor cell killing can be measured using a specific assay that uses NK-92 cells transfected with a high-affinity FcR. The results are compared to wild-type NK-92 cells that do not express FcR.

[0276] In some embodiments, the present invention considers anti-MUC16 antibody variants (such as full-length anti-MUC16 antibody variants) that contain an Fc region having some, but not all, effector functions. This makes said antibody variants desirable candidates for applications where the in vivo half-life of the anti-MUC16 antibody is important, and certain effector functions (such as CDC and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. Primary NK cells, which mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Fc expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in the following literature: U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Patent No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, for example, ACTI for flow cytometry). TM Non-radioactive cytotoxicity assays (Cell Technology, Inc., Mountain View, California); and CytoTox 96 TMNon-radioactive cytotoxicity assays (Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the target molecule can be assessed in vivo, for example, in animal models such as those disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody agent cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO2006 / 029879 and WO 2005 / 100402. To assess complement activation, CDC assays can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0277] Antibodies with reduced effector function include those with substitutions of one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more positions of amino acid positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant with substitutions to alanine residues 265 and 297 (US Patent No. 7,332,581).

[0278] Certain antibody variants with enhanced or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604(2001).)

[0279] In some embodiments, a variant of the anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) is provided, which includes a variant Fc region containing one or more amino acid substitutions that improve ADCC. In some embodiments, the variant Fc region contains one or more amino acid substitutions that improve ADCC, wherein said substitution is at positions 298, 333, and / or 334 (EU numbers of residues) in the variant Fc region. In some embodiments, the anti-MUC16 antibody agent (e.g., a full-length anti-MUC16 antibody) variant includes the following amino acid substitutions in its variant Fc region: S298A, E333A, and K334A.

[0280] In some implementations, alterations are made in the Fc region that result in changes (i.e., enhancement or reduction) of C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0281] In some embodiments, variants of anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) are provided, wherein the variant Fc region contains one or more amino acid substitutions that increase half-life and / or improve binding to nascent Fc receptors (FcRn). Antibodies with increased half-life and improved binding to FcRn are described in US2005 / 0014934A1 (Hinton et al.). Those antibodies contain an Fc region having one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include those Fc variants with substitutions at one or more residues in the following Fc region: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, the substitution of Fc region residue 434 (U.S. Patent No. 7,371,826).

[0282] Other examples of Fc region variants can also be found in Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0283] Consider anti-MUC16 antibody agents that include any Fc variants or combinations thereof described herein (such as full-length anti-MUC16 antibodies).

[0284] Glycosylation variants

[0285] In some embodiments, the anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) or its antigen-binding fragment provided herein is modified to increase or decrease the degree of glycosylation of the anti-MUC16 antibody agent. The addition or deletion of glycosylation sites in the anti-MUC16 antibody agent can be conveniently accomplished by altering the amino acid sequence of the anti-MUC16 antibody agent or its polypeptide moiety, thereby creating or removing one or more glycosylation sites.

[0286] When an anti-MUC16 antibody agent or its antigen-binding fragment contains an Fc region, the carbohydrates to which it is attached can be modified. Naturally occurring antibodies produced by mammalian cells typically contain branched, bianthraquinone oligosaccharides, which are usually attached to the Asn297 of the CH2 domain of the Fc region via an N-bond. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “stem” of the bianthraquinone oligosaccharide structure. In some embodiments, the oligosaccharides in the anti-MUC16 antibody agent of this technology can be modified to produce anti-MUC16 antibody agent variants with certain improved properties.

[0287] The N-glycans attached to the CH2 domain of Fc are heterogeneous. Antibodies or Fc fusion proteins generated in CHO cells undergo fucosylation via fucosyltransferase activity. See Shoji-Hosaka et al., J. Biochem. 140:777-83 (2006). Typically, a small percentage of naturally occurring defucosylated IgG is detectable in human serum. N-glycosylation of Fc is crucial for binding to FcγR; and defucosylation of N-glycans increases the ability of Fc to bind FcγRIIIa. Increased FcγRIIIa binding can enhance ADCC, which can be advantageous in certain therapeutic applications of antibody agents requiring cytotoxicity.

[0288] In some embodiments, enhanced effector function may be detrimental when Fc-mediated cytotoxicity is not desired. In some embodiments, the Fc fragment or CH2 domain is unglycosylated. In some embodiments, the N-glycosylation site in the CH2 domain is mutated to prevent glycosylation.

[0289] In some embodiments, variants of anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) containing an Fc region are provided, wherein the carbohydrate structure attached to the Fc region has reduced or absent fucose, which can improve ADCC function. Specifically, this document considers anti-MUC16 antibody agents having reduced fucose relative to the amount of fucose on the same anti-MUC16 antibody agent produced in wild-type CHO cells. That is, characterized by a lower amount of fucose than in other cases originally produced by native CHO cells (e.g., CHO cells producing a native glycosylation pattern, such as CHO cells containing a native FUT8 gene). In some embodiments, the anti-MUC16 antibody agent is an antibody agent in which less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycan on the antibody agent comprises fucose. For example, the amount of fucose in such an anti-MUC16 antibody agent can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. In some embodiments, the anti-MUC16 antibody agent is an antibody agent in which none of the N-linked glycans on the antibody agent contain fucose, i.e., the anti-MUC16 antibody agent is completely fucose-free or fucose-free or defucosylated. The amount of fucose is determined, for example, by calculating the average amount of fucose within the glycan chain at Asn297 relative to the sum of all sugar structures (e.g., complex, heterogeneous, and high-mannose structures) attached to Asn 297, as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU number of Fc region residues) in the Fc region; however, Asn297 can also be located approximately ±3 amino acids upstream or downstream of position 297 due to minor sequence variations in the antibody, i.e., between positions 294 and 300. Such fucosylated variants can possess improved ADCC function. See, for example, US Patent Publication Nos. 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa HakkoKogyo Co., Ltd.).Examples of publications involving “defucosylated” or “fucosylated” antibody agent variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO 2002 / 031140; Okazaki et al., J.Mol.Biol.336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech.Bioeng.87:614 (2004). Examples of cell lines capable of producing defucosylation antibodies include Lec13 CHO cells with protein fucosylation defects (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312A1, Adams et al., especially in Example 11), and knockout cell lines, such as CHO cells with α-1,6-fucosylation gene FUT8 knocked out (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0290] Variants of anti-MUC16 antibodies (such as full-length anti-MUC16 antibodies) with branched oligosaccharides are also provided, for example, wherein the biantennary oligosaccharide attached to the Fc region of the anti-MUC16 antibody is branched by GlcNAc. Such anti-MUC16 antibody variants (such as full-length anti-MUC16 antibodies) may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in the following literature: WO 2003 / 011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umana et al.); US 2005 / 0123546 (Umana et al.); and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Variants of anti-MUC16 antibodies (such as full-length anti-MUC16 antibodies) having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. These anti-MUC16 antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.) and WO 1999 / 22764 (Raju, S.).

[0291] In some embodiments, anti-MUC16 antibody variants containing the Fc region (such as full-length anti-MUC16 antibodies) are capable of binding to FcγRIII. In some embodiments, anti-MUC16 antibody variants containing the Fc region (such as full-length anti-MUC16 antibodies) exhibit ADCC activity in the presence of human effector cells (e.g., T cells) or increased ADCC activity in the presence of human effector cells, compared to otherwise identical anti-MUC16 antibody variants containing the Fc region of human wild-type IgG1.

[0292] Cysteine ​​engineered variants

[0293] In some embodiments, it may be desirable to generate cysteine-engineered anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) or antigen-binding fragments thereof, wherein one or more amino acid residues are substituted with cysteine ​​residues. In some embodiments, the substituted residues are located at accessible sites of the anti-MUC16 antibody agent or antigen-binding fragments thereof. By substituting those residues with cysteine, a reactive thiol group is thereby positioned at an accessible site of the anti-MUC16 antibody agent and can be used to conjugate the anti-MUC16 antibody agent to other portions, such as pharmaceutical portions or linker-pharmaceutical portions, to generate anti-MUC16 immunoconjugates, as further described herein. Cysteine-engineered anti-MUC16 antibody agents (such as anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies) can be generated, as described, for example, in U.S. Patent No. 7,521,541.

[0294] derivative

[0295] In some embodiments, the anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) provided herein, or their antigen-binding fragments, may be further modified to contain other non-protein moieties known in the art and readily available. Moieties suitable for deriving anti-MUC16 antibody agents 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), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly(1,3-dioxolane), poly(1,3,6-trioxane), ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to an anti-MUC16 antibody agent can vary, and if more than one polymer is attached, they can be the same or different molecules. Typically, the number and / or type of polymers used for derivation can be determined based on considerations including, but not limited to, the specific properties or functions of the anti-MUC16 antibody agent to be improved, and whether the anti-MUC16 antibody agent derivative will be used in a therapy under defined conditions.

[0296] In some embodiments, an anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) or its antigen-binding fragment is provided as a conjugate of a non-protein portion that can be selectively heated by exposure to radiation. In some embodiments, the non-protein portion is carbon nanotubes (Kam et al., Proc. Natl. Acad. Sci. USA 102:11600-11605 (2005)). The radiation can have any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but heat the non-protein portion to a temperature capable of killing cells near the anti-MUC16 antibody agent-non-protein portion.

[0297] Antibody conjugates

[0298] In some embodiments, this document provides an anti-MUC16 antibody agent or an antigen-binding fragment conjugate thereof, wherein the anti-MUC16 antibody agent or an antigen-binding fragment thereof is conjugated to one or more agents, such as imaging agents or cytotoxic agents. This document also provides bispecific antibody conjugates, wherein the bispecific antibody is conjugated to one or more agents, such as imaging agents or cytotoxic agents. This document also provides antibody heavy chain conjugates, wherein the antibody heavy chain is conjugated to one or more agents, such as imaging agents or cytotoxic agents. This document also provides antibody light chain conjugates, wherein the antibody light chain is conjugated to one or more agents, such as imaging agents or cytotoxic agents. This document also provides fusion protein conjugates, wherein the fusion protein is conjugated to an agent, such as an imaging agent or cytotoxic agent. In some embodiments, the agents are covalently or non-covalently conjugated.

[0299] In some implementations, the developing agent is a detectable marker, such as a chromogenic agent, an enzyme catalyst, a radioactive isotope agent, an isotope agent, a fluorescent agent, a toxic agent, a chemiluminescent agent, a nuclear magnetic resonance contrast agent, or other marker.

[0300] The detectable group can be any material with detectable physical or chemical properties. Such detectable labels have been well-developed in the fields of immunoassays and imaging. Generally, almost any label that can be used in such methods can be applied to this technique. Therefore, the label can be any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Labels useful in the practice of this technique include magnetic beads (e.g., Dynabeads). TM ), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.), radioactive labeling (e.g., 3 H, 14 C 35 S, 125 I, 121 I, 131 I, 112 In、99 mTc), and other imaging agents such as microbubbles (used for ultrasound imaging). 18 F, 11 C 15 O、 89 Zr、 89 Zr-DFO (used for positron emission tomography) 99m TC 111 In (used in single-photon emission computed tomography), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and calorimetric labels (such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)) beads. Patents describing the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, each of which is incorporated herein by reference in its entirety and for all purposes. See also Handbook of Fluorescent Probes and Research Chemicals (6th Edition, Molecular Probes, Inc., Eugene, Oregon).

[0301] The label can be coupled directly or indirectly to the desired component to be determined using methods well known in the art. As mentioned above, a variety of labels can be used, and the choice of label depends on factors such as the required sensitivity, ease of conjugation to the compound, stability requirements, available instruments, and disposal procedures.

[0302] Non-limiting examples of suitable chromophores include diaminobenzidine and 4-hydroxyazobenzyl-2-carboxylic acid.

[0303] Non-restrictive examples of suitable enzyme labels include malate dehydrogenase, staphylococcal nuclease, δ-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucosylamylase, and acetylcholinesterase.

[0304] Suitable radioactive isotopes are well known to those skilled in the art and include beta emitters, gamma emitters, positron emitters, and X-ray emitters. Non-limiting examples of suitable radioactive isotope labeling include… 3 H, 18 F, 111 In、 125 I, 131 I, 32 P, 33P, 35 S, 11 C 14 C 51 Cr 57 To 58 Co、 59 Fe、 75 Se、 152 Eu、 90 Y、 67 Cu、 217 Ci、 211 At、 212 Pb, 47 Sc、 223 Ra、 223 Ra、 89 Zr、 177 Lu and 109 Pd. In some implementations, 111 In is the preferred isotope for in vivo imaging because it avoids... 125 I or 131 The problem of debromination of I-labeled anti-MUC16 antibodies or their antigen-binding fragments in the liver. Furthermore, 111 In has a more favorable gamma emission energy for imaging (Perkins et al., Eur. J. Nucl. Med. 70: 296-301 (1985); Carasquillo et al., J. Nucl. Med. 25: 281-287 (1987)). For example, conjugated with a monoclonal antibody having 1-(p-isothiocyanobenzyl)-DPTA 111 In shows very little uptake in non-tumor tissues (particularly the liver), thus enhancing the specificity of tumor localization (Esteban et al., J. Nucl. Med. 28: 861-870 (1987)).

[0305] Suitable, non-limiting examples of non-radioactive isotope labeling include 157Gd, 55 Mn, 162 Dy、 52 Tr and 56 Fe.

[0306] Non-limiting examples of suitable fluorescent labels include 152 Eu labeling, fluorescein labeling, isothiocyanate labeling, rhodamine labeling, phycoerythrin labeling, phycocyanin labeling, allophycocyanin labeling, green fluorescent protein (GFP) labeling, phthalaldehyde labeling, and fluorescent amine labeling.

[0307] Non-limiting examples of chemiluminescent labeling include luminol labeling, isoluminol labeling, aromatic acridine ester labeling, imidazole labeling, acridine salt labeling, oxalate ester labeling, luciferin labeling, luciferase labeling, and jellyfish luminescent protein labeling.

[0308] Non-limiting examples of MRI contrast agents include heavy metal nuclei such as Gd, Mn, and iron.

[0309] Techniques known to those skilled in the art for conjugating the aforementioned labels to the anti-MUC16 antibody agent or its antigen-binding fragment, bispecific antibody, antibody heavy chain, antibody light chain, and fusion protein are described, for example, in the following literature: Kennedy et al., Clin. CMm. Acta 70:1-31 (1976); and Schurs et al., Clin. CMm. Acta 81:1-40 (1977). The conjugation techniques mentioned in the latter literature are the glutaraldehyde method, the periodate method, the bismaleimide method, and the m-maleimide benzyl-N-hydroxy-succinimide ester method, all of which are incorporated herein by reference.

[0310] Non-limiting examples of cytotoxic agents include cell inhibitors or cytokillants, radioactive metal ions (e.g., alpha emitters), and toxins (e.g., Pseudomonas exotoxin A, abrin, cholera toxin, ricin A, and diphtheria toxin).

[0311] In some embodiments, the agent is a diagnostic agent. A diagnostic agent is an agent that can be used to diagnose or detect a disease by targeting cells containing the antigen. Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and magnetic resonance imaging (MRI) enhancers (e.g., paramagnetic ions). U.S. Patent No. 6,331,175 describes MRI techniques and the preparation of antibodies conjugated with MRI enhancers, and is incorporated herein by reference in its entirety. Preferably, the diagnostic agent is selected from radioisotopes, enhancers for magnetic resonance imaging, and fluorescent compounds. To load an anti-MUC16 antibody agent or its antigen-binding fragment with a radioactive metal or paramagnetic ion, it may be necessary to react the anti-MUC16 antibody agent or its antigen-binding fragment with a reagent having a long tail with various chelating groups for binding ions. This tail can be a polymer, such as polylysine, a polysaccharide, or other derivatized or derivatizable chains, having side groups that can bind to chelating groups, such as ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, thioureas, polyoximes, and similar groups known to be suitable for this purpose. The chelate is chemically coupled to the antibody using standard methods. Chelates are typically linked to antibodies via groups that can bond with the molecule with minimal loss of immunoreactivity and minimal aggregation and / or internal cross-linking. Other, less common methods and reagents for conjugating chelates to antibodies are disclosed in U.S. Patent No. 4,824,659, entitled "Antibody Conjugates," published April 25, 1989, by Hawthorne, the disclosure of which is incorporated herein by reference in its entirety. Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogues used in conjunction with diagnostic isotopes for radiographic imaging. When combined with non-radioactive metals (such as manganese, iron, and gadolinium), the same chelates can be used for MRI when used with the anti-MUC16 antibody agent or its antigen-binding fragment provided herein.

[0312] Macrocyclic chelates such as NOTA, DOTA, and TETA are used with a variety of metals and radioactive metals (most specifically with radionuclides of gallium, yttrium, and copper). These metal-chelate complexes can be made very stable by adapting the ring size to the target metal. This article covers the use of chelates for stably binding nuclides (e.g., for RAIT). 223 Ra) is interested in other cyclic chelates, such as macrocyclic polyethers.

[0313] Pharmaceutical Composition

[0314] This document also provides compositions comprising an anti-MUC16 antibody (such as a full-length anti-MUC16 antibody) or an antigen-binding fragment thereof (such as pharmaceutical compositions, also referred to herein as formulations), nucleic acids encoding said antibody, vectors comprising nucleic acids encoding said antibody, or host cells comprising said nucleic acids or vectors. In some embodiments, pharmaceutical compositions are provided comprising an anti-MUC16 antibody and optionally a pharmaceutically acceptable vector.

[0315] Suitable formulations of anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) or their antigen-binding fragments are obtained by mixing an anti-MUC16 antibody agent of the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10). (Residues) 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, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as TWEEN. TM PLURONICS TM Or polyethylene glycol (PEG). Exemplary formulations are described in WO 98 / 56418, which is expressly incorporated herein by reference. Lyophilized formulations suitable for subcutaneous administration are described in WO 97 / 04801. Such lyophilized formulations can be reconstituted to high protein concentrations using suitable diluents, and the reconstituted formulations can be administered subcutaneously to the individuals to be treated herein. Lipofectin or liposomes can be used to deliver the anti-MUC16 antibody agent of this technology into cells.

[0316] The formulations described herein may contain, in addition to an anti-MUC16 antibody (such as a full-length anti-MUC16 antibody) or its antigen-binding fragment, one or more active compounds necessary for the specific indication being treated, preferably those compounds having complementary activities that do not adversely affect each other. For example, in addition to an anti-MUC16 antibody or its antigen-binding fragment, it may be desirable to further provide an antitumor agent, growth inhibitor, cytotoxic agent, or chemotherapeutic agent. Such molecules are suitably present in a combination of amounts effective for the intended purpose. The effective amount of such other agents depends on the amount of anti-MUC16 antibody present in the formulation, the type of disease or disorder or treatment, and other factors discussed above. These agents are typically used at the same dose and via the route of administration as described herein or at approximately 1% to 99% of the doses used to date.

[0317] Anti-MUC16 antibody agents (such as anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies) or their antigen-binding fragments can also be encapsulated in microcapsules (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared by cohesive drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or encapsulated in crude emulsions. Sustained-release formulations can be prepared.

[0318] Sustained-release formulations of anti-MUC16 antibody agents (such as anti-MUC16 antibodies, e.g., full-length anti-MUC16 antibodies) or their antigen-binding fragments can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing said antibody agent (or fragments thereof), said matrix being in the form of a molded article (e.g., a film or microcapsule). Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (US Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamic acid esters, non-degradable ethylene-vinyl acetate, and degradable lactic acid-glycolic acid copolymers (such as LUPRON DEPOT). TM(Injectable microspheres composed of lactic-glycolic acid copolymer and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins in a shorter timeframe. When encapsulated antibody agents are kept in vivo for extended periods, they can denature or aggregate due to exposure to humidity at 37°C, leading to loss of biological activity and possible alterations in immunogenicity. Rational strategies can be designed to stabilize anti-MUC16 antibody agents based on the mechanisms involved. For example, if the aggregation mechanism is found to be the formation of intermolecular SS bonds through thio-disulfide exchange, stabilization can be achieved by modifying thiol residues, lyophilizing with acidic solutions, controlling humidity levels, using appropriate additives, and developing specific polymer matrix compositions.

[0319] In some embodiments, the anti-MUC16 antibody (such as a full-length anti-MUC16 antibody) or its antigen-binding fragment is formulated in a buffer containing citrate, NaCl, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination thereof. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment is formulated in a buffer containing about 100 mM to about 150 mM glycine. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment is formulated in a buffer containing about 50 mM to about 100 mM NaCl. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment is formulated in a buffer containing about 10 mM to about 50 mM acetate. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment is formulated in a buffer containing about 10 mM to about 50 mM succinate. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is formulated in a buffer containing about 0.005% to about 0.02% polysorbate 80. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is formulated in a buffer having a pH between about 5.1 and 5.6. In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is formulated in a buffer containing 10 mM citrate, 100 mM NaCl, 100 mM glycine, and 0.01% polysorbate 80, wherein the formulation is at pH 5.5.

[0320] Preparations intended for internal use must be sterile. This can be easily achieved, for example, by filtration through a sterile filter membrane.

[0321] Treatment with anti-MUC16 antibody agents

[0322] In some embodiments, this document provides a method for treating a subject's cancer (specifically, a subject's MUC16-positive cancer), comprising administering to the subject in need a therapeutically effective amount of an anti-MUC16 antibody agent or an antigen-binding fragment thereof. In some embodiments, the anti-MUC16 antibody agent or an antigen-binding fragment thereof is administered at a therapeutically effective dose (as described herein). In some embodiments, the anti-MUC16 antibody agent or an antigen-binding fragment thereof is administered according to the methods described herein. In some embodiments, the anti-MUC16 antibody agent or an antigen-binding fragment thereof is administered in combination with one or more additional pharmaceutically active agents.

[0323] For use in subjects of a specific species, an anti-MUC16 antibody or its antigen-binding fragment that binds to MUC16 of that specific species is used. For example, for treating humans, an anti-MUC16 antibody or its antigen-binding fragment that binds to human MUC16 is used. In some embodiments, the anti-MUC16 antibody or its antigen-binding fragment is an immunoglobulin.

[0324] Additionally, for use in subjects of a specific species, the anti-MUC16 antibody or its antigen-binding fragment is derived from that specific species. For example, for treating humans, the anti-MUC16 antibody or its antigen-binding fragment may comprise an immunoglobulin containing a human constant region. In some embodiments, the subject is a human.

[0325] In some implementations, MUC16-positive cancers are ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine cancer (e.g., endometrial cancer), primary peritoneal cancer, or any other cancer that expresses the MUC16 receptor.

[0326] In some implementations, treatment may be used to achieve beneficial or desired clinical outcomes, including but not limited to symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. In one specific implementation, "treatment" may also be used to prolong survival compared to expected survival without treatment. In some implementations, administration of the anti-MUC16 antibody agent or its antigen-binding fragment, or the pharmaceutical composition described herein, to a subject suffering from cancer (e.g., ovarian cancer, lung cancer, pancreatic cancer, breast cancer, fallopian tube cancer, uterine cancer (e.g., endometrial cancer), primary peritoneal cancer, or any other tissue expressing the MUC16 receptor) achieves at least one, two, three, four, or more of the following effects: (i) reducing or alleviating the severity of one or more cancer symptoms; (ii) reducing the duration of one or more cancer-related symptoms; (iii) preventing the recurrence of cancer-related symptoms; (iv) reducing hospitalizations of the subject; (v) reducing the length of hospitalizations; (vi) increasing the survival of the subject; (vii) enhancing or improving the efficacy of another therapy; (viii) suppressing the development or onset of one or more cancer-related symptoms; (ix) reducing the number of cancer-related symptoms; and (x) improving quality of life as assessed by methods well known in the art. (x) Inhibit tumor recurrence; (xi) Resolve tumor and / or one or more tumor-related symptoms; (xii) Inhibit the progression of tumor and / or one or more tumor-related symptoms; (xiii) Reduce tumor growth; (xiv) Reduce tumor size (e.g., volume or diameter); (xv) Reduce the formation of new tumors; (xvi) Prevent, eradicate, remove, or control primary, regional, and / or metastatic tumors; (xvii) Reduce the number or size of metastases; (xviii) Reduce mortality; (xix) Increase recurrence-free survival; (xx) Maintain tumor size and prevent or reduce its increase to less than the increase in tumor size after administration of standard therapy, as measured by conventional methods available to those skilled in the art, such as magnetic resonance imaging (MRI), dynamic contrast-enhanced MRI (DCE-MRI), X-ray and computed tomography (CT) scans, or positron emission tomography (PET) scans; and / or (xxi) Increase the duration of patient remission. Treatment may achieve one or more of the foregoing.

[0327] Subjects treated according to the methods provided herein can be any mammal, such as rodents, cats, dogs, horses, cattle, pigs, monkeys, primates, or humans. In some embodiments, the subject is a human. In some embodiments, the subject is a dog. As used herein, the terms "subject" and "patient" are used interchangeably.

[0328] In some implementations, subjects treated according to the methods provided herein have been diagnosed with MUC16-positive cancer, including but not limited to ovarian cancer, lung cancer, pancreatic cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer, or cancer of any other tissue expressing MUC16.

[0329] Diagnostic uses

[0330] In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment described herein may be used for diagnostic purposes to detect, diagnose, or monitor the conditions described herein (e.g., conditions involving MUC16-positive cancer cells). In some embodiments, the anti-MUC16 antibody agent or its antigen-binding fragment is labeled for diagnostic purposes.

[0331] In some embodiments, this document provides a method for detecting the condition described herein, comprising (a) measuring the expression of MUC16 or a fragment thereof in a cell or tissue sample of a subject using one or more of the anti-MUC16 antibody agents or antigen-binding fragments thereof described herein; and (b) comparing the expression level of MUC16 or a fragment thereof with a control level, such as the level in a normal tissue sample (e.g., from a subject who does not have the condition described herein or from the same patient prior to the onset of the condition), whereby an increase or decrease in the measured level of MUC16 or a fragment thereof expression compared with the control level of MUC16 or a fragment thereof expression indicates the condition described herein.

[0332] The antibodies described herein can be used to determine the level of MUC16 or fragments thereof in biological samples using classical immunohistochemical methods as described herein or known to those skilled in the art (see, for example, Jalkanen et al., J. Cell. Biol. 101:976-985 (1985); and Jalkanen et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods that can be used to detect protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay markers are known in the art and include enzyme markers such as glucose oxidase; radioisotopes such as iodine ( 125 I, 121I), carbon ( 14 C), sulfur 35 S), tritium ( 3 H), Indium 121 In) and technetium ( 99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, as well as biotin. In some embodiments, the assay label is conjugated to an anti-MUC16 antibody agent or its antigen-binding fragment provided herein for direct detection. In some embodiments, the assay label is conjugated to a secondary antibody that binds to an anti-MUC16 antibody agent or its antigen-binding fragment provided herein. The type of secondary antibody is selected based on the class of the primary antibody (e.g., IgG or IgM), the source host, and the preferred type of label. In some embodiments, the secondary antibody is a class or isotype-specific antibody (e.g., IgG, IgM, IgA, IgE, or IgG). In some embodiments, the secondary antibody is a subclass-specific antibody (e.g., IgG1, IgG2, IgG4, IgA1, or IgA2). In some embodiments, the secondary antibody binds to one or more classes or subclasses of antibodies. In some embodiments, the secondary antibody binds to the heavy chain of the primary antibody. In some embodiments, the secondary antibody binds to the light chain of the primary antibody. In some embodiments, the secondary antibody binds to the κ light chain of the primary antibody. In some embodiments, the secondary antibody binds to the λ light chain of the primary antibody. In some embodiments, the secondary antibody is an anti-Fc, anti-F(ab), or anti-(Fab')2 fragment antibody. In some embodiments, the secondary antibody is a rabbit, mouse, goat, donkey, or chicken antibody.

[0333] In some implementations, the condition described herein (e.g., MUC16-positive cancer) is monitored by repeating the diagnostic methods used for a period of time after the initial diagnosis.

[0334] The presence of labeled molecules in a subject (i.e., in vivo) is detected using methods known in the art for in vivo scanning. Those skilled in the art will be able to determine the appropriate method for detecting a particular label. Methods and apparatus that can be used in the diagnostic methods of this invention include, but are not limited to, computed tomography (CT), whole-body scanning (such as positron emission tomography (PET)), magnetic resonance imaging (MRI), and ultrasound scanning.

[0335] This document also discloses a method for detecting cancer in a subject in vivo, comprising (a) administering an effective amount of any of the anti-MUC16 constructs disclosed herein to the subject, wherein the anti-MUC16 construct is configured to target MUC16-expressing cancer cells and is labeled with a radioactive isotope; and (b) detecting the presence of a tumor in the subject by detecting a level of radioactivity emitted by the anti-MUC16 construct above a reference value, optionally wherein the radioactive isotope is... 89Zr-deferromin B (DFO). In some embodiments, the subject is diagnosed with or suspected of having cancer. Alternatively or additionally, in some embodiments, positron emission tomography or single-photon emission computed tomography is used to detect the level of radioactivity emitted by the anti-MUC16 construct. In any of the foregoing embodiments, the method further includes administering to the subject an effective amount of an immunoconjugate comprising an anti-MUC16 construct of the present technology conjugated with a radionuclide. The radionuclide may be an isotope emitting alpha particles, an isotope emitting beta particles, an Auger emitter, or any combination thereof.

[0336] Delivery of anti-MUC16 antibody agents

[0337] Anti-MUC16 antibody agents or antigen-binding fragments thereof, as described herein, or compositions containing said antibodies or antigen-binding fragments thereof, or cells expressing said antibodies or antigen-binding fragments thereof, can be delivered to subjects via a variety of routes. These routes include, but are not limited to, parenteral, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, and subcutaneous routes. Lung administration can also be employed, for example, by using an inhaler or nebulizer and formulating it as an aerosol for use as a spray. In one embodiment, the anti-MUC16 antibody agent or antigen-binding fragment or composition thereof described herein is administered parenterally to the subject. In some embodiments, the parenteral administration is intravenous, intramuscular, or subcutaneous.

[0338] The amount of anti-MUC16 antibody agent or its antigen-binding fragment or combination that will be effective in treating and / or preventing the condition will depend on the nature of the disease and can be determined by standard clinical techniques.

[0339] The precise dosage to be used in the composition will also depend on the route of administration and the type of cancer, and should be determined based on the practitioner's judgment and the individual subject's circumstances. For example, the effective dosage can also be varied depending on the method of administration, target site, patient's physiological condition (including age, weight, and health), whether the patient is human or animal, other medications administered, or whether the treatment is prophylactic or therapeutic. Optimally titrating the therapeutic dose is crucial to optimizing safety and efficacy.

[0340] In some implementations, in vitro assays are used to help identify the optimal dose range. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[0341] For anti-MUC16 antibody agents or their antigen-binding fragments, the dosage can range from about 0.0001 to 100 mg / kg of patient body weight, and more typically from 0.01 to 15 mg / kg of patient body weight. For example, the dosage could be 1 mg / kg body weight, 10 mg / kg body weight, or in the range of 1-10 mg / kg, or in other words, for a 70 kg patient, it could be 70 mg or 700 mg, or in the range of 70-700 mg. Generally, due to the immune response to foreign peptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, lower doses of human antibodies and less frequent administration are usually possible.

[0342] In some embodiments, such as in the administration of engineered cells expressing the antibody or its antigen-binding fragment or CAR, the subject is administered cells ranging from about 1 million to about 100 billion, such as, for example, 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 600 million cells, about 600 million cells, about 1 ... The total number of cells is approximately 10 million, approximately 70 million, approximately 80 million, approximately 90 million, approximately 10 billion, approximately 25 billion, approximately 50 billion, approximately 75 billion, approximately 90 billion, or a range defined by any two of the foregoing values, and in some cases approximately 100 million to approximately 50 billion cells (e.g., approximately 120 million, approximately 250 million, approximately 350 million, approximately 450 million, approximately 650 million, approximately 800 million, approximately 900 million, approximately 3 billion, approximately 30 billion, approximately 45 billion) or any value between these ranges. In some embodiments, the dose of total cells and / or the dose of individual cell subpopulations is at or approximately 10 4 With or about 10 9 Within the range of cells / kg body weight, such as in 10 5 With 10 6 Between cells per kg body weight, for example, is or approximately 1 x 102 5 Cells / kg, 1.5 x 10 5 Cells / kg, 2x10 5 cells / kg, or 1x10 6 Cells / kg, 2x10 6 Cells / kg, 5x10 6 cells / kg, or 10x10 6Cells / kg body weight. For example, in some embodiments, cells are administered at a dose of approximately 10... 4 With or about 10 9 The T cells / kg body weight ratio is between 10 and 10. 5 With 10 7 The T cells per kg of body weight, or within a certain margin of error.

[0343] Anti-MUC16 antibody agents or their antigen-binding fragments can be administered under various conditions. The interval between single doses can be 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, or 2 years.

[0344] Combination therapy

[0345] In some embodiments, the method provided herein for treating a subject's cancer (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine cancer (e.g., endometrial cancer), or primary peritoneal cancer) comprising administering to a subject in need a pharmaceutical composition containing an anti-MUC16 antibody agent or its antigen-binding fragment described herein further comprises administering one or more additional therapeutic agents to the subject. In some embodiments, the additional therapeutic agent is used to treat the subject's cancer (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine cancer (e.g., endometrial cancer), or primary peritoneal cancer). In some embodiments, the additional therapeutic agent is used to treat any side effects of treatment using the anti-MUC16 antibody agent or its antigen-binding fragment described herein.

[0346] In some embodiments, the additional agent is an agent for treating ovarian cancer. In some embodiments, the additional agent is an agent for treating pancreatic cancer. In some embodiments, the additional agent is an agent for treating lung cancer. In some embodiments, the additional agent is an agent for treating breast cancer. In some embodiments, the additional agent is an agent for treating fallopian tube cancer. In some embodiments, the additional agent is an agent for treating uterine cancer (e.g., endometrial cancer). In some embodiments, the additional agent is an agent for treating primary peritoneal cancer.

[0347] The anti-MUC16 antibody or its antigen-binding fragment described herein can be administered simultaneously or sequentially (before and / or after) with other therapeutic agents. The antibody or its antigen-binding fragment and other therapeutic agents can be administered in the same or different compositions and via the same or different routes of administration. The first therapy (which is the anti-MUC16 antibody agent or its antigen-binding fragment or other therapeutic agent described herein) may be administered to a subject with cancer (e.g., ovarian cancer, pancreatic cancer, lung cancer, breast cancer, fallopian tube cancer, uterine cancer (e.g., endometrial cancer) or primary peritoneal cancer) before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks), simultaneously with (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks) the second therapy (the anti-MUC16 antibody agent or its antigen-binding fragment or other therapeutic agent described herein). In some embodiments, the additional therapeutic agent, when administered to a subject in combination with the anti-MUC16 antibody agent or its antigen-binding fragment described herein, is administered as the same composition (pharmaceutical composition). In other embodiments, the additional therapeutic agent, when administered in combination with the anti-MUC16 antibody agent or its antigen-binding fragment described herein, is administered to the subject as a composition different from the anti-MUC16 antibody agent or its antigen-binding fragment described herein (e.g., using two or more pharmaceutical compositions).

[0348] Products and reagent kits

[0349] In some embodiments of this technology, articles containing materials that can be used to treat cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) or to deliver an anti-MUC16 antibody agent (such as a full-length anti-MUC16 antibody) to cells expressing MUC16 on their cell surface are provided. The articles may include a container and a label or packaging instructions on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from various materials (e.g., glass or plastic). Typically, the container contains a composition effective for treating the disease or disorder described herein and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an anti-MUC16 antibody agent of this technology. The label or packaging instructions indicate that the composition is intended to treat a specific condition. The label or packaging instructions will also include instructions for administering the anti-MUC16 antibody agent composition to a patient. Articles and kits containing combination therapies described herein are also contemplated.

[0350] Instructions for use (IPA) are instructions typically included in the commercial packaging of therapeutic products. They contain information regarding the indications, usage, dosage, administration, contraindications, and / or warnings for using such therapeutic products. In some embodiments, the IPA indicates that the composition is intended for the treatment of cancers such as HCC, melanoma, squamous cell carcinoma of the lung, ovarian cancer, yolk sac tumor, choriocarcinoma, neuroblastoma, hepatoblastoma, nephroblastoma, non-seminomatous germ cell tumor of the testis, gastric cancer, or liposarcoma.

[0351] Additionally, the article may include a second container containing pharmaceutically acceptable buffer solutions, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may also include other materials required from a commercial and user perspective, including additional buffer solutions, diluents, filters, needles, and syringes.

[0352] Kits are also provided for use for various purposes, optionally in combination with the articles, such as for treating cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer) or for delivering anti-MUC16 antibody agents (such as full-length anti-MUC16 antibodies) to cells expressing MUC16 on their cell surface. Kits of this technology include one or more containers containing an anti-MUC16 antibody agent composition (or unit dosage form and / or article), and in some embodiments, also include another agent (as described herein) and / or instructions for use according to any method described herein. The kits may also include a description of individuals suitable for treatment. Instructions supplied with kits of this technology are typically written instructions on a label or package insert (e.g., paper pages included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0353] For example, in some embodiments, the kit comprises a composition containing an anti-MUC16 antibody (such as a full-length anti-MUC16 antibody). In some embodiments, the kit comprises a) a composition containing an anti-MUC16 antibody, and b) an effective amount of at least one other agent, wherein the other agent enhances the effect of the anti-MUC16 antibody (e.g., therapeutic effect, detection effect). In some embodiments, the kit comprises a) a composition containing an anti-MUC16 antibody, and b) instructions for administering the anti-MUC16 antibody composition to an individual for the treatment of cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). In some embodiments, the kit comprises a) a composition containing an anti-MUC16 antibody; b) an effective amount of at least one other agent, wherein the other agent enhances the effect of the anti-MUC16 antibody (e.g., therapeutic effect, detection effect); and c) instructions for administering the anti-MUC16 antibody composition and the one or more other agents to an individual for the treatment of cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). The anti-MUC16 antibody and the one or more other agents may be present in separate containers or in a single container. For example, the kit may contain one different composition or two or more compositions, wherein one composition contains an anti-MUC16 antibody and another composition contains another agent.

[0354] In some embodiments, the kit comprises a nucleic acid (or nucleic acid set) encoding an anti-MUC16 antibody (such as a full-length anti-MUC16 antibody). In some embodiments, the kit comprises a) a nucleic acid (or nucleic acid set) encoding an anti-MUC16 antibody and b) a host cell for expressing said nucleic acid (or nucleic acid set). In some embodiments, the kit comprises a) a nucleic acid (or nucleic acid set) encoding an anti-MUC16 antibody and b) instructions for: i) expressing an anti-MUC16 antibody in a host cell, ii) preparing a composition comprising the anti-MUC16 antibody, and iii) administering the composition comprising the anti-MUC16 antibody to an individual for the treatment of cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer). In some embodiments, the kit comprises a) a nucleic acid (or nucleic acid group) encoding an anti-MUC16 antibody; b) a host cell for expressing the nucleic acid (or nucleic acid group); and c) instructions for use in: i) expressing the anti-MUC16 antibody in the host cell, ii) preparing a composition containing the anti-MUC16 antibody, and iii) administering the composition containing the anti-MUC16 antibody to an individual for the treatment of cancers characterized by high MUC16 expression and / or high aerobic glycolysis (e.g., kidney cancer, cervical cancer, or prostate cancer).

[0355] This document also discloses a kit comprising an anti-MUC16 construct of the present technology, a mouse anti-MUC16 antibody or an antigen-binding fragment thereof, and instructions for use, wherein the mouse anti-MUC16 antibody or antigen-binding fragment comprises (a) a variable heavy (VH) chain comprising heavy chain complementarity-determining regions 1 (HC-CDR1), HC-CDR2, and HC-CDR3 of SEQ ID NO: 17, 18, and 19, and a variable light (VL) chain comprising light chain complementarity-determining regions 1 (LC-CDR1), LC-CDR2, and LC-CDR3 of SEQ ID NO: 14, 15, and 16, respectively; or (b) a variable heavy (VH) chain comprising heavy chain complementarity-determining regions 1 (HC-CDR1), HC-CDR2, and HC-CDR3 of SEQ ID NO: 35, 36, and 37, and a variable light (VL) chain comprising light chain complementarity-determining regions 1 (LC-CDR1), LC-CDR2, and LC-CDR3 of SEQ ID NO: 32, 33, and 34, respectively. The mouse anti-MUC16 antibody or antigen-binding fragment (e.g., those described in US 9,169,328) can be used to identify patients who respond to treatment using the anti-MUC16 construct. In some embodiments, the mouse anti-MUC16 antibody or antigen-binding fragment is used to detect tumors expressing MUC16 in samples obtained from the patient via Western blotting, immunohistochemistry, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or immunoelectrophoresis.

[0356] The kit for this technology is packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouth flasks, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kit may optionally include additional components, such as buffer solutions and explanatory information. Therefore, this application also provides articles of manufacture including vials (e.g., sealed vials), bottles, wide-mouth flasks, flexible packaging, etc.

[0357] Instructions for use related to the use of anti-MUC16 antibody compositions typically include information on the intended therapeutic dose, dosing schedule, and route of administration. Containers may be single-dose, bulk (e.g., multi-dose packs), or subunit doses. For example, kits may be available containing sufficient doses of an anti-MUC16 antibody as disclosed herein (e.g., a full-length anti-MUC16 antibody) to provide an individual with an extended period of effective treatment, such as one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or longer. Kits may also include multiple unit doses of the anti-MUC16 antibody and pharmaceutical composition, along with instructions for use, and are packaged in sufficient quantities for storage and use in a pharmacy (e.g., a hospital pharmacy and a dispensing pharmacy).

[0358] Those skilled in the art will recognize that several implementations are possible within the scope and spirit of this technology. The technology will now be described in more detail with reference to the following non-limiting embodiments. These embodiments further illustrate the technology, but should not, of course, be construed as limiting the scope of the technology in any way.

[0359] Example

[0360] The present technology is further illustrated by the following examples, which should not be construed as limiting the invention in any way. The following examples show the preparation, characterization, and use of illustrative anti-MUC16 antibodies of the present technology. The following examples demonstrate the generation of human antibodies and bispecific antibodies of the present technology, and the characterization of their binding specificity and in vivo biological activity.

[0361] Example 1: In vitro characterization of antibodies binding to the carboxyl terminus of MUC16

[0362] Previously, mouse monoclonal antibodies were developed targeting a peptide sequence (referred to herein as "peptide-2") in the extracellular (juxtamembrane) region of the carboxyl terminus of MUC16. This embodiment describes the testing of six of these mouse monoclonal antibodies, which showed differential binding to MUC16. The goal of this study was to identify the best antibody candidates for clinical use in humanization and potential translation. The study was divided into three phases. In the first phase, the six antibodies were evaluated based on the results of a comparative in vitro screening process. The goal of this phase was to select two lead candidates for in vivo assays based on three parameters: 1) antibody compliance with the bioconjugate; 2) results for the radiolabeled antibody, including radiochemical yield and molar activity; and 3) performance of the radioimmunoconjugate in cell-based assays, including determination of immunoreaction fraction, saturation binding, and internalization.

[0363] In the second phase of the study, the in vivo tumor-targeting and radiopharmacological characteristics of the two lead candidates identified from the in vitro screening process were evaluated (see Example 2).

[0364] Finally, in the third phase of the study, the lead MUC16 carboxyl-terminal binding antibody identified in the previous phase was humanized. The humanized variant was then evaluated for in vivo tumor targeting and radiopharmacological characteristics (see Example 3).

[0365] The three antibodies selected in this study, 9C9, 4H11, and 4A5, had previously demonstrated robust high-affinity binding to the C-terminus of MUC16 using various analytical methods, including enzyme-linked immunosorbent assay (ELISA) using supernatant from their respective hybridoma cultures, Western blotting using purified recombinant pFUSE MUC16c114 (containing an extracellular domain fused to the C-terminus of the human Fc antibody domain at 58 amino acid residues), flow cytometry, and saturation binding assays using MUC16-expressing OVCAR3 cells. Two other antibodies, 4C7 and 29G9, bound to MUC16 were also observed by ELISA and Western blotting. The final antibody, 4A2, was positive only by ELISA.

[0366] 89 The ideal characteristics of Zr (including its physical (decay) half-life, which matches the biological half-life well) 1 / 2 =72.4h) and its ability to remain in cells after internalization, combined with its in vivo pharmacokinetics via antibody tumor targeting, make it an isotope selected for radiolabeling various MUC16 carboxyl-terminal binding antibodies and evaluating the in vitro and in vivo radiopharmacological characteristics of said antibodies. 89 Zr was transported via the TR19 / 9 cyclotron (Ebco Industries Inc.) at Memorial Sloan Kettering Cancer Center. 89 Y(p,n) 89 Zr is produced by the reaction and purified to produce a specific activity of 196-496 MBq / mg. 89 Zr. Activity measurements were performed using a CRC-15R dosimeter (Capintec). For quantification of activity, samples were counted on an automated Wizard gamma counter (Perkin Elmer). Radiolabeling of the ligands was monitored using instant thin-layer chromatography paper (Agilent Technologies) and analyzed using Winscan Radio-TLC software (Bioscan Inc.) on a Bioscan AR-2000 radiometric ITLC reader.

[0367] In order to use 89 Zr radiolabeled antibodies were conjugated with a deferrosensitive isothiocyanate-functionalized variant (p-SCN-Bn-DFO) under the same reaction conditions to produce DFO-immunoconjugates for all six antibodies. The goal was to facilitate the bioconjugation of the bifunctional chelator to one or more ε-amines on lysine residues randomly distributed throughout the antibody structure. Briefly, the antibodies were suspended at an average concentration of 2–3 mg / mL in citrate buffer (25 mM sodium citrate, 150 mM sodium chloride). The antibodies were buffer-exchanged using a disposable Sephadex G-25PD10 desalting column (17085101; GE Healthcare, Life Sciences) pre-equilibrated with Chelex-treated PBS and concentrated using a centrifuge filter unit with a 50,000 molecular weight cutoff (Amicon Ultra 4 centrifuge filter unit, Millipore) to obtain a final concentration of 12–15 mg / mL. The pH of the antibody solution was adjusted to 8.5–9.0 using 0.1 M Na₂CO₃. Next, 10 molar equivalents of isothiocyanate-deferromin (p-SCN-Bn-DFO) (B-705; Macrocyclics, Inc.) were dissolved in DMSO (41640; Sigma Aldrich) at a concentration of 10 mg / mL. The reaction was incubated at 37°C for 1 h at 500 rpm on a thermostat. The DFO-conjugated antibody was purified using a PD10 desalting column and concentrated using the centrifugation filter unit described above.

[0368] 89 Zr is derived from the radiochemistry and molecular imaging probe core of MSKCC, dissolved in 1M oxalic acid. 89 ZrOxalate was used as the base. The solution was neutralized with 1M sodium carbonate to achieve a pH of approximately 7. Each immunoconjugate dissolved in Chelex-treated PBS (pH 7.4) was then added to the neutralized solution. 89 Zr was incubated together at 37°C for 1 hour. The progress of the radiolabeling reaction was monitored by radioactive transient thin-layer chromatography (radioactive ITLC) by spotting 0.5 μL of the crude reaction mixture onto silica gel-impregnated glass microfiber paper tape (iTLC-SG; Varian), and analyzed using 50 mM EDTA (pH 5.5) as the mobile phase solvent on an ITLC scanner (AR-2000; Bioscan Inc.). On ITLC, 89Zr-labeled radioimmunoconjugate complexes remained at the origin, while free... 89 Zr was absorbed by EDTA in the mobile phase and migrated along with the solvent front. The crude radiochemical yield was calculated using radiometric ITLC data. Purification was then performed using a PD10 desalting column via size exclusion chromatography. 89 Zr-radioimmunoconjugates were then centrifuged and filtered to concentrate the final volume for preparing tracer doses. The radiochemical purity of the purified radioimmunoconjugates was confirmed by radioactive ITLC, and they were then used in animal experiments.

[0369] To determine serum stability, 100 μL of each radioimmunoconjugate was incubated with 900 μL of human serum (H4522; Sigma Aldrich) in a thermostatic mixer at 37°C with constant agitation. Samples were taken from each microcentrifuge tube on days 0, 1, 3, 5, and 7 and analyzed by radioactive ITLC. All samples were analyzed in triplicate. Serum stability of the radioimmunoconjugates was measured as the value retained at the origin of the radioactive ITLC band. 89 The percentage of Zr is reported as a complete %.

[0370] The cell binding assay was determined using a modified procedure following the procedure described in Lindmo et al. (1984) J Immunol Methods 72:77-89. 89 The immune response score of Zr-DFO antibody. Therefore, SKOV3... c114 Cells with a range of 5.0 x 10 5 -5.0x10 6 Cells / mL were suspended in 500 μL of PBS (pH 7.4) supplemented with 1% BSA in microcentrifuge tubes. Aliquots of various radioimmunoconjugates (50 μL 1 μCi / mL stock solution) were added to each tube, and the total volume of cells and radioimmunoconjugates in each tube was brought to 500 μL. The samples were incubated in a thermostatic mixer at 37°C and 500 rpm for 60 min. The treated cells were then pelleted by centrifugation (1400 rpm for 4 min), the supernatant was aspirated, and the pellet was washed three times with ice-cold PBS. The supernatant was then removed, and the radioactivity associated with the cell pellet was counted. Activity data were background-corrected and compared with the total counts in appropriate control samples. Immunoreactivity fractions were determined by linear regression analysis plotting the total / bound radioactivity against the reciprocal of the normalized cell concentration. Additionally, the lead antibody was evaluated in a bead-based binding assay. 89 Immunoreactivity of Zr-DFO-4H11 (see Figure 5).

[0371] To generate SKOV3c114 SKOV3 cells, purchased from the American Type Culture Collection (ATCC, Manassas, Virginia), were used with plasmid phrGFP-MUC16. c114 Transfection was performed using a plasmid encoding 114 amino acids from the C-terminus of MUC16. Untransfected SKOV3 (wild-type) cells were also cultured and used as a negative control in the experiments. The cells were cultured in RPMIM McCoy's 5A medium, which was modified to contain 1.5 mM L-glutamine, 100 units / mL penicillin G, 100 μg / mL streptomycin, 10% fetal bovine serum, and 800 μg / mL genimycin G418. The cells were maintained in a water-jacketed incubator at 37°C with a 5% CO2 supply. The cell line was passaged weekly in split T-150 flasks (1:5) using 0.25% trypsin / 0.53 mM EDTA in a calcium- and magnesium-free Hank buffer solution. MUC16 cells expressing OVCAR3 were obtained from ATCC and cultured in RPMI 1640 medium supplemented with heat-inactivated fetal bovine serum (20% v / v, GIBCO, Life Technologies), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4.5 g / L glucose, 1.5 g / L sodium bicarbonate, 0.01 mg / mL bovine insulin (Gemini Bio-Products, 700-112P), 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0372] For saturation binding studies, six concentrations (0.1, 1, 5, 10, 25, 50, and 100 nM) of MUC16 carboxyl-terminus-bound antibody were used. 89 Zr-labeled variants were compared with 500,000 SKOV3 molecules suspended in PBS supplemented with 1% BSA at 37°C. c114 Cells were incubated together for 1 hour. Simultaneously, parallel setups were prepared and analysis of the antibody and SKOV3 was performed. c114 Non-specific binding to cells. The latter is achieved by binding the antibody to the carboxyl terminus of MUC16. 89 Zr-radioimmunoconjugate variants and 100 nM of the corresponding unlabeled antibody were added to SKOV3. c114 The mixture of cells was prepared. All experiments were performed in triplicate.

[0373] Using SKOV3 c114 Cellular studies were conducted to investigate the internalization of various radioimmunoconjugates. Approximately 1 × 10⁻⁶ [units of measurement missing]. 5 Cells were seeded in 12-well plates and incubated overnight. 2 mL of the radioimmunoconjugate (1 μCi / mL SKOV3) was added. c114Culture medium was added to each well. Plates were incubated at 37°C and 4°C for 1, 4, 12, and 24 h. After each incubation period, cell supernatant was collected, and cells were washed twice with 1 mL of ice-cold phosphate-buffered saline (PBS). Surface binding activity was collected by washing cells in 1 mL of 100 mM acetic acid + 100 mM glycine (1:1, pH 3.5). Adhering cells were then lysed with 1 mL of 1 M sodium hydroxide. Each wash was collected, and activity was counted. The percentage of internalized activity was calculated as the ratio of the activity of the lysate to the total activity from the culture medium, PBS, acid, and alkaline washes.

[0374] Although the bioconjugation and radiolabeling of immunoconjugates were performed under the same conditions, the variable radiochemical yields and molar activities of the radioimmunoconjugates ranged from 1.16 MBq / nmol (0.21 mCi / mg) to 31.8 MBq / nmol (5.74 mCi / mg) (Table 4). One antibody, 4A2, may not have been radiolabeled under the same conditions used for the bioconjugation and radiolabeling of the other five antibodies and was discarded in further analysis. Antibodies 29G9 and 4A5 were used... 89 Zr radiolabeling produces low molar activity. Specifically, [ 89 The extremely low molar activity of Zr-DFO-4A5 was poor compared to radioiodinated variants of the same antibody that previously showed high binding affinity (KD = 7.3 ± 1.1 nM) to MUC16 cells expressing OVCAR3 (Dharma et al. (2010) Appl Immunohistochem Mol Morphol. 18:462-472). It seems possible that the solvent accessibility and compliance with amine-based conjugations of lysine residues in 4A5 are less than those of tyrosine residues, which are more readily accessible by radioiodination of 4A5, leading to the difference in radiochemical yield between the two methods used for radiolabeling antibodies. The low immunoreactivity fraction may be associated with low molar activity (higher unlabeled fraction), which could prevent radiolabeled antibodies from reaching the target. In conclusion, 29G9 and 4A5 were further investigated in this study due to their low radiochemical yields and poor molar activity.

[0375] Table 4. In vitro characterization of six MUC16 CTD-binding mouse monoclonal antibodies

[0376]

[0377] ++++ indicates a positive result for MUC16 binding in ELISA, Western blotting, flow cytometry, and saturation binding assays.

[0378] ++ indicates a positive result for MUC16 binding only in ELISA and Western blot.

[0379] + indicates a positive result for MUC16 binding only in ELISA.

[0380] ---The instruction is not applicable and has not been performed.

[0381] All data presented are expressed as mean ± SD. Where applicable, statistical differences were analyzed by unpaired two-tailed Student's t-test (with Welch correction when mentioned). Differences at the 95% confidence level (P < 0.05) were considered statistically significant and indicated by an asterisk.

[0382] Of the other three antibodies, 9C9, 4H11, and 4C7, the radioimmunoconjugate of 4C7 produced the lowest molar activity and exhibited the lowest immunoreactivity score. These results, combined with its relatively high binding affinity (>10 nM) derived from the saturation binding assay, led to the exclusion of 4C7 from the second stage of the screening workflow.

[0383] Therefore, 9C9 and 4H11 were identified as two lead MUC16 carboxyl-terminal binding antibodies. 89 Variants of the Zr tag [ 89 Zr]Zr-DFO-9C9 and [ 89 Zr-DFO-4H11 exhibits favorable in vitro characteristics, including high molar activity, ≥80% immunoreactivity fraction, and binding affinity in the 5–11 nM range (Table 4 and Figure 3B). The binding affinity values ​​derived from the saturation binding assays in this study are consistent with previously reported values ​​obtained in similar experiments using radioiodinated variants of 9C9 and 4H11 (Dharma et al. (2010) Appl Immunohistochem Mol Morphol. 18:462–472).

[0384] It is noteworthy that, although they possess comparable binding affinity for peptide-2 in the carboxyl-terminal domain of MUC16, 9C9 and 4H11... 89 Zr-radioimmunoconjugates exhibited different rates of cellular uptake (Fig. 3C). At 4°C, the two radioimmunoconjugates were reacted with SKOV3... c114 Cell binding was low and comparable. However, at 37°C, [ 89 Zr-DFO-4H11 exhibits higher radioactive uptake, with 12.47% ± 3.02% of the normalized applied activity internalized within 4 hours, followed by slow accumulation, reaching 14.09% ± 1.34% at 24 hours (Figure 3C). On the other hand, [ 89Zr-DFO-9C9 exhibited relatively slow cellular uptake, indicated by 5.22% ± 1.34% internalized up to 1 hour, followed by gradual accumulation, reaching 11.89 ± 2.21% at 24 hours. These differences in cellular uptake rates and maximum internalization values ​​achieved by the two lead antibody candidates can be attributed to more than one factor, including the total charge of the radioimmunoconjugates, which may be influenced by differences in the amino acid composition of the antibody classes or the geometry of binding to the MUC16 peptide-2, which can be deduced from the crystal structure of one or more binding pockets of 9C9 with 4H11 scFv. Upon incubation in serum, the radioimmunoconjugates of both lead antibody candidates showed comparable high stability (Figure 3D). Interestingly, an unrelated study exploring the development of CAR T cells using single-chain variable fragments (scFv) of 9C9 and 4H11 revealed that the scFv sequences from these two antibodies are identical (data not shown), suggesting that the two antibodies may have the same molecular footprint on MUC16 due to binding to the same epitope in the peptide-2 sequence within the juxtamembrane region of the extracellular domain of MUC16. This was achieved in this study by […]. 89 The blocking of the binding of Zr]Zr-DFO-mu4H11 to biotinylated peptide-2 was experimentally verified (Figure 3E).

[0385] Example 2: In vivo characterization of antibodies binding to the carboxyl terminus of MUC16

[0386] After identifying 9C9 and 4H11 as two lead candidates, a second phase of investigation was conducted using these antibodies. This second phase involved in vivo characterization of the tumor-targeting ability and overall radiopharmacological properties of these antibodies. For this purpose, radioimmunoconjugates with high radiochemical purity and high molar activity were synthesized. 89 Zr]Zr-DFO-9C9 and [ 89 Zr]Zr-DFO-4H11, and carrying subcutaneous xenograft SKOV3 c114 The tumor was tested in mice via PET imaging and biodistribution studies.

[0387] Eight to ten-week-old nu / nu female mice were purchased from the Charles River Laboratory. Animals were housed in ventilated cages with free access to food and water and allowed to acclimatize for approximately one week before being inoculated with tumor cells. The cells were administered subcutaneously in a 1:1 mixture of 150 μL fresh culture medium and BD Matrigel (356234, BD Biosciences) at a concentration of 5 × 10⁶ cells / mL. 6 One cell was implanted into the right shoulder of each mouse with SKOV3. c114 Tumor. During SKOV3 injection. c114Experiments were conducted approximately 3 weeks after cell culture. To generate a bilateral tumor model, 5x10 [cells / plants / etc.] were [used / implants / etc.]. 6 One SKOV3 cell was inoculated into the right shoulder of 8-10 week old female nu / nu mice, and 5 x 10 cells were injected two weeks later. 6 SKOV3 c114 Cells were seeded on the left shoulder. 10x10 6 One OVCAR3 cell was implanted into the right shoulder of a 6-8 week old female nude mouse. The seed for the xenograft from the HGSOC patient was provided by the MSKCC Antitumor Assessment Core and passaged and expanded into a subcutaneous xenograft.

[0388] PET imaging experiments were performed on an Inveon PET / CT scanner (Siemens Healthcare). SKOV3 was injected intravenously via tail vein into the right shoulder carrying a subcutaneous xenograft. c114 Mice with tumors were administered a lead MUC16 carboxyl-terminal binding antibody. 89 Zr-labeled radioimmunoconjugate variants (150–260 μCi; 5.55–9.62 MBq in 200 μL of Chelex-treated PBS). Animals were anesthetized by inhalation of a mixture of 2% isoflurane (Baxter Healthcare) and medical air gas and placed on a scanning bed. PET data for each mouse were recorded via static scanning at different time points following injection of the radioimmunoconjugate. Images were analyzed using ASIPro VM software (Concorde Microsystems). PET images of bilateral tumor models were acquired using a mouse hotel in an Inveon PET / CT scanner and analyzed using AMIDE software. Briefly, images reconstructed using 3D ordered subset expectation-maximization (3D OSEM) were calibrated for the tracer injection dose and smoothed using a Gaussian function with a full width at half maximum (FWHM) of 1.5 before PET images were overlaid with CT images.

[0389] Using the carboxyl terminus of the lead MUC16 antibody 89 Zr-marked variants in carrying SKOV3 c114+Biodistribution of subcutaneous xenografts of tumors was studied in female nude mice. Each radioimmunoconjugate was administered to the mice via lateral tail vein injection at a concentration of 21–30 μCi; 0.77–1.11 MBq in 200 μL PBS. For the blockade group, animals were co-injected with a 50-fold overdose of unlabeled antibody. Animals (n = 4 / group) were euthanized by CO2 asphyxiation to analyze the biodistribution of the radioimmunoconjugates in mice at different time points following injection. After euthanasia, vital organs (e.g., blood, heart, lungs, liver, spleen, stomach, pancreas, large intestine, small intestine, reproductive organs (including ovaries, fallopian tubes, and uterus), kidneys, bones, muscles, tail, axillary lymph nodes) and one or more tumors were harvested, weighed, and analyzed against... 89 Radioactivity was determined on a Zr-calibrated gamma counter. Counts were converted to activity using a calibration curve generated from known standards. Count data were corrected for background and decay at injection time, and the injection dose % / g (%ID / g) for each tissue sample was calculated by normalizing the total activity injected per mouse.

[0390] The administration was performed at 24-hour intervals following the injection of the radioimmunoconjugate (pi). 89 Serial PET imaging of Zr-DFO-9C9 (Fig. 4A) depicts the outline of a subcutaneous tumor over 24 hours, after which activity gradually accumulates within the tumor over time up to 96 hp.i. 89 Zr-DFO-9C9 showed high radioactivity concentrations in the liver as early as 24 hours post-infection, and the signal from this tissue was significantly cleared at subsequent time points. Most notably, [ 89 Zr-DFO-9C9 exhibits relatively high radioactivity concentrations in the kidneys. This is an unexpected observation for radioimmunoconjugates based on full-length antibodies, whose molecular weight exceeds the renal filtration cutoff for bodily clearance. Furthermore, mouse kidneys are known not to express MUC16 or analogs of the MUC16 juxtamembranous carboxyl-terminal domain, justifying the presence of the antibody in this organ. Additionally, from injection […]. 89 Maximum density projection (MIP) images from a longitudinal PET study of Zr-DFO-9C9 in mice indicate gradual clearance of activity from the kidneys between 24 and 96 h pI for the radioimmunoconjugate. It appears possible that the high radioactive concentrations in the liver and kidneys of mice at early time points are due to the combination of these highly perfused organs with the slow in vivo pharmacokinetics of the 9C9 antibody. Despite the existence of a tumor-provided target library, […]. 89The persistence of activity in systemic circulation, confirmed by PET signals in the heart and aortic arch as seen in MIP images of Zr-DFO-9C9 at 72 and 96 hpi, indicates the extremely slow in vivo pharmacokinetic characteristics of this antibody. These in vivo observations confirm the relatively slow in vitro cellular uptake characteristics exhibited by the 9C9 antibody in previous phases of this study.

[0391] On the other hand, [the procedure was performed at 24-hour intervals after the injection of the radioimmunoconjugate]. 89 Serial PET imaging of Zr-DFO-4H11 (Fig. 2B) depicting subcutaneous SKOV3 at 24 h pi c114 The outline of the tumor. 89 Zr]Zr-DFO-9C9 and [ 89 Direct head-to-head comparison of sequential PET images of Zr-DFO-4H11 shows that at the earliest time point [ 89 Zr]Zr-DFO-4H11 in SKOV3 c114 The radioactivity concentration in the tumor is higher than [ 89 Zr]Zr-DFO-9C9. 89 Zr-DFO-4H11 also showed radioactive uptake in the liver at 24 h pi; however, the concentration of activity in this organ decreased at subsequent time points, while the activity gradually increased in the tumor until 96 h pi for the radioimmunoconjugate. Besides the tumor (the main target library of antibodies) and the liver (the clearance site of exogenous immunoglobulins), injection […]. 89 MIP images of Zr-DFO-4H11 mice showed bilateral symmetrical PET lesions in the axillary lymph nodes. In addition to this abnormality, [ 89 The in vivo radiopharmacological characteristics of Zr-DFO-4H11 are compared to its counterparts. 89 Zr-DFO-9C9 is more advantageous. 89 Zr]Zr-DFO-4H11 was already in SKOV3 24 hours ago c114 The relatively rapid uptake of the medium produces high-contrast PET images, and leaves minimal activity in systemic circulation and background organs other than the liver at subsequent time points.

[0392] Independent biodistribution studies confirmed observations from PET imaging, including subcutaneous SKOV3. c114 Tumors and target organs were harvested 96 hpi from xenografts using radioimmunoconjugates (Figure 6C). To verify the specificity of antibody binding and active uptake in tumors, a blockade group was included in the in vivo biodistribution study of the two radioimmunoconjugates. Subcutaneous SKOV3 was used.c114 Xenografts were co-injected with 50-fold excess (by weight) of unlabeled 9C9 or 4H11 antibodies to block [the virus]. 89 Zr]Zr-DFO-9C9 and [ 89 Specific uptake of ZrZr-DFO-4H11. While a decrease in target-mediated uptake and associated activity of radioimmunoconjugates is expected, the active concentrations in tissues exhibiting nonspecific uptake are expected to remain unchanged. However, blockade of target-mediated specific uptake of radioimmunoconjugates in tumors often manifests as a slightly increased active concentration in well-perfused non-target background organs, including the heart, lungs, liver, spleen, and kidneys. This can be attributed to the blockade of a large number of persistently circulating radioimmunoconjugates from a tumor-rich target library.

[0393] In short, [ 89 Zr]Zr-DFO-9C9 and [ 89 The biodistribution profile of Zr-DFO-4H11 is consistent with that of the two radioimmunoconjugates on PET imaging. With the exception of a few case-specific exceptions documented in PET imaging studies, active uptake in most non-target background organs was comparable and low (≤5% ID / g). Specifically, [ 89 Zr-DFO-9C9 showed high activity concentrations in the kidneys (11.2 ± 2.35% ID / g), which were significantly higher than [ 89 Zr]Zr-DFO-4H11 (4.3±1.00% ID / g; p=0.0016) and [ 89 Zr-DFO-isotype IgG (4.9 ± 0.57% ID / g; p = 0.002) uptake in this organ. SKOV3 co-injected with a 50-fold excess of unlabeled 9C9 antibody. c114 Xenografts did not show any effect on the kidneys [ 89 The uptake of ZrZr-DFO-9C9 was blocked (13.3 ± 2.64% ID / g), suggesting that uptake in this organ may be nonspecific.

[0394] Interestingly, unlike PET images, comparative biodistribution studies confirmed that in both MUC16-targeting radioimmunoconjugates ([ 89 Zr]Zr-DFO-9C9 9.1±2.49% ID / g and [ 89 The concentrations of Zr-DFO-4H11 (6.3 ± 1.41% ID / g; p = 0.099) or isotype IgG (6.4 ± 1.02% ID / g; p = 0.091) in the liver at 96 h pi were between 6.3 ± 1.41% ID / g and 6.4 ± 1.02% ID / g; p = 0.091, respectively. c114The radioactivity concentrations of xenografts were not significantly different. Although relative to [ 89 Zr]Zr-DFO-9C9 (4.2±0.48% ID / g; p=0.002) or isotype IgG (3.4±0.40% ID / g; p=0.001), in comparison with [ 89 SKOV3 injected together with Zr]Zr-DFO-4H11 c114 Increased active concentrations of xenografts were observed in axillary lymph nodes (11.5 ± 2.80% ID / g), but uptake in these tissues was nonspecific, as it was not blocked in mice co-injected with a 50-fold overdose of unlabeled 4H11 antibody (13.2 ± 2.72% ID / g; p = 0.43). [The text abruptly ends here, likely due to an incomplete translation or missing information.] 89 Zr]Zr-DFO-4H11's SKOV3 c114 Further histopathological analysis of H&E-stained sections of PET-positive and biodistribution-positive lymph nodes harvested from xenografts did not reveal the presence of neoplastic cells. Finally, [ 89 Zr]Zr-DFO-9C9 (18.7±2.37%ID / g) and [ 89 Tumor uptake of Zr-DFO-4H11 (17.4 ± 2.51% ID / g) was comparable and significantly higher than that of isotype IgG (4.7 ± 0.42% ID / g; p = 0.00002 and 0.00006, respectively). Furthermore, co-injection of the MUC16-targeted radioimmunoconjugate with a 50-fold overdose of its corresponding unlabeled variant blocked […]. 89 Zr]Zr-DFO-9C9 and [ 89 Tumor uptake of Zr-DFO-4H11 (6.8 ± 1.81% ID / g and 7.4 ± 2.18% ID / g) (Figure 4C).

[0395] In summary, comparing SKOV3 using PET imaging and biodistribution studies... c114 Results of in vitro and in vivo evaluations of the radioactive uptake ratio of tumors to background organs associated with two lead antibodies targeting the extracellular domain of MUC16 (Figure 4D) revealed [ 89 Zr-DFO-4H11 produces relatively better in vivo radiopharmacological characteristics. These findings increase the credibility of the clinical applicability of a murine variant of the 4H11 antibody, which previously showed excellent performance in immunohistochemical staining of formalin-fixed paraffin-embedded surgical samples obtained from HGSOC and lobular breast cancer patients.

[0396] Example 3: Humanized 4H11 antibody

[0397] Humanize mouse 4H11 to ensure compatibility with humanized variants and MUC16. c114 Minimal impairment of binding affinity. The sequence of the humanized heavy chain is provided as SEQ ID NO:4, and the sequence of the humanized light chain is provided as SEQ ID NO:2. To verify retention of in vitro target binding capacity and in vivo pharmacological characteristics in a preclinical setting, a humanized variant of 4H11 (“hereinafter referred to as hu4H11”) was conjugated with DFOs (Figure 5A), as previously described for the mouse variant. MALDI-ToF analysis of the immunoconjugates revealed one DFO for each antibody conjugate. Prior to radiolabeling, the DFOs were analyzed by flow cytometry using SKOV3. c114 Cells were used to test the target binding of DFO immunoconjugates to SKOV3 cells.

[0398] The rightward shift of the fluorescence peak on the X-axis of the histogram indicates the interaction between the DFO-hu4H11 immunoconjugate and SKOV3. c114 Positive binding to cells was observed, but the lack of a fluorescence peak shift compared to unstained cells and cells stained with secondary antibody only indicated the absence of binding to SKOV3(wt) cells (Fig. 5B). Notably, the SKOV3(wt) cell line is more representative of ovarian clear cell carcinoma, a known OvCa subtype independent of MUC16 expression. Furthermore, using… 89 The Zr radiolabeled DFO-hu4H11 consistently provides high radiochemical yields and purity of radioimmunoconjugates, with a molar activity of 23.6 MBq / nmol (n = 9) (Figures 5C-5D). [The following text appears to be unrelated and possibly a separate excerpt: "Found [ 89 The target binding fraction of Zr-DFO-hu4H11 was 96 ± 0.53%, and in a bead-based radioligand binding assay, the binding of the radioimmunoconjugate to biotinylated MUC16 peptide-2 captured on streptavidin beads could be partially blocked in the presence of a significant excess of unlabeled 4H11 antibody (Figure 5E).

[0399] After characterizing its in vitro target binding capacity, the hu4H11 immunoconjugate was further tested to evaluate its in vivo biodistribution and radiopharmacological properties. Consistent with the in vivo characteristics demonstrated through longitudinal PET imaging studies of its mouse precursor, hu4H11 was able to clearly delineate subcutaneous xenografted SKOV3 at an early time point after injection of the radioconjugate. c114 Tumor outline (Fig. 6A). Although some persistence of activity in systemic circulation was observed at the midpoint of 72 h pi, the vast majority of injected activity was found in the tumor by 144 h pi. The results from the biodistribution study are consistent with observations from PET images and show that activity in SKOV3 decreased with the reduction of background activity in the blood pool. c114Gradually accumulating within the tumor (Figure 6B). 89 Tumor uptake of Zr-DFO-hu4H11 was inhibited in the blockade group, in which mice were co-injected with a 40-fold overdose (by weight) of unlabeled hu4H11 antibody and in vivo biodistribution was evaluated at 72 h p. (22.4 ± 3.65 vs 14.3 ± 1.50% ID / g; p = 0.006). Unlike its mouse precursor, [ 89 Zr]Zr-DFO-hu4H11 in SKOV3 c114 No PET-positive axillary lymph nodes were observed in the xenografts. However, biodistribution studies of harvested bilateral axillary lymph nodes revealed active concentrations ranging from 7.9 ± 1.23% ID / g at 36 h pi to 10.3 ± 4.04% ID / g at 144 h pi. Notably, the absence of inhibited radioactive concentrations in the axillary lymph nodes of the blockade group mice at 72 h pi suggests that uptake in this tissue may be nonspecific. As a radiotracer, [ 89 Zr-DFO-hu4H11 exhibits an excellent tumor-to-background organ ratio (Figure 6C). This favorable in vivo characteristic suggests a promising future for the development of hu4H11-based drugs, including radiopharmaceuticals, for use in immuno-PET and targeted radiotherapy.

[0400] In a bilateral tumor model, [the following was performed] 89 Further investigation into the in vivo specificity of Zr-DFO-hu4H11 binding to MUC16 C-terminal expression cells, including SKOV3 implantation in the left shoulder of nu / nu mice. c114 Cells were implanted, and an SKOV3 tumor was implanted in the right shoulder. 89 Zr]Zr-DFO-hu4H11 shows the relationship between SKOV3 and c114 The binding of cell-expressed targets exhibits excellent specificity, as demonstrated by the high tumor uptake of radioactive materials in this tumor and the minimal nonspecific uptake in SKOV3 tumors (Figure 7A). The latter is generally attributed to the enhanced in vivo penetration and retention of full-length antibody-based imaging agents in poorly vascularized solid tumor tissues.

[0401] For injection [ 89 In vitro analysis of bilateral tumors in mice containing Zr-DFO-hu4H11 revealed radioactive expression of SKOV3 at the carboxyl terminus of MUC16. c114 Focal accumulation of tumor cells in the perivascular space and in areas rich in healthy tumor cells (Figs. 7B and 7C; dashed circles). On the other hand, SKOV3... c114 The necrotic areas of the tumor (Figs. 7B and 7C; dashed triangles) reveal the absence of radioactivity. This contrasts with the bilateral tumor model.89 PET images of Zr-DFO-hu4H11 were consistent, and the target-negative SKOV3 tumor showed minimal radioactivity accumulation (Fig. 7B). Histopathological analysis of bilateral tumors via H&E staining revealed SKOV3... c114 The tumor carries an architecture and morphological features significantly different from the typical cell morphology of clear cytoplasm, a characteristic cell line of SKOV3 cells representing ovarian clear cell carcinoma (Fig. 7D). Previous reports have shown that expression of the C-terminal domain of MUC16 in NIH / 3T3 cells induces transformation and enhances metastatic properties in this cell line, while ectopic expression of the C-terminal domain in SKOV3 cells increases cell motility, invasiveness, and tumorigenicity in vitro and in vivo.

[0402] To test the applicability of a 4H11-based radiotracer in a model that does not artificially overexpress MUC16 peptide-2, a subcutaneous xenograft model was developed using OVCAR3 cells, a representative cell line for HGSOC that naturally expresses high levels of MUC16. In this model, [ 89 PET images of Zr-DFO-hu4H11 show high radioactivity concentrations in OVCAR3 tumors at the midpoint of 72 hpi (Fig. 8A). Finally, to explore its clinical use... 89 The feasibility and proof-of-concept demonstration of Zr-DFO-hu4H11 as an immunochemical PET agent were presented using a patient-derived xenograft (PDX) model representing HGSOC. In this model... 89 PET images of Zr-DFO-hu4H11 revealed high radioactivity concentrations in PDX tumors at 72 hpi. Notably, PET images from OVCAR3 and the PDX model revealed persistent background activity in mouse blood pools (BP) (including the heart and descending aorta). This is consistent with observations from previous experiments using humanized 4H11 antibodies (Figs. 5–7) and appears to be independent of the target library volume provided by one or more tumors. The persistent activity in systemic circulation could indicate characteristic features of the antibody's pharmacokinetics and biological half-life in vivo.

[0403] This study highlights the practicality of radiopharmacological screening in identifying optimal candidates during antibody-based drug development. While several nuclear and label-free biophysical and biochemical analytical methods have been developed to characterize key antibody features, including binding affinity and cellular internalization rate, these techniques remain limited in their ability to predict or characterize the in vivo behavior of lead antibody candidates. Results derived from in vitro radiometric measurements can be combined with in vivo radiopharmacological features revealed through non-invasive nuclear imaging and biodistribution studies as a strategy for better screening and characterizing lead antibody candidates. Radiopharmacological screening offers the unique benefits of visualization, traceability, and quantitative evaluation of antibody pharmacokinetics in vivo, all achievable with high sensitivity at microdose doses. Obtaining this information can facilitate a better understanding and characterization of antibody accessibility and binding to their homologous targets to define their efficacy as drugs, while also outlining any anomalous interactions that may occur with non-target organs that could lead to potential toxicity.

[0404] Example 4: Surface plasmon resonance (SPR) characterization of humanized 4H11 antibody

[0405] The relative binding affinity of 4H11 humanized antibodies (H1L1, H1L2, H2L1, H2L2) was compared using a BIACore-X100 instrument (GE Healthcare) with a biotinylated CAP chip via surface plasmon resonance. The assay consisted of capturing biotinylated MUC16 peptide-2 (TLDRSSVLVDGYSPNRNE; SEQ ID NO:52) onto a streptavidin-coated sensor, followed by single-cycle kinetics to allow the antibody to flow through at various concentrations. Ligand capture was performed by flowing 0.5 μg / ml of biotinylated MUC16 peptide-2 at a rate of 5 μl / min for 65 seconds. The 4H11 humanized antibodies were then flowed through at various test concentrations (150 nM, 75 nM, 37.5 nM, 18.8 nM, and 9.4 nM). The assay conditions used to measure the effect of the added antibody were as follows: association time of 2 minutes, dissociation time of 10 minutes, and flow rate of 30 μl / min.

[0406] The relative binding affinity of all four antibodies was comparable, ranging from 1 to 3 nM K. D (Table 5). The sensory map of mouse IgG4H11 showed a similar trend to that of the humanized antibody (not shown). However, the viscosity of the kinetic dissociation step was observed, especially at high concentrations.

[0407] Table 5: Binding parameters of 4H11 antibody

[0408]

[0409] Example 5: Surface plasmon resonance (SPR) characterization of humanized 18C6 antibody

[0410] The relative binding affinity of 18C6 humanized antibodies (H1L1, H1L2, H2L1, H2L2) was compared using a BIACore-X100 instrument (GE Healthcare) with a CM5 chip via surface plasmon resonance. The assay consisted of capturing four humanized antibodies (H1L1, H1L2, H2L1, H2L2) on a CM5 chip coated with anti-mouse IgG under near-saturation conditions, or capturing parental mouse 18C6 IgG on a CM5 chip coated with anti-human IgG, and passing MUC16 peptide-2 glycopeptide (TLDRSSVLVDGYSPNRNE; SEQ ID NO:52) through the chip at various concentrations.

[0411] Ligand capture was achieved by allowing humanized anti-Muc16 antibodies (18C6 H1L1, 18C6 H1L2, 18C6 H2L1, 18C6 H2L2) and parental 18C6 mouse antibodies to flow onto the corresponding chips under saturation conditions. MUC16 peptide-2 was then flowed through the chips at various test concentrations (150 nM, 75 nM, 37.5 nM, 18.8 nM, and 9.4 nM). The assay conditions for measuring the effects of the glycopeptides were as follows: association time of 2 min, dissociation time of 10 min, and flow rate of 30 μl / min.

[0412] The relative binding affinity of all four antibodies tested was comparable to that of the parental 18C6 mouse antibodies (Table 6).

[0413] Table 6: Binding parameters of 18C6 antibody

[0414]

[0415] Example 6: Characterization of humanized 4H11 and 18C6 antibodies by fluorescence-activated cell sorting analysis

[0416] In this embodiment, the ability of the antibody to bind to MUC16-positive cells (OVCAR3) or cells expressing the MUC16 peptide (SKOV3 and A2780 transfectants) was evaluated.

[0417] OVCAR3, SKOV3, and A2780 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia) and maintained in culture according to the supplier's instructions. MUC16-expressing cell lines were generated by transfecting MUC16-negative human ovarian cancer cell lines (SKOV3 and A2780) with the C-terminal MUC16 sequence element (essential for tumor promotion) using the Vitality phrGFP vector expression system (which generates a green fluorescent protein fusion protein) (Stratagene, La Jolla, California). (A2780-phrGFP-MUC16c344 and SKOV3-phrGFP-MUC16c344). Stable cell lines were selected using genistein (G418, Invitrogen, Grand Island, NY) in their respective media and isolated based on green fluorescent protein expression. Stable transfectants were maintained in G418 media in a standard manner. The ΔMUC16c114 transfectant showed cell surface expression of the MUC16 protein from the presumed cleavage site to the carboxyl terminus (amino acids 1776 to 1890). The ΔMUC16c344 transfectant showed cell surface expression of the MUC16 protein from amino acid 1547 to the carboxyl terminus (amino acid 1890).

[0418] Adhesive target cells were removed by washing with 0.05% trypsin and 0.1% EDTA, and the cells were counted using a hemocytometer. Cells were dispensed into multiple Eppendorf tubes, each containing at least 0.5–1 × 10⁻⁶ cells. 6Cells were washed with phosphate-buffered saline (PBS) containing 1% FCS and 0.025% sodium azide (FACS buffer). For internal FACS staining, cells in Eppendorf tubes were permeated at room temperature for 10 min with FACS permeation solution 2 diluted 1:10 (BD BioSciences, San Jose, CA), followed by two washes with ice-cold FACS buffer. For surface FACS staining, cells were incubated on ice for 30 min with 1 mg / tube of Alex Fluor 647-conjugated 4H11 or 18C6 mouse mAb or 4H11 or 18C6 humanized antibody, either unconjugated or in tube. All cells were washed three times with FACS buffer. Cells labeled with 4H11 or 18C6 mouse mAb were further incubated on ice for 30 min with 1 mg / tube of the secondary antibody goat anti-mouse IgG2b-PE (phycoerythrin), followed by three washes with FACS buffer. Cells were analyzed using a FACS Calibur instrument. The mean PE fluorescence and percentage of PE-positive cells measured by 4H11 or 18C6 mouse mAbs are shown in Figures 9A and 9B. The mean Alexa-647 fluorescence and percentage of Alexa-647-positive cells measured by 4H11 or 18C6 humanized antibodies are shown in Figures 9C and 9D. Data for H1L2 humanized 4H11 antibody and H1L1 humanized 18C6 antibody are also shown.

[0419] In individual experiments, full-length humanized 4H11 antibodies (IgG1-Fc) containing different combinations of humanized heavy and light chain variable regions of the 4H11 antibody were measured. Fluorescence staining of MUC16-positive OVCAR3 or SKOV3 transfected daughter cells expressing the MUC16 peptide was performed using anti-human IgG1-Fc-PE antibody. Figures 10A and 10B provide data on mean PE fluorescence and the percentage of PE-positive cells for the measured 4H11 H1L1, H1L2, H2L1, and H2L2 antibodies.

[0420] The amino acid sequences of the L1, L2, H1, and H2 variable regions of 4H11 are provided herein as SEQ ID NO:2, 3, 4, and 5, respectively. The amino acid sequences of the L1, L2, H1, and H2 variable regions of 18C6 are provided herein as SEQ ID NO:20, 21, 22, and 23, respectively.

[0421] Example 7: Characterization of humanized 4H11 and 18C6 antibodies by Matrigel invasion assay

[0422] Antibody inhibition of basement membrane invasion was determined in the Matrigel invasion chamber, as previously described by Rao et al. (2017) ACS Chem. Biol. 12(8):2085-2096, which is incorporated herein by reference in its entirety. SKOV3 cell lines expressing the C-terminal portion of MUC16 required for invasion were generated as described above. Transfected cells and wild-type MUC16-expressing ovarian cancer cells (OVCAR3, OVCA-433, and CAOV3) were pretreated, either untreated or with a full-length humanized 4H11 antibody (IgG1-Fc), which contained different combinations of humanized heavy and light chain variable regions of the 4H11 antibody, prior to exposure to the Matrigel invasion chamber. The number of invading cells was counted. Figure 11 shows exemplary data for MUC16-positive OVCAR, VCA-433, and CAOV3 cell lines, and Figure 12 shows exemplary data for MUC16-expressing SKOV3 cell lines and parental SKOV3 cell lines. SKOV3 cell lines expressing the mutant MUC16 peptide N123mut c114 were used as negative controls for invasion.

[0423] Example 8: Generation of anti-MUC16 bispecific antibody

[0424] This embodiment describes the generation of anti-MUC16 bispecific antibodies (BsAbs) from humanized 4H11 and 18C6 anti-MUC16 scFv. A single-stranded BsAb containing an N-terminal anti-MUC16 scFv and a C-terminal mouse monoclonal antibody against human CD3εscFv is generated. 4H11 anti-MUC16 BsAbs and 18C6 anti-MUC16 BsAbs are generated using standard DNA techniques by cloning DNA fragments encoding anti-MUC16 scFv and anti-human CD3εscFv antibodies derived from the parental clone L2K into an expression vector. A hexahistine (His) tag is inserted downstream of the C-terminal anti-MUC16 BsAb for antibody purification and detection. The sequences of exemplary 4H11 anti-MUC16 BsAbs are provided in SEQ ID NO:44, 69-71, and 88-91. An exemplary sequence of 18C6 anti-MUC16BsAb is provided in SEQ ID NO:72-75 and 92-95.

[0425] Chinese hamster ovary (CHO) cells were transfected with an anti-MUC16 BsAb expression vector, and stable expression was achieved using standard drug selection with methionine thioimide (MSX) (a glutamine synthase (GS)-based method) (Fan et al., Biotechnology Bioengineering. 109(4), 1007-1005(2012)). CHO cell supernatants containing secreted anti-MUC16 BsAb molecules were collected. Anti-MUC16 BsAb was purified using a HisTrap HP column (GE Healthcare) via an FPLC AKTA system. Briefly, CHO cell cultures were clarified and loaded onto a column with a low imidazole concentration (20 mM), followed by elution of the bound anti-MUC16 bispecific antibody protein using an isocratic high imidazole concentration elution buffer (500 mM). BsAb banding was observed by SDS-PAGE, indicating successful purification of the BsAb.

[0426] Example 9: Anti-MUC16 BsAb-MUC16+ cell specificity

[0427] In this embodiment, the specificity of 4H11 and 18C6 anti-MUC16 BsAb binding to MUC16-expressing cancer cells was evaluated. In one study, two target cell lines, MUC16, were used. + OVCAR3 cell line and MUC16 - SKOV3 cell line. OVCAR3 and SKOV3 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, Virginia) and maintained in culture according to ATCC literature. FACS analysis was performed to confirm the binding of the anti-MUC16 antibody to both target cell lines; binding was observed only with the MUC16+OVCAR3 cell line. SKOV3 or OVCAR3 cell lines were incubated with anti-MUC16 Ab followed by secondary antibody, or incubated with secondary antibody alone as a control. Increased binding was expected in the MUC16+OVCAR3 cell line relative to the control, while SKOV3 showed low or no signal.

[0428] Example 10: Anti-MUC16 BsAb targeted cytotoxicity

[0429] In this embodiment, the ability of 4H11 and 18C6 anti-MUC16 BsAb to induce MUC16-specific cytotoxicity was evaluated. 4H11 anti-MUC16 BsAb and 18C6 anti-MUC16 BsAb were reacted with MUC16 at a concentration of approximately 0.2 μg / ml. + OVCAR3 target cell line or MUC16 -SKOV3 t...

Claims

1. An anti-mucin 16 (MUC16) construct comprising an antibody moiety that specifically recognizes a mucin 16 (MUC16) polypeptide, wherein the antibody moiety comprises: (a) (i) A variable-weight (VH) chain sequence of SEQ ID NO: 4 or 5; and (ii) A variable light (VL) chain sequence of SEQ ID NO: 2 or 3; or (b) (i) A variable-weight (VH) chain sequence of SEQ ID NO: 22 or 23; and (ii) A variable light (VL) chain sequence of SEQ ID NO: 20 or 21, The VH and VL chains are humanized, and optionally, among them The MUC16 polypeptide is human MUC16, or the MUC16 c114 polypeptide containing the amino acid sequence of SEQ ID NO: 44; or MUC16 is glycosylated at N24 or N30 relative to SEQ ID NO:

44.

2. The anti-MUC16 construct according to claim 1, wherein the antibody portion is IgA, IgD, IgE, IgG, IgM, Fab, Fab', F(ab')2, Fv or single-chain Fv (scFv), optionally wherein the scFv comprises any one of SEQ ID NO: 53-68.

3. The anti-MUC16 construct according to claim 1 or 2, wherein the antibody portion comprises human-derived heavy and light chain constant regions, optionally wherein the heavy chain constant region has an isotype selected from γ1, γ2, γ3 and γ4, and optionally wherein the light chain constant region has an isotype selected from κ and λ.

4. The anti-MUC16 construct according to claim 1 or 2, wherein the antibody portion is an immunoglobulin comprising two identical heavy chains and two identical light chains, optionally wherein the immunoglobulin is IgG.

5. The anti-MUC16 construct according to claim 1 or 2, wherein the anti-MUC16 construct is monospecific or multispecific, optionally wherein the multispecific anti-MUC16 construct comprises an anti-CD3 antibody moiety.

6. The anti-MUC16 construct according to claim 5, wherein the multispecific anti-MUC16 construct is bispecific.

7. The anti-MUC16 construct according to claim 1 or 2, wherein the anti-MUC16 construct is (i) a tandem scFv, optionally wherein the tandem scFv comprises two scFvs linked by a peptide linker; (ii) a biantibody (Db); (iii) a single-chain biantibody (scDb); (iv) a dual-affinity retargeting (DART) antibody; (v) F(ab')2; (vi) a dual variable domain (DVD) antibody; (vii) a club-and-mortar structure (KiH) antibody; (viii) a docking-locked (DNL) antibody; (ix) a chemically crosslinked antibody; (x) a heteropolypeptide antibody; or (xi) a heteroconjugate antibody.

8. The anti-MUC16 construct according to claim 5, wherein the multispecific anti-MUC16 construct comprises a first antibody portion that specifically recognizes MUC16 and a second antibody portion that specifically recognizes a second antigen.

9. The anti-MUC16 construct according to claim 8, wherein the second antigen is an antigen expressed on the surface of a T cell, optionally wherein the second antigen is a CD3 polypeptide selected from CD3γ, CD3δ, CD3ε and CD3ζ and / or optionally wherein the anti-MUC16 construct comprises any one of SEQ ID NO: 42, 69-75 and 88-95.

10. The anti-MUC16 construct according to claim 1 or 2, wherein the anti-MUC16 construct is a chimeric antigen receptor (CAR) comprising at least one of the following: (i) a co-stimulatory domain, (ii) a CD3 zeta (ζ) chain cytoplasmic signaling domain, (iii) the scFv of any one of SEQ ID NO: 53-68, or (iv) any one of SEQ ID NO: 80-87 and 97-103.

11. The anti-MUC16 construct according to claim 1 or 2, further conjugated with a detection agent or a developer.

12. The anti-MUC16 construct of claim 11, wherein the anti-MUC16 construct is conjugated with an alpha emitter, an Auger emitter, a beta emitter, a gamma emitter, a positron emitter, or an x-ray emitter, optionally wherein the positron emitter is 89Zr-deferrosensitized B (DFO).

13. The anti-MUC16 construct of claim 11, wherein the anti-MUC16 construct comprises: a light chain sequence of SEQ ID NO: 10 or 11 and a heavy chain sequence of SEQ ID NO: 12 or 13.

14. The anti-MUC16 construct according to claim 1, wherein the anti-MUC16 construct comprises: a light chain sequence of SEQ ID NO: 28 or 29 and a heavy chain sequence of SEQ ID NO: 30 or 31.

15. A polynucleotide encoding an anti-MUC16 construct according to any one of claims 1-14.

16. A vector comprising the polynucleotide of claim 15 operably linked to a promoter.

17. A cell comprising an anti-MUC16 construct according to any one of claims 1-14, a polynucleotide according to claim 15, or a vector according to claim 16, wherein optionally the cell is a mammalian cell.

18. The cell of claim 17, wherein the cell is an immune cell.

19. The cell of claim 18, wherein the cell is a T cell or a B cell.

20. A pharmaceutical composition comprising: a therapeutically effective amount of the anti-MUC16 construct according to any one of claims 1-14, the polynucleotide according to claim 15, or the carrier according to claim 16 or the cell according to any one of claims 17-19; and a pharmaceutically acceptable carrier.

21. Use of a therapeutically effective amount of the anti-MUC16 construct according to any one of claims 1-14 or the pharmaceutical composition according to claim 20 in the preparation of a medicament for treating MUC16-positive cancers in patients of need, wherein the MUC16-positive cancer is selected from ovarian cancer, pancreatic cancer, lung cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer.

22. The use according to claim 21, wherein the MUC16-positive cancer is metastatic.

23. The use according to claim 21 or 22, wherein the pharmaceutical composition inhibits or reduces metastasis in the patient, optionally wherein the patient is a human patient.

24. A method for generating effector cells, comprising genetically modifying the cells with one or more nucleic acids encoding an anti-MUC16 construct according to any one of claims 1-14.

25. Use of the cell of claim 18 in the preparation of a medicament for patients with MUC16-positive cancer in need, wherein the immune cell is a lymphocyte, and wherein the MUC16-positive cancer is selected from ovarian cancer, pancreatic cancer, lung cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer.

26. The use according to claim 25, wherein the lymphocyte is a T cell.

27. The use according to claim 25 or 26, wherein the cells are amplified prior to administration to the patient.

28. The use according to claim 25 or 26, wherein the medicament further comprises a therapeutically effective amount of an additional therapeutic agent.

29. Use of the anti-MUC16 construct according to any one of claims 1-14 in the preparation of a kit for use in a method for detecting MUC16 in cancer samples, wherein the method comprises: (a) Contact the cancer sample with the anti-MUC16 construct; (b) Detecting direct or indirect binding between the anti-MUC16 construct and the MUC16 peptide in the cancer sample, wherein the cancer sample is selected from samples of ovarian cancer, pancreatic cancer, lung cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer. Optionally, the anti-MUC16 construct is conjugated with a detectable label selected from: chromogenic labels, enzyme-catalyzed labels, radioisotope labels, fluorescent labels, toxic labels, chemiluminescent labels, and nuclear magnetic resonance contrast agents.

30. Use of the anti-MUC16 construct according to any one of claims 1-9 in the preparation of a kit for use in a method for diagnosing an individual suspected of having MUC16-positive cancer, wherein the method comprises: a) Apply an effective amount of the anti-MUC16 construct to the individual; as well as b) Determine a direct or indirect binding between the anti-MUC16 construct and the MUC16 peptide in the individual, wherein a level of direct or indirect binding above a threshold level indicates that the individual has the MUC16-positive cancer, wherein the MUC16-positive cancer is selected from ovarian cancer, pancreatic cancer, lung cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer.

31. Use of the anti-MUC16 construct according to any one of claims 1-9 in the preparation of a kit for use in a method for diagnosing an individual suspected of having MUC16-positive cancer, wherein the method comprises: a) Contact a sample containing cells derived from said individual with the anti-MUC16 construct; as well as b) Determine the number of cells in the sample that bind to the anti-MUC16 construct, wherein a value for the number of cells that bind to the anti-MUC16 construct above a threshold level indicates that the individual has the MUC16-positive cancer, wherein the MUC16-positive cancer is selected from ovarian cancer, pancreatic cancer, lung cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer.

32. Use of the anti-MUC16 construct according to any one of claims 1-11 and 13-14 in a kit used in a method for preparing a method for detecting MUC16-positive cancers in a subject in vivo, wherein the method comprises (a) Administering an effective amount of the anti-MUC16 construct to the subject, wherein the anti-MUC16 construct is configured to target MUC16-expressing cancer cells and is labeled with a radioisotope; and (b) The presence of MUC16-positive cancer in the subject is detected by detecting a level of radioactivity emitted above a reference value by the anti-MUC16 construct, wherein optionally the radioactive isotope is 89Zr-deferromin B (DFO), and wherein the MUC16-positive cancer is selected from ovarian cancer, pancreatic cancer, lung cancer, breast cancer, uterine cancer, fallopian tube cancer, or primary peritoneal cancer.

33. The use according to claim 32, wherein positron emission tomography or single-photon emission computed tomography is used to detect the level of radioactivity emitted by the anti-MUC16 construct.

34. The use according to claim 32, further comprising administering an effective amount of an immunoconjugate to the subject, the immunoconjugate comprising an anti-MUC16 construct conjugated with a radionuclide according to any one of claims 1-11 and 13-14.

35. The use according to claim 34, wherein the radionuclide is an isotope that emits alpha particles, an isotope that emits beta particles, an Auger emitter, or any combination thereof.

Images