Antibodies to MUC1 and methods of use thereof

Abstract

MUC1 is overexpressed in many cancers, including those of the lung, colon, breast, ovary, and pancreas. Embodiments of the present invention are directed to the discovery of monoclonal antibodies and fragments thereof, such as human single-chain variable fragments (scFv), that recognize human MUC1-SEA.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 861,619, filed June 14, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] All patents, patent applications, and publications cited herein are incorporated by reference in their entirety. The disclosures of these publications are incorporated by reference into this application in order to more fully describe the state of the art known to those skilled in the art at the date of the invention described and claimed herein.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction of either the patent document or the patent disclosure, as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[0004] Government interests This invention was made with government support under Grant No. 5T32CA207201 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] FIELD OF THE INVENTION The present invention relates to antibodies against MUC1 and methods of use thereof. [Background technology]

[0006] Background of the Invention Mucins coat the apical surface of epithelial cells in the lungs, stomach, intestines, eyes, and several other organs, and they protect the body from infection by preventing pathogens from reaching the cell surface. Mucin 1 (MUC1) is a glycoprotein encoded by the MUC1 gene that fulfills its protective function by binding to pathogens. Summary of the Invention

[0007] The present invention provides an isolated monoclonal antibody, or antigen-binding fragment thereof, that binds to the MUC1-SEA domain (SEQ ID NO: 1) or a peptide corresponding to an epitope on said domain.

[0008] In embodiments, the antibody comprises a VH, wherein the VH corresponds to one or more amino acid sequences of SEQ ID NOs: 12, 13, 14, 15, 16, or a portion thereof.

[0009] In embodiments, the antibody comprises a VL, wherein the VL corresponds to one or more amino acid sequences of SEQ ID NOs: 17, 18, 19, 20, 21, or a portion thereof.

[0010] In embodiments, the antibody comprises a VH and a VL, wherein the VH corresponds to one or more amino acid sequences of SEQ ID NOs: 12, 13, 14, 15, 16, or a portion thereof, and the VL corresponds to one or more amino acid sequences of SEQ ID NOs: 17, 18, 19, 20, 21, or a portion thereof, or any combination thereof.

[0011] In embodiments, the antibody comprises one or more of the amino acid sequences set forth in Table 1. For example, the antibody comprises one or more of the CDRs set forth in Table 1. For example, the antibody can correspond to clone T4E3, G2-2-F8, G1-3-A3, G1-2-B10, G1-1-A1, or G3-1-D6.

[0012] In embodiments, the CDR3 of the antibody comprises one or more of the amino acid sequence GMDV at the end of the VH-CDR3, a VH-CDR3 that is 15-20 amino acids, a single amino acid insertion at the 3' end of the VL CDR3, or any combination thereof.

[0013] In embodiments, antibodies may be humanized or fully human.

[0014] In embodiments, antibodies may be monospecific, bispecific, trispecific, or multispecific.

[0015] In embodiments, the antibody may be a full chain antibody, a single chain antibody, or a Fab fragment antibody.

[0016] In embodiments, the antibody has a binding affinity in the range of 1 pM to 1 μM.

[0017] In embodiments, the antibody of any one of the preceding claims is linked to a therapeutic agent. For example, the therapeutic agent may be a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine. In a non-limiting example, the therapeutic agent is MMAE.

[0018] In embodiments, the antibody may be produced by a cell.

[0019] In embodiments, the antibody may be provided in a pharmaceutical composition comprising the antibody and a pharmaceutically acceptable excipient.

[0020] The present invention further provides cells that produce the antibodies described herein.

[0021] Still further, the present invention provides pharmaceutical compositions comprising an antibody described herein and a pharmaceutically acceptable excipient.

[0022] The present invention also provides nucleic acids encoding the antibodies described herein. For example, the nucleic acid encodes an isolated monoclonal antibody or antigen-binding fragment thereof that binds to the MUC1-SEA domain (SEQ ID NO: 1) or a peptide corresponding to an epitope on said domain. For example, the nucleic acid comprises one or more nucleotide sequences set forth in SEQ ID NOs: 23-32, a portion thereof, or any combination thereof.

[0023] In embodiments, the nucleic acid may be provided in a vector.

[0024] Also provided herein are vectors comprising the nucleic acids described herein.

[0025] In embodiments, the vector may be provided in a cell.

[0026] The present invention provides a cell comprising the vector described herein.

[0027] The cells can be provided in a pharmaceutical composition. For example, the pharmaceutical composition can include the cells and a pharmaceutically acceptable excipient.

[0028] Still further, the present invention provides a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment thereof described herein. In embodiments, the antigen-binding fragment of the CAR comprises an scFv or Fab.

[0029] In embodiments, the CAR comprises a bispecific CAR, a dual-targeting CAR, a trispecific CAR, or a multispecific CAR.

[0030] In embodiments, the CAR can be provided in an engineered cell, such as an engineered T cell.

[0031] In embodiments, the CAR is encoded by a nucleic acid.

[0032] Aspects of the present invention are also directed to nucleic acids encoding chimeric antigen receptors, such as CARs, described herein.

[0033] Aspects of the present invention are also directed to cells comprising a chimeric antigen receptor, such as a chimeric antigen receptor described herein. In embodiments, the cells comprise T cells.

[0034] In embodiments, the cells may be further engineered to secrete antibodies or fragments thereof, for example, the secreted antibodies include monoclonal antibodies.

[0035] In embodiments, the secreted antibody comprises a monospecific antibody, a bispecific antibody, a trispecific antibody, or a multispecific antibody.

[0036] In embodiments, the secreted antibodies include immune checkpoint blocking antibodies, e.g., the secreted antibodies can modulate the immune system of a subject.

[0037] Aspects of the present invention are also directed to pharmaceutical compositions of CAR T cells, such as the CAR T cells described herein, and a pharmaceutically acceptable excipient.

[0038] In embodiments, the engineered T cells comprise a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor is specific for the MUC1-SEA domain.

[0039] In embodiments, the CAR of an engineered cell, such as an engineered T cell, comprises an scFv or Fab.

[0040] In embodiments, the CAR of an engineered cell, such as an engineered T cell, comprises a monospecific CAR, a bispecific CAR, a trispecific CAR, or a multispecific CAR.

[0041] In embodiments, the engineered cells comprise nucleic acid encoding a chimeric antigen receptor and, optionally, further encoding a polypeptide, including an antibody, a fragment thereof, that can be secreted from the engineered cells.

[0042] The present invention is also directed to pharmaceutical compositions comprising the engineered T cells described herein and a pharmaceutically acceptable excipient.

[0043] Aspects of the present disclosure are also directed to methods for treating a subject suffering from cancer. In embodiments, the methods include administering to a subject suffering from cancer a composition comprising the antibody or cells described herein. In exemplary embodiments, the cancer comprises cancer cells that express MUC1, mesothelin, and / or other tumor-associated antigens. In embodiments, the antibody or cells induce apoptosis of cancer cells, such as MUC1-expressing cancer cells.

[0044] In embodiments, the cancer comprises an epithelial cancer. Non-limiting examples of epithelial cancers that can be treated according to aspects of the present invention include breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, mouth cancer, esophageal cancer, small intestine and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer and skin cancer, e.g., squamous cell and basal cell carcinoma, prostate cancer, renal cell carcinoma, and other known cancers that affect epithelial cells throughout the body.

[0045] In embodiments, the method can further comprise administering to the subject a chemotherapeutic agent.

[0046] In embodiments, the method may further comprise the step of selecting a subject with a MUC1-expressing cancer.

[0047] Still further, aspects of the present invention are drawn to methods for inducing apoptosis in cancer cells, for example, the methods comprising contacting cancer cells with an antibody or CAR as described herein.

[0048] In embodiments, the cancer cells contain MUC1-SEA on their surface.

[0049] [The present invention 1001] An isolated monoclonal antibody or antigen-binding fragment thereof that binds to the MUC1-SEA domain (SEQ ID NO: 1) or a peptide corresponding to an epitope on said domain. [The present invention 1002] V corresponding to one or more amino acid sequences of SEQ ID NOs: 12, 13, 14, 15, 16, or a portion thereof H 、 V corresponding to one or more amino acid sequences, or portions thereof, of SEQ ID NOs: 17, 18, 19, 20, and 21; L 、 or any combination thereof 1001. The antibody of the present invention, comprising: [The present invention 1003] 1001. An antibody of the present invention comprising one or more of the amino acid sequences set forth in Table 1. [The present invention 1004] 1001. An antibody of the present invention corresponding to clone T4E3, G2-2-F8, G1-3-A3, G1-2-B10, G1-1-A1, or G3-1-D6. [The present invention 1005] the CDR3 of the antibody is V H - The amino acid sequence at the end of CDR3, GMDV, is 15 to 20 amino acids, V H -CDR3, V L 1001. The antibody of the present invention, comprising one or more of: a single amino acid insertion at the 3' end of the CDR3; or any combination thereof. [The present invention 1006] 1001. An antibody of the invention that is humanized or fully human. [The present invention 1007] The antibody of the present invention is monospecific or bispecific. [The present invention 1008] The antibody of the present invention is a single-chain antibody. [The present invention 1009] 1001. An antibody of the present invention, including a Fab fragment antibody. [The present invention 1010] 1001. The antibody of the present invention, having a binding affinity in the range of 1 pM to 1 μM. [The present invention 1011] Any of the preceding antibodies of the invention linked to a therapeutic agent. [The present invention 1012] The antibody of the present invention 1010, wherein said therapeutic agent is a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine. [The present invention 1013] The antibody of the present invention, wherein the therapeutic agent is MMAE. [The present invention 1014] A cell that produces any one of the antibodies 1001 to 1013 of the present invention. [The present invention 1015] A pharmaceutical composition comprising any one of the antibodies of the present inventions 1001 to 1013 and a pharmaceutically acceptable excipient. [The present invention 1016] A nucleic acid encoding any one of the antibodies of the present inventions 1001 to 1013. [The present invention 1017] A nucleic acid encoding an isolated monoclonal antibody or antigen-binding fragment thereof that binds to the MUC1-SEA domain (SEQ ID NO: 1) or a peptide corresponding to an epitope on said domain. [The present invention 1018] A nucleic acid of the present invention 1017, comprising one or more nucleotide sequences set forth in SEQ ID NOs: 23 to 32, a part thereof, or any combination thereof. [The present invention 1019] A vector comprising the nucleic acid of the present invention. [The present invention 1020] A cell comprising the vector of the present invention. [The present invention 1021] A pharmaceutical composition comprising the cells of the present invention. [The present invention 1022] A chimeric antigen receptor (CAR) comprising any one of the antibodies or antigen-binding fragments thereof of the present inventions 1001 to 1013. [The present invention 1023] The CAR of the present invention, wherein the antigen-binding fragment comprises an scFv or Fab. [The present invention 1024] CARs of the present invention 1022, including bispecific or dual-targeting CARs. [The present invention 1025] A cell comprising a CAR of the present invention 1022 to 1024. [The present invention 1026] 1025. Cells of the invention, including T cells. [The present invention 1027] The cell of the present invention 1023, which further secretes an antibody or fragment thereof. [The present invention 1028] 1027. The cell of claim 1027, wherein the secreted antibody comprises a monoclonal antibody. [The present invention 1029] 1027. The cell of claim 1027, wherein the secreted antibody comprises an immune checkpoint blocking antibody. [The present invention 1030] The cell of claim 1027, wherein said secreted antibodies modulate the immune system of a subject. [The present invention 1031] A pharmaceutical composition comprising the cells of the present invention and a pharmaceutically acceptable excipient. [The present invention 1032] A nucleic acid encoding a CAR of the present invention 1022 to 1024. [The present invention 1033] An engineered T cell comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor is specific for the MUC1-SEA domain. [The present invention 1034] 1033. The engineered T cell of the present invention, wherein said CAR comprises an scFv or Fab. [This invention 1035] 1033. The engineered T cell of the present invention, wherein the CAR comprises a bispecific CAR. [The present invention 1036] The nucleic acid Polypeptides, including antibodies, fragments thereof, capable of being secreted from said engineered cells. The engineered T cell of the present invention further encodes: [This invention 1037] A pharmaceutical composition comprising the engineered T cells of any of the present inventions 1033 to 1036 and a pharmaceutically acceptable excipient. [The present invention 1038] A method for treating a subject suffering from cancer, comprising administering to the subject a composition comprising any one of the antibodies of the present inventions 1001 to 1013 or any one of the cells of the present inventions 1033 to 1036. [This invention 1039] The method of claim 1038, wherein said cancer expresses MUC1, mesothelin, and / or other tumor-associated antigens. [The present invention 1040] The method of claim 1038, wherein the cancer comprises an epithelial cancer. [This invention 1041] The method of the present invention 1040, wherein said epithelial cancer includes breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, mouth cancer, esophageal cancer, small intestine and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer and skin cancer, e.g., squamous cell and basal cell carcinoma, prostate cancer, renal cell carcinoma, and other known cancers affecting epithelial cells throughout the body. [The present invention 1042] The method of claim 1038, further comprising administering to said subject a chemotherapeutic agent. [This invention 1043] The method of claim 1038, further comprising selecting a subject with a MUC1-expressing cancer. [This invention 1044] The method of claim 1038, wherein said antibody or cell induces apoptosis of MUC1-expressing cancer cells. [This invention 1045] A method for inducing apoptosis in cancer cells, comprising contacting the cancer cells with any one of the antibodies of the present inventions 1001 to 1013 or any one of the CARs of the present inventions 1022 to 1024. [The present invention 1046] 1045. The method of claim 1045, wherein said cancer cells contain MUC1-SEA on their surface. Other objects and advantages of the present invention will become readily apparent from the description that follows. [Brief explanation of the drawings]

[0050] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0051] [Figure 1] Dose-response curves for two anti-MUC1-C scFv-Fc antibodies are shown. T4E3 scFv-Fc exhibits a 6-fold lower EC50 than 3D1 scFv-Fc. Dose-response curves were generated by incubating serial dilutions of scFv-Fc with HCT116-MUC1 (MUC1-C+) or HCT116-v (MUC1-) colon cancer cell lines, followed by incubation with a secondary anti-human Fc-FITC antibody and detecting binding by flow cytometry. [Figure 2]This figure shows tumor cell killing by anti-MUC1-C CAR T cells 3D1-ZsGreen and T4E3-ZsGreen. The cell killing assay utilizes a Celigo imaging cytometer to visualize cell killing. Fluorescent protein-expressing cancer cell lines: HCT116-v, HCT116-MUC1, and COV362, were seeded in 96-well plates (3000 cells / well) and incubated with T cells at an effector-to-target (E:T) ratio of 10:1 or 2:1. The plates were imaged after 22 and 42 hours. Cell viability was calculated by counting mCardinal+ cells. Loss of mCardinal signal indicates cancer cell death. The colon cancer cell line HCT116-v does not express MUC1-C, but HCT116-MUC1 and COV362 are 100% MUC1-C positive. X48-ZsGreen are CAR T cells that target CXCR4, which is not found in any of the cancer cell lines utilized. Note - Due to T cell availability, T4E3-ZsGreen CAR T cells were not incubated with HCT116-MUC1 cancer cells. [Figure 3-1] Figure 3 shows the experimental design for T4E3 tumor cell killing. Cancer cells were seeded at 3000 per well in 200 μL of RPMI-1640 + 10% FBS + 20 mM HEPES. They were spun down at 300 g for 5 minutes and then imaged using a Celigo imaging cytometer. 100 μL of medium was removed from each well, and T cells were added at an E:T ratio of 10:1 or 2:1 according to the plate map below. Due to a shortage of T4E3 cells, T4E3 was added at the specified E:T ratio only to two wells of COV362 and one well of HCT116-v. IL-21 (30 ng / mL) was also added to the cultures to ensure T cell longevity. Plates were imaged immediately after T cell addition. Plates were also imaged 7, 22, and 42 hours after T cell addition. Included herein is a plate map, with Xs indicating wells receiving the indicated CAR T cells. [Figure 3-2] See description of Figure 3-1. [Figure 3-3] See description of Figure 3-1. [Figure 4] 1 shows the discovery of anti-MUC-1-SEA scFv. [Figure 5-1] Figure 5 shows T4E3 scFv CAR T versus 3D1 scFv CAR T killing. [Figure 5-2] See description of Figure 5-1. [Figure 6] Dose-dependent killing is observed. [Figure 7-1] Figure 7 shows the gene assignments. [Figure 7-2] See description of Figure 7-1. [Figure 7-3] See description of Figure 7-1. [Figure 8] The annotated structure of MUC1-SEA (SEQ ID NO: 1) is shown. [Figure 9-1] FIG. 9 shows an alignment of anti-MUC1 antibody amino acid sequences (SEQ ID NOs: 135, 12, 13, 15, 14, 136, 16, 137, 17, 18, 21, 20, 138, 19, 139, 48, and 140-145, respectively, in order of appearance). [Figure 9-2] See description of Figure 9-1. [Figure 9-3] See description of Figure 9-1. [Figure 9-4] See description of Figure 9-1. [Figure 10-1] Figure 10 shows an analysis of the anti-MUC1 antibody CDR3 region. The figure discloses SEQ ID NOS: 146-158 and 120, respectively, in order of appearance. [Figure 10-2] See description of Figure 10-1. [Figure 11-1]Figure 11 provides accurate information on epithelial ovarian cancer. https: / / www.cancer.org / content / dam / cancer-org / research / cancer-facts-and-statistics / annual-cancer-facts-and-figures / 2018 / cancer-facts-and-figures-special-section-ovarian-cancer-2018.pdf [Figure 11-2] See description of Figure 11-1. [Figure 12-1] FIG. 12 shows the treatment of epithelial ovarian cancer. [Figure 12-2] See description of Figure 12-1. [Figure 13-1] Figure 13 shows that the immunosuppressive microenvironment of ovarian cancer impedes CAR T cell efficacy. [Figure 13-2] See description of Figure 13-1. [Figure 14] We show that Tregs in the ovarian cancer microenvironment impede CAR T cell efficacy. Tregs contribute to tumor progression in ovarian cancer by suppressing tumor-specific T cells and dendritic cells, blocking T cell proliferation by secreting TGF-beta and IL-10, and transmitting inhibitory signals to TILs through high expression of CTLA-4 on Tregs. [Figure 15-1] Figure 15 shows a CAR T cell factory for the treatment of solid tumors. [Figure 15-2] See description of Figure 15-1. [Figure 15-3] See description of Figure 15-1. [Figure 16-1] Figure 16 shows the selection of ovarian cancer CAR T cell factory components. In one embodiment, the targeting component comprises an anti-MUC1-C-CAR and the payload comprises an anti-CCR4 antibody. Without wishing to be bound by theory, MUC1-C CAR T cell factories will migrate into MUC1+ tumors and reverse the immunosuppressive ovarian cancer tumor microenvironment, resulting in tumor cell death. [Figure 16-2] See description of Figure 16-1. [Figure 17-1] Figure 17 shows the development of anti-CCR4 CAR T cells that can be used in combination with MUC1-C CAR T cell therapy. [Figure 17-2] See description of Figure 17-1. [Figure 18-1] FIG. 18 shows a schematic diagram of the MUC1-C-terminal domain and binding epitope of the anti-MUC1-C antibody h3D1 IgG (provided by the Kufe lab). [Figure 18-2] See description of Figure 18-1. [Figure 19-1] Figure 19 shows the effect of CAR affinity and the effect of CAR epitope.For example, the affinity of CAR affects T cell activation, killing rate, and the ability of CAR to distinguish between healthy tissue and cancerous tissue.In addition, even if membrane-proximal CAR has lower binding efficiency, when CAR recognizes membrane-proximal epitope on antigen, more efficient T cell activation occurs. [Figure 19-2] See description of Figure 19-1. [Figure 20-1] Figure 20 shows the reduction in affinity after conversion of h3D1 IgG to scFv. Loss of affinity of scFv-Fc after binding to cell lines hindered early MUC-1C CAR T cell efforts. Quantification of affinity of 3D1 scFv-Fc and 3D1 IgG. The figure discloses SEQ ID NO: 159. [Figure 20-2] See description of Figure 20-1. [Figure 21] The amino acid sequence of the construct containing MUC1-SEA (SEQ ID NO: 1) is shown. [Figure 22-1] Figure 22 shows full-length MUC1, including the MUC1-SEA domain. The MUC1-SEA domain undergoes autoproteolytic self-cleavage at the conserved GVVS sequence (SEQ ID NO: 33). As indicated by the arrows, cleavage occurs after the G and before the V. The figure discloses SEQ ID NOs: 160, 34, and 161, respectively, in order of appearance. [Figure 22-2] See description of Figure 22-1. [Figure 23]Muc1-SEA purification is shown. Muc1-SEA was expressed from the pET28a vector in BL21(DE3) cells. The protein was purified via an N-terminal His tag (Ni-NTA resin). 125 ml of BL21(DE3) harboring pET28a-MUC1(sea) was subcultured to an OD of 0.6 and induced with either 0.1 or 0.5 mM IPTG. The culture was grown overnight at 30°C and pelleted at 9000 rpm. The pellet was resuspended in 4 mL of B-PER and then sonicated for 15 minutes (30 / 59 s on / off cycle). The lysed cells were spun down, and the supernatant was diluted 50:50 with Ni-NTA binding buffer (20 mM imidazole) and then incubated with Ni-NTA resin for 1 hour. The resin was collected, washed with binding buffer (20 mM imidazole), and then eluted with 250 mM imidazole. Collected proteins were buffer-exchanged into PBS for long-term storage. For visualization, all samples were prepared with 4x LDS loading dye. Samples 1-5 were unreduced, and sample 6 was reduced with 10% BME. [Figure 24] Showing Muc1-SEA autocleavage. 4-12% Bolt gels were run in MES buffer. Samples were non-reducing (10% BME) unless otherwise specified. Approximately 5 μg was loaded per well, except for MBP-Muc1 (approximately 4 μg). The very faint band at approximately 6 kDa may be a cleavage product. [Figure 25] Figure 1 shows h3D1 binding. Nunc maxisorb plates were coated with 1 μg / ml Muc1-SEA in PBS overnight at 4°C. The next day, plates were blocked with 4% milk-PBS for 2 hours at 37°C. The blocking solution was then discarded and replaced with the appropriate antibody dilution in 2% milk-PBST. The antibody solution was incubated for 1 hour at 37°C and then washed six times with PBST. 3D1 binding was detected using an anti-human Fc-HRP secondary (1:100k dilution in 2% milk-PBST). After a 1 hour incubation at 37°C, plates were washed six times with PBST and TMB substrate was added. The reaction was quenched by the addition of stop buffer and read at 450 nm. [Figure 26-1]FIG. 26 shows the MUC1 panning summary. [Figure 26-2] See description of Figure 26-1. [Figure 27] Dose-response curves for two anti-MUC1-C scFv-Fc antibodies are shown. T4E3 scFv-Fc exhibits a 6-fold lower EC50 than 3D1 scFv-Fc. Dose-response curves were generated by incubating serial dilutions of scFv-Fc with HCT116-MUC1 (MUC1-C+) or HCT116-v (MUC1-) colon cancer cell lines, followed by incubation with a secondary anti-human Fc-FITC antibody and detecting binding by flow cytometry. [Figure 28] Binding properties of anti-MUC1-SEA scFv are shown. [Figure 29] FR1-CDR2 alignment is shown. The figure discloses FR1-CDR2 of SEQ ID NOs: 135, 12, 13, 15, 14, 136, 16, 137, 17, 18, 21, 20, 138, and 19, respectively, in order of appearance. [Figure 30] FR3-FR4 alignment is shown. The figure discloses FR3-FR4 of SEQ ID NOs: 135, 12, 13, 15, 14, 136, 16, 137, 17, 18, 21, 20, 138, and 19, respectively, in order of appearance. [Figure 31-1] Figure 31 shows T4E3 scFv CAR T versus 3D1 scFv CAR T killing. Colon cancer cell line HCT116-v is a control MUC1- cell line. COV362 is a MUC1+ cell line. mAb2-3 is a control anti-CCR4 antibody. [Figure 31-2] See description of Figure 31-1. [Figure 32] Dose-dependent killing is shown. HCT116-v is a control MUC1- cell line. COV362 is a MUC1+ cell line. mAb2-3 is a control anti-CCR4 antibody. [Figure 33]This figure shows tumor cell killing by anti-MUC1-C CAR T cells 3D1-ZsGreen and T4E3-ZsGreen. The cell killing assay utilizes a Celigo imaging cytometer to visualize cell killing. Fluorescent protein-expressing cancer cell lines: HCT116-v, HCT116-MUC1, and COV362, were seeded in 96-well plates (3000 cells / well) and incubated with T cells at an effector-to-target (E:T) ratio of 10:1 or 2:1. The plates were imaged after 22 and 42 hours. Cell viability was calculated by counting mCardinal+ cells. Loss of mCardinal signal indicates cancer cell death. The colon cancer cell line HCT116-v does not express MUC1-C, but HCT116-MUC1 and COV362 are 100% MUC1-C positive. X48-ZsGreen is a CAR T cell that targets CXCR4, which is not found in any of the cancer cell lines utilized. [Figure 34] CAR insert map of a MUC1-CCR4 dual-targeted CAR that kills Tregs and tumor cells alike is shown. [Figure 35-1] Figure 35 shows the vector map of a MUC1-CCR4 dual-targeted CAR that kills Tregs and tumor cells alike. [Figure 35-2] See description of Figure 35-1. [Figure 36-1] Figure 36 shows the use of F2A to generate a Fab construct. The figure discloses an alignment of the sequences as SEQ ID NOS: 163-174, respectively, in order of appearance, and "RAKRSGSG" as SEQ ID NOS: 162. [Figure 36-2] See description of Figure 36-1. [Figure 36-3] See description of Figure 36-1. [Figure 37-1] Figure 37 shows an analysis of the initial design construct design. [Figure 37-2] See description of Figure 37-1. [Figure 38]Figure 1 shows the strategy for Fab design using the F105 leader. In this construct, the leader sequence was changed based on the observation that initial B cell receptor designs used the F105 leader in the pHAGE vector instead of the VH leader for heavy chain expression. Additionally, the identity of the Fab was changed to the commercially available anti-hemagglutinin antibody Medi8852, which binds to the HA stem. The figure discloses "RAKRSGSG" as SEQ ID NO: 162. [Figure 39] We show that Medi8852 binds to the HA stem but not to MUC1-SEA. [Figure 40] mAb2-3 Fab and 3D1 Fab were cloned into the Medi8852 Fab F105 construct and evaluated for binding and expression. Both mAb2-3 and 3D1 Fab were observed to have higher EC50 values ​​than their respective scFvs. [Figure 41] mAb2-3 Fab and 3D1 Fab cloned into the Medi8852 Fab F105 construct and assessed for binding and expression by flow cytometry are shown. Each value represents the average of three replicates. [Figure 42] Figure 1 shows the Media8852 construct expanded to generate a Fab with a lambda light chain (anti-Muc1 T4E3 Fab). The results show an example of a Fab with lower affinity than the scFv. [Figure 43] Figure 1 shows Fab CAR T cell killing. Fab CAR T cells kill MUC1+ tumor cells less efficiently than scFv CAR T cells. [Figure 44-1] Figure 44 shows an embodiment of a bispecific crossover Fab. L1, L2, L3, and L4 refer to non-limiting examples of linkers. The figure discloses "RAKRSGSG" as SEQ ID NO: 162, "(Gly) 7 " as SEQ ID NO: 175, and "(Gly) 5 " as SEQ ID NO: 176. [Figure 44-2] See description of Figure 44-1. [Figure 45-1]Figure 45 shows an embodiment of a bispecific crossover Fab. All values ​​are the average of three replicates. [Figure 45-2] See description of Figure 45-1. [Figure 46-1] Figure 46 shows the dose response curve for binding. [Figure 46-2] See description of Figure 46-1. [Figure 47] (A) Lentiviral constructs of monospecific anti-MUC1-C and (B) anti-mesothelin CAR T cells. (C) Lentiviral constructs of bispecific anti-MUC1-C / mesothelin CAR T cells. (D) Lentiviral constructs of bispecific anti-MUC1-C / mesothelin CAR T cell factories. (E) Schematic representation of the mechanism of action of anti-MUC1-C / mesothelin CAR T factories. [Figure 48] (Top panel) Flow cytometry histogram of Ovcar-4 and COV362 stained with anti-mesothelin APC (blue) revealing that Ovcar-4 is 11.4% mesothelin+ and COV362 is 54.2% mesothelin+ compared to unstained controls (red). (Bottom panel) Flow cytometry histogram of Ovcar-4 and COV362 stained with 233 nM anti-MUC1-C h3D1 IgG, followed by anti-human Fc-FITC secondary staining, revealing that Ovcar-4 is 46.1% MUC1-C+ and COV362 is 85.5% MUC1-C+. [Figure 49] (A) A schematic representation of an orthotopic mouse model of ovarian cancer highlighting its usefulness in demonstrating ovarian cancer metastasis. (B) Tumors and ovaries from five mice bearing orthotopic ovarian tumors. The tumor in the top mouse was generated from the high-grade serous ovarian cancer (HGSC) cell line Ovcar-4 (350,000 cells / mouse), while the bottom four ovaries and tumors were generated from the HGSC cell line COV362 (500,000 cells / mouse). [Figure 50-1]Figure 50 shows (A) a schematic representation of an orthotopic humanized mouse model that allows for the study of metastasis and the tumor microenvironment, (B) a t-SNE 2D scatter plot showing the mapping of CD45+ leukocytes in the humanized NSG-SGM3 mouse model of renal cell carcinoma, and (C) a violin diagram showing the presence of CCR4 within clusters 1 and 2, indicating the presence of Treg and Th2 cells. [Figure 50-2] See description of Figure 50-1. [Figure 51(A)-1] Figure 51(A) shows the amino acid sequences of anti-MUC1 antibodies. Figure 51(A) and Figure 51(B) disclose SEQ ID NOs: 47, 15, 13, 48-57, 73, 20, 18, and 74-83, respectively, in order of appearance. [Figure 51(A)-2] See the explanation for Figure 51(A)-1. [Figure 51(A)-3] See the explanation for Figure 51(A)-1. [Figure 51(A)-4] See the explanation for Figure 51(A)-1. [Figure 51(B)-1] Figure 51(B) shows the amino acid sequences of anti-MUC1 antibodies. Figure 51(A) and Figure 51(B) disclose SEQ ID NOs: 47, 15, 13, 48-57, 73, 20, 18, and 74-83, respectively, in order of appearance. [Figure 51(B)-2] See the explanation for Figure 51(B)-1. [Figure 52(A)-1] Figure 52(A) shows the nucleotide sequences of anti-MUC1 antibodies. Figures 52(A) through 52(D) disclose SEQ ID NOs: 35, 36, 24, 37-46, and 60-72, respectively, in order of appearance. [Figure 52(A)-2] See the explanation for Figure 52(A)-1. [Figure 52(B)-1] Figure 52(B) shows the nucleotide sequences of anti-MUC1 antibodies. Figures 52(A) through 52(D) disclose SEQ ID NOs: 35, 36, 24, 37-46, and 60-72, respectively, in order of appearance. [Figure 52(B)-2] See the explanation for Figure 52(B)-1. [Figure 52(B)-3] See the explanation for Figure 52(B)-1. [Figure 52(B)-4]See the explanation for Figure 52(B)-1. [Figure 52(C)-1] Figure 52(C) shows the nucleotide sequences of anti-MUC1 antibodies. Figures 52(A) through 52(D) disclose SEQ ID NOs: 35, 36, 24, 37-46, and 60-72, respectively, in order of appearance. [Figure 52(C)-2] See the explanation for Figure 52(C)-1. [Figure 52(D)-1] Figure 52(D) shows the nucleotide sequences of anti-MUC1 antibodies. Figures 52(A) through 52(D) disclose SEQ ID NOs: 35, 36, 24, 37-46, and 60-72, respectively, in order of appearance. [Figure 52(D)-2] See the explanation for Figure 52(D)-1. [Figure 53(A)] Figure 53(A) shows the amino acid sequences of anti-MUC1 antibodies. Figures 53(A) through 53(C) disclose, in order of appearance, SEQ ID NOs: 16, 58, 51, 14, 48, 177, 59, 21, 84, 85, 19, 74, 86, and 87, respectively. [Figure 53(B)] Figure 53(B) shows the amino acid sequences of anti-MUC1 antibodies. Figures 53(A) through 53(C) disclose, in order of appearance, SEQ ID NOs: 16, 58, 51, 14, 48, 177, 59, 21, 84, 85, 19, 74, 86, and 87, respectively. [Figure 53(C)] Figure 53(C) shows the amino acid sequences of anti-MUC1 antibodies. Figures 53(A) through 53(C) disclose, in order of appearance, SEQ ID NOs: 16, 58, 51, 14, 48, 177, 59, 21, 84, 85, 19, 74, 86, and 87, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0052] Detailed Description of the Invention MUC1 is a member of the mucin family and encodes a membrane-bound, glycosylated phosphoprotein. MUC1 is a heterodimeric protein complex encoded by a single transcript. It has a core protein mass of 120-225 kDa, which increases to 250-500 kDa upon glycosylation. It extends 200-500 nm beyond the cell surface. The protein is anchored to the apical surface of many epithelia by a transmembrane domain. Beyond the transmembrane domain is an SEA domain, which contains a cleavage site for release of the large extracellular domain.

[0053] After translation, the MUC1 polypeptide precursor undergoes autocleavage into two subunits, which then form a stable noncovalent complex. The large MUC1 N-terminal subunit, designated MUC1-N, is the mucin component of the MUC1 dimer, possessing a characteristic variable number of tandem repeats extensively decorated with O-linked glycans. MUC1-N extends far beyond the cellular glycocalyx and is tethered to the cell surface through its association with the transmembrane MUC1 C-terminal subunit (MUC1-C). This allows MUC1-N to contribute to a physical barrier protecting epithelial cell layers from exposure to toxins, microorganisms, and other forms of stress from the external environment. See Kufe, Donald W. "Targeting the human MUC1 oncoprotein: a tale of two proteins." Cancer biology & therapy 7.1 (2008):81-84.

[0054] MUC1-C has a 58-amino acid extracellular domain, a 28-amino acid transmembrane domain, and a 72-amino acid cytoplasmic tail. It is involved in intracellular signaling. MUC1-C functions as an oncoprotein, especially given its involvement in diverse signaling pathways linked to tumorigenesis. In this context, overexpression of MUC1-C blocks the induction of apoptosis in response to DNA damage, oxidative stress, and hypoxia. Overexpression of MUC1-C has been shown to result in anchorage-independent growth and tumorigenicity. MUC1-C stabilizes β-catenin, and the interaction between MUC1-C and β-catenin contributes in part to MUC1-induced transformation. MUC1-C also leads to constitutive activation of the anti-apoptotic IKKβ->NFκB pathway, as seen in a variety of carcinomas and hematopoietic malignancies. Importantly, overexpression of the MUC1-C cytoplasmic domain is sufficient to induce anchorage-independent growth and tumorigenicity, indicating that the shed MUC1-N mucin subunit is not important for transformation. See Kufe, Donald W. "Targeting the human MUC1 oncoprotein: a tale of two proteins." Cancer biology & therapy 7.1 (2008): 81-84.

[0055] MUC1 overexpression and aberrant glycosylation are associated with many cancers, including human carcinomas and hematological malignancies. See, for example, Sritama and Mukherjee, "MUC1: a multifaceted oncoprotein with a key role in cancer progression." Trends in molecular medicine 20.6 (2014):332-342. The ability of chemotherapy drugs to access cancer cells is hindered by heavy glycosylation of the extracellular domain of MUC1. Glycosylation creates a highly hydrophilic region that prevents hydrophobic chemotherapy drugs from passing through. This prevents the drugs from reaching their targets, which are normally located intracellularly. Similarly, glycosylation has been shown to bind growth factors. This allows cancer cells that produce large amounts of MUC1 to concentrate growth factors near their receptors, enhancing receptor activity and cancer cell growth. MUC1 also prevents immune cells from interacting with receptors on the surface of cancer cells through steric hindrance. This inhibits antitumor immune responses.

[0056] MUC1 is cleaved immediately after synthesis within the SEA domain, a highly conserved 120-amino acid domain. Cleavage of MUC1 within the SEA domain results in two unequal chains: a large extracellular N-terminal domain containing a tandem repeat array specifically bound by strong noncovalent interactions to a smaller C-terminal domain containing the transmembrane and cytoplasmic domains of the molecule. MUC1 cleavage can render targeting somewhat intractable, as the shed components can sequester many anti-MUC1 antibodies. For example, the shed domain has been shown to sequester circulating anti-tandem repeat antibodies, limiting their ability to reach MUC1+ tumor cells. However, a region called the SEA domain remains tethered to the cell surface even after MUC1 cleavage. The MUC1 SEA domain is formed by the interaction of the N-terminal subunit with the extracellular portion of the C-terminal subunit after cleavage, thereby remaining anchored to the cell surface. Importantly, the SEA domain contains a stable targeting structure for anti-cancer antibodies.

[0057] Aspects of the present invention are directed to the discovery of monoclonal antibodies and fragments thereof that recognize human MUC1-SEA. For example, embodiments can include fully human or humanized antibodies that recognize MUC1-SEA, but can also include antibody fragments such as human single-chain variable fragments (scFv). For example, in the scFv-Fc format, the MUC1-SEA scFv T4E3 binds to MUC1+ cells with six-fold higher affinity than the anti-MUC1-C antibody 3D1. Those skilled in the art will recognize that antibodies can be utilized in various forms and formats, including CAR T cells and CAR T factories, or as bispecific antibodies. When T4E3 is utilized as the targeting portion of CAR T cells, T4E3 CAR T cells preferentially kill MUC1+ tumor cells and not MUC1- cells. CAR T cells that recognize activated T cells and CXCR4 from the same human leukocyte donor do not kill either MUC1+ or MUC1- tumor cell lines that lack CXCR4.

[0058] A detailed description of one or more preferred embodiments is provided herein. However, it is understood that the present invention can be embodied in various forms. Therefore, the specific details disclosed herein should not be construed as limiting, but as a basis for the claims and as a representative basis for teaching one skilled in the art how to use the present invention in any suitable manner.

[0059] Abbreviations and Definitions The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The use of the words "a" or "an" when used in conjunction with the word "comprising" in the claims and / or specification can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0060] Whenever any of the phrases "for example," "such as," "including," etc. are used herein, unless expressly stated otherwise, it is understood that the phrase "without limitation" is also involved. Similarly, "one example," "exemplary," etc. are understood to be non-limiting.

[0061] The term "substantially" permits deviations from the descriptor that do not adversely affect the intended purpose. It is understood that a descriptor is modified by the term "substantially" even if the word "substantially" is not explicitly recited.

[0062] Terms such as "comprising," "including," "having," and "involving" (and similarly, "comprises," "includes," "has," and "involves") are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprising" and, therefore, is construed as an open term meaning "at least the following," and not excluding additional features, limitations, aspects, etc. Thus, for example, "a process comprising steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the terms "a" or "an" are used, they are understood to mean "one or more," unless such interpretation is meaningless in the context.

[0063] As used herein, the term "about" is used herein to mean approximately, roughly, around, or within a range. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify numerical values ​​above and below the stated value by a variance of 20 percent above or below (high or low).

[0064] The MUC1 gene undergoes conformational stress and loses the GSVVV motif located within the sea urchin sperm protein enterokinase and agrin (SEA) domain. (SEQ ID NO: 34) (See underlined and bolded text below) encodes a single polypeptide chain that is autoproteolytically cleaved immediately after translation into two peptide fragments: a longer N-terminal subunit (MUC1-N) and a shorter C-terminal subunit (MUC1-C). Outside the cell, the two subunits remain associated through stable hydrogen bonds.

[0065] TIFF0007742311000001.tif72150

[0066] MUC1-N is composed of a proline, threonine, and serine-rich (PTS) domain and an SEA domain. Embodiments of the present invention provide isolated monoclonal antibodies specific for MUC1, particularly MUC1-SEA. See, for example, Figure 8 for the annotated structure of MUC1-SEA.

[0067] MUC1 antibodies were identified through the use of a 27 billion human single-chain antibody (scFv) phage display library by using soluble human MUC1 as the library selection target. These antibodies represent a new class of monoclonal antibodies against MUC1.

[0068] For example, embodiments can include one or more of the nucleic acid and amino acid sequences described herein.

[0069] Heavy chain nucleotide sequence TIFF0007742311000002.tif178150TIFF0007742311000003.tif224150TIFF0007742311000004.tif169150

[0070] Heavy chain amino acid sequence (CDR1 is shown in bold, CDR2 is underlined, and CDR3 is shown in bold and underlined) TIFF0007742311000005.tif19150TIFF0007742311000006.tif225150TIFF0007742311000007.tif226150TIFF0007742311000008.tif87150

[0071] Light chain nucleotide sequence TIFF0007742311000009.tif123150TIFF0007742311000010.tif228150TIFF0007742311000011.tif132150

[0072] Light chain amino acid sequence (CDR1 is shown in bold, CDR2 is underlined, and CDR3 is shown in bold and underlined) TIFF0007742311000012.tif68150TIFF0007742311000013.tif219150TIFF0007742311000014.tif223150TIFF0007742311000015.tif17150

[0073] In the sequences contained herein, an asterisk "*" can represent an amber / stop codon. For example, TG1 bacterial cells can be mutated so that the TAG stop codon is read as Q (glutamine). When IMGT is used to resolve a DNA sequence into FW and CDR regions, the TG1 bacterial cells are unaware of the presence of an amber suppressor, so the cells assume it is a stop codon while it is read as Q in the phage. In embodiments, the sequences can be recloned so that the TAG is changed to a Q codon.

[0074] Embodiments also feature antibodies having a particular percentage of identity or similarity to the amino acid or nucleotide sequence of the anti-MUC1 antibodies described herein. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity when compared to a specific region or the entire length of any one of the anti-MUC1 antibodies described herein. Sequence identity or similarity to the nucleic acids and proteins of the invention can be determined by sequence comparison and / or alignment using methods known in the art. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine the percent sequence identity or similarity of the nucleic acids and proteins of the invention.

[0075] With respect to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, delete, or substitute a single amino acid or a small percentage of amino acids in the encoded sequence are collectively referred to herein as "conservatively modified variants." In some embodiments, the alteration results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants of the anti-MUC1 antibodies disclosed herein can exhibit increased cross-reactivity to MUC1 compared to unmodified MUC1 antibodies.

[0076] As used herein, the term "antibody" can refer to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), multivalent antibodies, monovalent antibodies, humanized antibodies, fully human antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity. "Specifically binds" or "immunoreacts" means that the antibody reacts more readily with one or more antigenic determinants of a desired antigen than with other polypeptides. Antibodies include polyclonal, monoclonal, chimeric, dAb (domain antibody), single-chain, F ab , F ab ', and F (ab’)2 Fragments, scFv, and F ab These include, but are not limited to, expression libraries.

[0077] The terms "antigen" and "antigenic molecule" can be used interchangeably and refer to any molecule that can be specifically bound by an antibody. The term "antigen" as used herein includes, for example, proteins, different epitopes on proteins (as different antigens within the meaning of the present invention), and polysaccharides. This primarily includes parts of bacteria, viruses, and other microorganisms (coatings, capsules, cell walls, flagella, pili, and toxins). Lipids and nucleic acids are antigenic only when combined with proteins and polysaccharides. Non-microbial exogenous (non-self) antigens can include pollen, egg white, and proteins from transplanted tissues and organs or on the surface of transfused blood cells. Preferably, the antigen is selected from the group consisting of cytokines, cell surface proteins, enzymes and receptors.

[0078] The term "chimeric antibody" can refer to an antibody, usually prepared by recombinant DNA techniques, that contains a variable region, i.e., a binding region, derived from one source or species and at least a portion of a constant region derived from a different source or species. For example, a chimeric antibody can contain a murine variable region and a human constant region. Other non-limiting forms of "chimeric antibodies" encompassed by the present invention are those in which the constant region has been modified or altered from that of the original antibody to generate particular properties, such as Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as "class-switched antibodies." Chimeric antibodies are the product of expressed immunoglobulin genes containing DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques well known in the art. See, for example, Morrison, SL, et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855, U.S. Patent No. 5,202,238 and U.S. Patent No. 5,204,244.

[0079] The term "humanized antibody" can refer to an antibody in which the framework or "complementarity-determining regions" (CDRs) have been modified to contain CDRs from an immunoglobulin of different specificity compared to that of the parent immunoglobulin. In one embodiment, murine CDRs are grafted into the framework regions of a human antibody to prepare a "humanized antibody." See, for example, Riechmann, L., et al., Nature 332 (1988) 323-327, and Neuberger, MS, et al., Nature 314 (1985) 268-270. Particularly preferred CDRs correspond to those representing sequences that recognize the antigens described herein. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant regions have been further modified or altered from those of the original antibody to generate specific properties according to the present invention, such as Fc receptor (FcR) binding.

[0080] The term "human antibody" as used herein can include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the state of the art (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that, upon immunization, can produce a full or partial repertoire of human antibodies in the absence of endogenous immunoglobulin production. Introduction of the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of 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, P., et al., J. Immunol. 147 (1991) 86-95). As already mentioned for the chimeric and humanized antibodies according to the present invention, the term "human antibody" as used herein also includes such antibodies that are modified in the constant region to generate particular properties, such as with respect to FcR binding, for example by "class switching", i.e., alteration or mutation of the Fc portion (e.g., IgG1 to IgG4 and / or IgG1 / IgG4 mutation).

[0081] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies isolated from host cells such as NSO or CHO cells, or from animals (e.g., mice) transgenic for human immunoglobulin genes or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have variable and constant regions in a rearranged form. Recombinant human antibodies according to the invention have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0082] Single-chain Fv ("scFv") polypeptide molecules comprise covalently linked V H :V L heterodimer, which consists of V linked by a peptide-encoded linker H and V L The protein can be expressed from a gene fusion containing the encoding gene. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883.) For example, referring to FIG. 9, one embodiment is a T4E3 V L (Sequence number 17 ) a T4E3 V described herein linked to an amino acid sequence H (Sequence number 12 ) amino acid sequence.

[0083] Many methods have been described for identifying chemical structures for converting naturally aggregated, but chemically separated, light and heavy polypeptide chains from antibody V regions into scFv molecules that will fold into a three-dimensional structure substantially similar to that of an antigen-binding site (see, e.g., U.S. Patent Nos. 5,091,513, 5,132,405, and 4,946,778).

[0084] Very large naive human scFv libraries have been and can be generated to provide a large source of rearranged antibody genes against a large number of target molecules. Smaller libraries can be constructed from individuals with infectious diseases to isolate disease-specific antibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43 (1992); Zebedee et al., Proc. Natl. Acad. Sci. USA 89:3 175-79 (1992)).

[0085] Generally, antibody molecules obtained from humans belong to any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from one another in the nature of the heavy chains present in the molecule. Certain classes also have subclasses, such as IgG1, IgG2, IgG3, and IgG4, as well as others. Furthermore, in humans, the light chain may be a kappa chain or a lambda chain. For example, with reference to FIG. 7, the light chain of the embodiments herein may be a kappa chain or a lambda chain. An "antibody" according to the present invention may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG or IgE), or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, preferably IgG1).

[0086] The term "antigen-binding site" or "binding portion" can refer to the portion of an immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent sections within the V regions of the heavy and light chains, called "hypervariable regions," are interposed between more conserved adjacent sections known as "framework regions" or "FRs." Thus, the term "FR" refers to the amino acid sequences naturally found between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are positioned relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are called "complementarity-determining regions" or "CDRs." For example, the V regions containing the CDRs of an exemplary antibody are shown in Figure 1. H and V L The regions are shown in Figure 9.

[0087] [Table 1] TIFF0007742311000017.tif127149

[0088] As used herein, the term "epitope" can include any protein determinant capable of specific binding to an immunoglobulin, scFv, or T-cell receptor. The variable region enables an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, the VL and VH domains of an antibody, or a subset of complementarity-determining regions (CDRs), combine to form the variable regions that define the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. Epitope determinants typically consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and typically have specific three-dimensional structural and charge characteristics. For example, antibodies can be raised against N- or C-terminal peptides of a polypeptide. As another example, the epitope of an antibody can be within the panning antigen MUC1, as described herein. As further described herein, anti-MUC1 antibodies can be directed against the SEA domain of MUC1.

[0089] As used herein, the terms "immunological binding" and "immunological binding properties" can refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K D ) and can be expressed in terms of smaller (K D ) represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, both "on rate constants" (K on ) and "off rate constant" (K off ) can be determined by calculation of the concentration and the actual rates of association and dissociation. (See Nature 361:186-87 (1993)). K off / Kon The ratio allows for the compensation of all parameters unrelated to affinity and is equal to the dissociation constant 3 / 4. (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473.) An antibody of the present invention is said to specifically bind to a MUC1 epitope if the equilibrium binding constant is sufficient to induce a therapeutic effect. In many cases, the equilibrium binding constant is ≦10 μM, preferably ≦10 nM, and most preferably ≦100 pM to about 1 pM, as measured by an assay such as a radioligand binding assay or a similar assay known to those skilled in the art, such as BIAcore. Alternatively, a moderate affinity is sufficient to induce a therapeutic effect.

[0090] "Specifically binds" or "having specificity for" can refer to an antibody that binds to an epitope via its antigen-binding domain, and that the binding involves some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope if it binds to that epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope.

[0091] Functionally, the binding affinity of anti-MUC1 antibodies is 10 -5 M~10 -12 For example, the binding affinity of an anti-MUC1 antibody is in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10-11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10-9M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M, or 10 -5 M~10 -6 I am M.

[0092] For example, anti-MUC1 antibodies can be monovalent or bivalent and include single or two chains. For example, a monovalent antibody has affinity for one antigen and / or one epitope, such as the MUC1-SEA peptide or a fragment thereof.

[0093] The term "bivalent" or "bispecific" antibody can refer to an antibody that can specifically bind to two different antigens simultaneously. For example, a bivalent antibody can contain two pairs of heavy and light chains (HC / LC) that specifically bind to different antigens, i.e., a first heavy chain and a first light chain (derived from an antibody against the first antigen) specifically bind together to the first antigen, and a second heavy chain and a second light chain (derived from an antibody against the second antigen) specifically bind together to the second antigen. Such bivalent, bispecific antibodies can specifically bind to two different antigens simultaneously, but not three or more antigens, in contrast to monospecific, monovalent antibodies, which can only bind to one antigen, and tetravalent, tetraspecific antibodies, which can simultaneously bind, for example, four antigen molecules.

[0094] The MUC1 protein, MUC1-SEA peptide, or derivatives, fragments, analogs, homologs, or orthologs thereof can be used as immunogens in the generation of antibodies that immunospecifically bind to these protein components. The MUC1 protein, MUC1-SEA peptide, or derivatives, fragments, analogs, homologs, or orthologs thereof bound to proteoliposomes can be used as immunogens in the generation of antibodies that immunospecifically bind to these protein components.

[0095] Those skilled in the art will recognize that it is possible to determine, without undue experimentation, whether a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by determining whether the former prevents the latter from binding to MUC1. If the human monoclonal antibody being tested competes with a human monoclonal antibody of the invention, as indicated by reduced binding by the human monoclonal antibody of the invention, then it is likely that the two monoclonal antibodies bind to the same, or closely related, epitopes.

[0096] Another method for determining whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the invention is to preincubate the human monoclonal antibody of the invention with the MUC1 protein or MUC1-SEA peptide with which it is normally reactive, then add the human monoclonal antibody to be tested and determine whether the human monoclonal antibody to be tested is inhibited in its ability to bind to MUC1. If the human monoclonal antibody to be tested is inhibited, it likely has the same, or a functionally equivalent, epitopic specificity as the monoclonal antibody of the invention. Screening of human monoclonal antibodies of the invention can also be performed by utilizing MUC1 and / or MUC1-SEA and determining whether the test monoclonal antibody can neutralize MUC1 and / or MUC1-SEA.

[0097] Various procedures known in the art can be used for the production of polyclonal or monoclonal antibodies directed against the proteins of the invention, or against their derivatives, fragments, analogs, homologs, or orthologs (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0098] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provide the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen that is the target of the desired immunoglobulin, or an epitope thereof, can be immobilized on a column, and immune-specific antibodies can be purified by immunoaffinity chromatography. Immunoglobulin purification is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia, PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0099] As used herein, the term "monoclonal antibody" or "MAb" or "monoclonal antibody composition" can refer to a population of antibody molecules containing only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity-determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with a particular epitope of an antigen characterized by a unique binding affinity for it.

[0100] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to induce lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0101] The immunizing agent will typically contain the protein antigen, a fragment thereof, or a fusion protein thereof. Generally, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). The immortalized cell line is usually a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, or human origin. Rat or mouse myeloma cell lines are usually used. The hybridoma cells can be cultured in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent growth of HGPRT-deficient cells.

[0102] Preferred immortalized cell lines are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are mouse myeloma lines, available, for example, from the Salk Institute Cell Distribution Center, San Diego, California, and the American Type Culture Collection, Manassas, Virginia. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

[0103] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of a monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in the therapeutic use of monoclonal antibodies, it is important to identify antibodies with high specificity and high binding affinity for the target antigen.

[0104] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103.) Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.

[0105] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0106] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567. DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the present invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0107] A fully human antibody is an antibody molecule in which the entire sequence of both the light chain and the heavy chain, including the CDRs, originates from human genes. Such antibodies are referred to herein as "humanized antibodies," "human antibodies," or "fully human antibodies." Human monoclonal antibodies can be prepared using trioma technology, human B-cell hybridoma technology (see Kozbor, et al., 1983 Immunol Today 4:72), or EBV hybridoma technology producing human monoclonal antibodies (see Cole, et al., 1985 In:MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp.77-96). Human monoclonal antibodies are available and can be produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B cells in vitro with Epstein-Barr virus (Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0108] In addition, human antibodies can also be produced using other techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. After challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as Marks et al., Bio / Technology 10, 779-783 (1992), Lonberg et al., Nature 368, 856-859 (1994), Morrison, Nature 368, 812-13 (1994), Fishwild et al., Nature Biotechnology 14, 845-51 (1996), Neuberger, Nature Biotechnology 14, 826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

[0109] Human antibodies can also be produced using transgenic non-human animals that are modified to produce fully human antibodies in response to antigen challenge rather than the animal's endogenous antibodies. (See PCT Publication WO94 / 02602.) Endogenous genes encoding heavy and light immunoglobulin chains in the non-human host are disabled, and active loci encoding human heavy and light immunoglobulin chains are inserted into the host's genome. Human genes are incorporated, for example, using yeast artificial chromosomes containing the necessary human DNA segments. Animals providing all the desired modifications are then obtained as offspring by breeding intermediate transgenic animals containing fewer than the total number of modifications required. A preferred embodiment of such a non-human animal is a mouse, referred to as a Xenomouse™, as disclosed in PCT Publications WO96 / 33735 and WO96 / 34096. The animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from animals after immunization with an immunogen of interest, e.g., as polyclonal antibody preparations, or alternatively, from immortalized B cells derived from animals, such as hybridomas, that produce monoclonal antibodies. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly or further modified to obtain antibody analogs, such as, for example, single-chain Fv (scFv) molecules.

[0110] An example of a method for producing a non-human host, exemplified as a mouse, lacking expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Patent No. 5,939,598. This can be obtained by a method comprising deleting a J segment gene from at least one endogenous heavy chain locus in embryonic stem cells to prevent rearrangement of the locus and the formation of transcripts of the rearranged immunoglobulin heavy chain locus, the deletion being carried out by a targeting vector containing a gene encoding a selectable marker, and producing a transgenic mouse from the embryonic stem cells, the somatic and germ cells of which contain the gene encoding the selectable marker.

[0111] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method involves introducing an expression vector containing a nucleotide sequence encoding the heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding the light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses the antibody containing the heavy and light chains.

[0112] In a further improvement of this procedure, methods for identifying clinically relevant epitopes on immunogens and correlating methods for selecting antibodies that immunospecifically bind with high affinity to the relevant epitopes are disclosed in PCT Publication WO 99 / 53049.

[0113] The antibody can be expressed by a vector containing a DNA segment encoding the single chain antibody described above. For example, the vector can be 23 to 27, 35, 36, 24, or 37 to 46 (heavy chain sequence) or SEQ ID NOs: 28 to 32 or 60 to 72 (light chain sequence) may include one or more of:

[0114] In embodiments, the antibody or fragment thereof may be provided as a nucleic acid construct encoding the antibody or fragment. See, e.g., U.S. Application No. 15 / 537,779, which is incorporated herein by reference in its entirety.

[0115] These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene guns, catheters, etc. Vectors include chemical conjugates such as those described in WO93 / 64701, which have a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in PCT / US95 / 02140 (WO95 / 22618), which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., protamine), plasmids, phages, etc. Vectors can be chromosomal, non-chromosomal, or synthetic.

[0116] Preferred vectors include viral vectors, fusion proteins, and chemical conjugates. Retroviral vectors include Moloney murine leukemia virus. DNA viral vectors are preferred. These vectors include pox vectors, such as orthopox or avipox vectors, herpes virus vectors, such as herpes simplex virus type I (HSV) vectors (see Geller, AI et al, J. Neurochem, 64:487 (1995); Lim, F., et al, in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al, Proc Natl. Acad. Sci.: USA 90:7603 (1993); Geller, AI, et al, Proc Natl. Acad. Sci. USA 87:1149 (1990)), adenovirus vectors (LeGal LaSalle et al, Science, 259:988 (1993); Davidson, et al, Nat. Genet. 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995)), and adeno-associated virus vectors (Kaplitt, MG. et al., Nat. Genet. 8:148 (1994)).

[0117] Poxvirus vectors transfer genes into the cytoplasm of cells. Avipoxvirus vectors only result in short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors are preferred for transferring nucleic acids into neural cells. Adenovirus vectors result in shorter-term expression (approximately 2 months) than adeno-associated virus (approximately 4 months), which in turn results in shorter expression than HSV vectors. The specific vector selected will depend on the target cell and the condition being treated. Introduction can be by standard techniques, such as infection, transfection, transduction, or transformation. Examples of modes of gene transfer include, for example, naked DNA, CaP04 precipitation, DEAE-dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0118] Vectors can be used to target essentially any desired target cell. For example, stereotactic injection can be used to guide vectors (e.g., adenovirus, HSV) to the desired location. In addition, particles can be delivered by intracerebroventricular (icv) injection using a minipump infusion system such as the SynchroMed Infusion System. A method based on bulk flow, called convection, has also proven effective in delivering large molecules to extended areas of the brain and may be useful for delivering vectors to target cells. (See Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injection, as well as oral or other known administration routes.

[0119] These vectors can be used to express large amounts of antibodies that can be used in a variety of ways, such as to detect the presence of MUC1 in a sample. The antibodies can also be used to attempt to bind to and destroy MUC1 activity.

[0120] Techniques can be adapted to produce single chain antibodies specific to antigenic proteins of the invention (see, e.g., U.S. Pat. No. 4,946,778). In addition, methods can be used to ab Monoclonal Fs with desired specificity for the protein, or derivatives, fragments, analogs, or homologs thereof, are suitable for constructing expression libraries (see, e.g., Huse, et al., 1989 Science 246:1275-1281). ab Antibody fragments containing the idiotype to a protein antigen can be produced by techniques known in the art, including, but not limited to, (i) F(ab')2 fragments produced by pepsin digestion of the antibody molecule, (ii) F(ab')2 fragments produced by reducing the disulfide bridges of the F(ab')2 fragments, and (iii) F(ab')2 fragments produced by reducing the disulfide bridges of the F(ab')2 fragments. ab (iii) F fragments produced by treating antibody molecules with papain and a reducing agent. ab fragments, and (iv) F v Contains fragments.

[0121] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (see U.S. Pat. No. 4,676,980) and for the treatment of HIV infection (see WO91 / 00360, WO92 / 200373, EP03089). It is contemplated that antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, and those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0122] In certain embodiments, antibodies are engineered or modified to alter the antibody's function for clinical use. For example, antibody effector function may be the focus of such engineering efforts to enhance the antibody's effectiveness in cancer treatment. Those skilled in the art will recognize that whether such modifications are employed depends on the use of the antibody. The Fc domain, in particular, is important for antibody function and has been the focus of many engineering efforts. These efforts to modify antibody function can generally be categorized into efforts to increase effector function, efforts to decrease effector function, and / or efforts to extend the antibody's serum half-life. When an antibody or fragment thereof is used to target CART cells, the Fc is not involved, and such Fc modifications are not utilized. On the other hand, for soluble antibodies, it may be desirable to use mutations to enhance Fc effector function.

[0123] One of the primary mechanisms of action of anti-cancer antibodies is the targeted killing of tumor cells through the recruitment of immune system components. This activity is achieved through the interaction of the Fc domain of the anti-cancer antibody with the complement component C1q or Fcγ receptor. However, many anti-cancer antibodies have failed in clinical trials due to insufficient efficacy. This has led to efforts to increase antibody potency through enhancing the antibody's ability to mediate cytotoxic functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP).

[0124] For example, such engineering efforts have focused on increasing the affinity of the Fc domain of anti-cancer antibodies for the low-affinity receptor FcγIIIa. Many mutations in the Fc domain have been identified that directly or indirectly enhance Fc receptor binding, thereby significantly enhancing cytotoxicity (see, for example, Lazar, GA, et al. (2006) PNAS 103, 4005-4010; Shields, RR et al. (2001) J. Biol. Chem. 276, 6591-6604; Stewart, R. et al. (2011) Protein Engineering, Design and Selection 24, 671-678; and Richards, JO et al. (2008) Mol Cancer Ther 7, 2517-2527). For example, such mutations include the mutations S239D / A330L / I332E (also called 3M), F243L, and G236A.

[0125] An alternative approach focuses on glycosylation of the Fc domain. FcγRs interact with carbohydrates on the CH2 domain, and the composition of these glycans has a substantial effect on effector function activity. One example of this is defucosylated (non-fucosylated) antibodies, which exhibit significantly enhanced ADCC activity through increased binding to FcγRIIIa.

[0126] In other embodiments, it is desirable to provide antibodies that cannot activate effector functions. For these purposes, IgG4 is commonly used. Furthermore, Fc engineering approaches have been used to determine the primary interaction sites of the Fc domain with Fcγ receptors and C1q, and then mutate these positions to reduce or abolish binding. Through alanine scanning, the C1q binding site was isolated to the region covering the hinge and upper CH2 of the Fc domain. Mutations such as K322A, L234A, and L235A combined are sufficient to almost completely abolish FcγR and C1q binding. Similarly, a set of three mutations, L234F / L235E / P331S (also known as TM), has a very similar effect.

[0127] An alternative approach is to modify the glycosylation of asparagine 297 in the Fc domain, which is known to be required for optimal FcR interaction. Loss of FcR binding has been observed with N297 point mutations, enzymatically deglycosylated Fc domains, recombinantly expressed antibodies in the presence of glycosylation inhibitors, and expression of the Fc domain in bacteria.

[0128] Embodiments of the present invention can further include antibodies or fragments with enhanced serum half-life of IgG through Fc engineering. IgG naturally persists in serum for extended periods due to FcRn-mediated recycling, giving it a typical half-life of approximately 21 days. Despite this, many efforts have been made to manipulate the pH-dependent interaction of the Fc domain with FcRn to increase affinity at pH 6.0 while maintaining minimal binding at pH 7.4. For example, the mutations T250Q / M428L resulted in an approximately two-fold increase in IgG half-life in rhesus monkeys, and the mutations M252Y / S254T / T256E (also known as YTE) resulted in an approximately four-fold increase in IgG half-life in cynomolgus monkeys.

[0129] As will be apparent to those skilled in the art, any number of such mutations can be made to provide engineered antibodies or fragments thereof with altered function and / or half-life. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, antibodies can be engineered with dual Fc regions, thereby providing enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)).

[0130] In certain embodiments, antibodies of the present invention can include Fc variants containing amino acid substitutions that alter the antigen-independent effector function of the antibody, particularly its circulating half-life. Such antibodies exhibit either increased or decreased binding to FcRn and therefore have increased or decreased serum half-lives, respectively, when compared with antibodies lacking these substitutions. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where a long half-life of the administered antibody is desired, for example, to treat chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful, for example, for administration to mammals where a shortened circulation time would be advantageous, for example, for in vivo diagnostic imaging, or in situations where the starting antibody has toxic side effects if present in the circulation for an extended period of time. Fc variants with reduced FcRn-binding affinity are also less likely to cross the placenta and are therefore useful in treating diseases or disorders in pregnant women. Additionally, other applications in which reduced FcRn-binding affinity may be desirable include applications in which localization to the brain, kidney, and / or liver is desired. In one exemplary embodiment, modified antibodies of the invention exhibit reduced transport from the vasculature across the epithelium of renal glomeruli. In another embodiment, modified antibodies of the invention exhibit reduced transport from the brain across the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, an antibody with altered FcRn binding comprises an Fc domain with one or more amino acid substitutions within the "FcRn-binding loop" of the Fc domain. The FcRn-binding loop is composed of amino acid residues 280-299 (EU numbering). Exemplary amino acid substitutions that alter FcRn-binding activity are disclosed in International PCT Publication No. WG05 / 047327, incorporated herein by reference. In certain exemplary embodiments, an antibody of the invention, or a fragment thereof, comprises an Fc domain with one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0131] In some embodiments, mutations are introduced into the constant region of the mAb to alter the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the mAb. For example, the mutation is a LALA mutation in the CH2 domain. In one aspect, the bsAb contains a mutation on one scFv unit of the heterodimeric mAb that reduces ADCC activity. In another aspect, the mAb contains mutations on both chains of the heterodimeric mAb that completely eliminate ADCC activity. For example, the mutation introduced into one or both scFv units of the mAb is a LALA mutation in the CH2 domain. These mAbs with variable ADCC activity can be optimized so that the mAb exhibits maximum selective killing toward cells expressing one antigen recognized by the mAb, but minimal killing toward a second antigen recognized by the mAb.

[0132] In other embodiments, antibodies for use in the diagnostic and therapeutic methods described herein have a constant region, e.g., an IgG1 or IgG4 heavy chain constant region, that has been altered to reduce or eliminate glycosylation. For example, antibodies of the invention can also include Fc variants containing amino acid substitutions that alter the glycosylation of the antibody. For example, the Fc variants can have reduced glycosylation (e.g., N-linked or O-linked glycosylation).

[0133] Exemplary amino acid substitutions resulting in reduced or altered glycosylation are disclosed in International PCT Publication No. WO 05 / 018572, which is incorporated herein by reference. In a preferred embodiment, the antibodies of the present invention, or fragments thereof, are modified to eliminate glycosylation. Such antibodies, or fragments thereof, may be referred to as "agly" antibodies, or fragments thereof (e.g., "agly" antibodies). Without being bound by theory, it is believed that "agly" antibodies, or fragments thereof, may have an improved safety and stability profile in vivo. An exemplary agly antibody, or fragment thereof, comprises a deglycosylated Fc region of an IgG4 antibody that lacks Fc effector function, thereby eliminating the potential for Fc-mediated toxicity to normal vital organs that express MUC1. In yet other embodiments, the antibodies of the present invention, or fragments thereof, comprise engineered glycans. For example, the antibodies may have a reduced number of fucose residues on the N-glycan at Asn297 of the Fc region, i.e., are defucosylated. In another embodiment, the antibody can have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.

[0134] The present invention also relates to immunoconjugates comprising antibodies conjugated to a cytotoxic agent, such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate).

[0135] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Re is included.

[0136] Conjugates of antibodies and cytotoxic agents are prepared using various bifunctional protein-binding agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as triene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies (see WO 94 / 11026).

[0137] Those skilled in the art will recognize that a wide variety of possible moieties can be attached to a given antibody or other molecule of the invention (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

[0138] Conjugation can be achieved by any chemical reaction that will link two molecules, so long as the antibody and other moiety retain their respective activities. This conjugation can involve many chemical mechanisms, such as covalent bonding, affinity bonding, intercalation, coordinate bonding, and complex formation. However, covalent bonding is preferred. Covalent bonding can be achieved by direct condensation of existing side chains or by incorporating an external crosslinking molecule. Many bivalent or polyvalent linking agents are useful for linking protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative linking agents include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of linking agents known in the art, but rather is illustrative of more common linking agents. (See Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987)). Preferred linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester).) See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives linked to antibodies by oligopeptide linkers.Particularly preferred linkers include (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G)), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat. #21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propianamido]hexanoate (Pierce Chem. Co., Cat. #2165-G), and (v) sulfo-NHS (-hydroxysulfo-succinimide) conjugated to EDC (Pierce Chem. Co., Cat. #2165-G). Chem. Co., Cat.#24510).

[0139] The above linkers contain components with different attributes, thereby resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically hindered disulfide bond, which can form conjugates with improved stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer available conjugates. In particular, sulfo-NHS can increase the stability of carbodiimide bonds. Carbodiimide bonds (such as EDC) when used in combination with sulfo-NHS form esters that are more resistant to hydrolysis than the carbodiimide bond reaction alone.

[0140] The antibodies disclosed herein can also be formulated as immunoliposomes.

[0141] Liposomes containing antibodies are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985), Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980), and U.S. Pat. Nos. 4,485,045 and 4,544,545.

[0142] Liposomes with extended circulation time are disclosed in US Pat. No. 5,013,556.

[0143] Particularly useful liposomes can be generated by reverse-phase evaporation with a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to generate liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to liposomes as described in Martin et al., J. Biol. Chem., 257:286-288 (1982) via a disulfide exchange reaction.

[0144] bispecific antibody A bispecific antibody (bsAb) is an antibody that contains two variable domains or scFv units, resulting in an antibody that recognizes two different antigens. The present invention provides bispecific antibodies that recognize MUC1-SEA and a second antigen. Exemplary second antigens include tumor-associated antigens (e.g., mesothelin, LINGO1), cytokines (e.g., IL-12, IL-15), and cell surface receptors. Bispecific antibodies in different formats are also provided herein. In some embodiments, each of the anti-MUC1 fragment and the second fragment is independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. In some embodiments, the bispecific antibody further comprises an Fc fragment. The bispecific antibodies of the present invention comprise a heavy and light chain combination or scFv of a MUC1 antibody disclosed herein. For example, SEQ ID NO: 23 to 27, 35, 36, 24, or 37 to 46 (heavy chain sequences) or SEQ ID NOs: 28 to 32 or 60 to 72 (light chain sequences) Please refer to.

[0145] In some embodiments, the second antigen of the bispecific antibody is mesothelin. Mesothelin is so named because of its expression in mesothelial cells. Mesothelin is a glycophosphatidylinositol (GPI)-linked cell surface glycoprotein synthesized as a 71 kD precursor protein. After synthesis, the precursor protein is then cleaved by the endoprotease furin, releasing a secreted N-terminal region called megakaryocyte-potentiating factor (MPF), while the 41 kD mature MSLN remains attached to the membrane.

[0146] Mesothelin expression is typically restricted to mesothelial cells lining the pleura, peritoneum, and pericardium, but is also highly expressed in many cancers and solid tumors, including malignant mesothelioma, pancreatic cancer, ovarian cancer, lung adenocarcinoma, endometrial cancer, biliary tract cancer, gastric cancer, and childhood acute myeloid leukemia. Higher expression in MSLN correlated with poor prognosis in patients with ovarian cancer, cholangiocarcinoma, lung adenocarcinoma, triple-negative breast cancer, and resectable pancreatic adenocarcinoma. See Hassan, Raffit, et al. "Mesothelin immunotherapy for cancer: ready for prime time?" Journal of Clinical Oncology 34.34 (2016):4171.

[0147] Thus, a bispecific antibody can include a first antibody that targets MUC1 and a second antibody that targets mesothelin.

[0148] In other embodiments, the second antigen of the bispecific antibody is CCR4. Chemokines are a family of secreted proteins primarily known for their role in leukocyte activation and chemotaxis. Their specific interaction with chemokine receptors on target cells induces a signal transduction cascade that leads to inflammatory mediator release, cell shape changes, and cell migration. CC chemokine receptor 4 (CCR4) is the cognate receptor for CC chemokines CCL17 and CCL22 and is expressed on functionally distinct subsets of T cells, including type 2 helper T cells (Th2) and regulatory T cells (Treg) (Iellem et al., 2001, and Imai et al., 1999). Increasing evidence indicates that CCL 17 / 22 secretion promotes increased numbers of tumor-infiltrating Tregs in malignant entities such as colorectal, ovarian, Hodgkin's lymphoma, and glioblastoma (Curiel et al., 2004; Wagsater et al., 2008; Niens et al., 2008; Jacobs et al., 2010; Hiraoka et al., 2006). Increased levels of Tregs in tumors hinder efficient antitumor immune responses (Wood et al., 2003; Levings et al., 2001) and are often associated with poor clinical outcomes and tumor progression (Hiraoka et al., 2006; Woo et al., 2001). Therefore, one major obstacle to successful cancer therapy may be caused by the migration of Tregs into tumors and their suppression of antitumor immune responses in the tumor microenvironment (Zou et al., 2006; Yu et al., 2005).

[0149] Thus, a bispecific antibody can comprise a first antibody that targets MUC1 and a second antibody that targets CCR4. One of skill in the art will recognize that any second antibody that targets CCR4 or a fragment thereof can be utilized in the present invention, including those contained in PCT / US2008 / 088435, PCT / US2013 / 039744, PCT / US2015 / 054202, and PCT / US2016 / 026232, which are incorporated by reference in their entireties.

[0150] Embodiments of the invention include multivalent antibodies and antigen-binding fragments that bind to MUC1 and one or more additional antigens. For example, the multivalent antibody or antigen-binding fragment can be specific for MUC1 and mesothelin. A multivalent antigen-binding protein has two or more antigen-binding sites. For purposes of this application, "valency" can refer to the number of antigen-binding sites. Thus, a bivalent antibody can refer to an antibody with two binding sites, a trivalent antibody can refer to an antibody with three binding sites, etc. The term "multivalent" can refer to any association, covalently or non-covalently, of two or more antigen-binding proteins, the association having two or more antigen-binding sites. The term "multivalent" encompasses bivalent, trivalent, tetravalent, etc.

[0151] Bispecific antibodies of the present invention can be constructed using methods known in the art. In some embodiments, bispecific antibodies are single polypeptides in which two scFv fragments are linked by a long linker polypeptide, the length of which is sufficient to allow intramolecular association between the two scFv units to form the antibody. In other embodiments, bispecific antibodies are two or more polypeptides linked by covalent or non-covalent bonds.

[0152] In another embodiment, bispecific antibodies are constructed using the "knobs-into-holes" method (Ridgway et al., Protein Eng 7:617-621 (1996)). In this method, Ig heavy chains of two different variable domains are reduced to selectively cleave heavy chain pairing while preserving the heavy-light chain pairing. Two heavy-light chain heterodimers that recognize two different antigens are mixed to promote heteroligation pairing mediated through engineered "knobs-into-holes" in the CH3 domains.

[0153] In another embodiment, bispecific antibodies can be constructed through the exchange of heavy-light chain dimers from two or more different antibodies to generate hybrid antibodies in which a first heavy-light chain dimer recognizes MUC1 and a second heavy-light chain dimer recognizes a second antigen. The mechanism of heavy-light chain dimerization is similar to the formation of human IgG4, which also functions as a bispecific molecule. The dimerization of IgG heavy chains is driven by intramolecular forces such as the pairing of the CH3 domains of each heavy chain with disulfide bridges. The presence of a specific amino acid (R409) in the CH3 domain has been shown to promote dimer exchange and the assembly of IgG4 molecules. Heavy chain pairing is also further stabilized by inter-heavy chain disulfide bridges in the hinge region of the antibody. Specifically, in IgG4, the hinge region is composed of amino acids 226-230 (the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 134) (relative to a stable IgG1 hinge region containing the amino acid sequence Cys-Pro-Ser-Cys) (SEQ ID NO: 133) This sequence difference at serine 229 is associated with the tendency of IgG4 to form novel intrachain disulfides in the hinge region (Van der Neut Kolfschoten, M. et al, 2007, Science 317:1554-1557 and Labrijn, A. F. et al, 2011, Journal of Immunol 187:3238-3246).

[0154] Thus, the bispecific antibodies of the invention comprise a nucleotide sequence comprising the R409 residue in the CH3 domain and the Cys-Pro-Ser-Cys sequence in the hinge region of an antibody that recognizes MUC1 or a second antigen. (SEQ ID NO: 133) Bispecific antibodies of the present invention can be created through the introduction of a heavy-light chain dimer, such that the heavy-light chain dimers are swapped to produce an antibody molecule having one heavy-light chain dimer that recognizes MUC1 and a second heavy-light chain dimer that recognizes a second antigen, where the second antigen is any antigen disclosed herein. As disclosed herein, known IgG4 molecules can also be modified so that the heavy and light chains recognize MUC1 or a second antigen. The use of this method to construct bispecific antibodies of the present invention can be beneficial due to the unique feature of IgG4 molecules, in that the Fc region differs from other IgG subtypes in that it interacts poorly with effector systems of the immune response, such as complement and Fc receptors expressed by certain leukocytes. This particular property makes these IgG4-based bispecific antibodies attractive for therapeutic applications, where the antibody is required to bind to a target and functionally modify a signaling pathway associated with the target, but does not induce effector activity.

[0155] In some embodiments, mutations are introduced into the constant region of the bsAb to alter the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the bsAb. For example, the mutation is a LALA mutation in the CH2 domain. In one aspect, the bsAb contains a mutation on one scFv unit of the heterodimeric bsAb that reduces ADCC activity. In another aspect, the bsAb contains mutations on both chains of the heterodimeric bsAb that completely eliminate ADCC activity. For example, the mutation introduced into one or both scFv units of the bsAb is a LALA mutation in the CH2 domain. These bsAbs with variable ADCC activity can be optimized so that the bsAb shows maximum selective killing toward cells expressing one antigen recognized by the bsAb, but minimal killing toward a second antigen recognized by the bsAb.

[0156] The bispecific antibodies disclosed herein may be useful in the treatment of a disease or condition, for example, cancer.

[0157] Chimeric antigen receptor (CAR) T-cell therapy Also provided herein are cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy. CAR T-cell therapy redirects a patient's T cells to kill tumor cells through exogenous expression of a CAR. CARs can be transmembrane fusion proteins linking the antigen-recognition domain of an antibody to the intracellular signaling domains of a T-cell receptor and co-receptor. Appropriate cells can be used that are contacted with the anti-MUC1 antibodies of the present invention (or engineered to express the anti-MUC1 antibodies as described herein). Solid tumors present unique challenges for CAR-T therapy. Unlike hematological cancers, tumor-associated target proteins are overexpressed between tumors and healthy tissues, resulting in on-target / off-tumor T-cell killing of healthy tissues. Furthermore, immunosuppression in the tumor microenvironment (TME) limits the activation of tumor-killing CAR-T cells. After such contact or engineering, the cells can be introduced into a cancer patient in need of treatment. The cancer patient may have any of the types of cancer disclosed herein. The cell (e.g., T cell) can be, but is not limited to, for example, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or a combination thereof. Exemplary CARS useful in embodiments of the present invention include, for example, those disclosed in PCT / US2015 / 067225 and PCT / US2019 / 022272, each of which is incorporated herein by reference in its entirety.

[0158] In certain cases, the lymphocyte comprises a chimeric, non-natural, at least partially engineered receptor. In certain cases, the engineered chimeric antigen receptor (CAR) has one, two, three, four, or more components, and in some embodiments, one or more components facilitate the lymphocyte's targeting or binding to one or more tumor antigen-containing cancer cells.

[0159] A CAR according to the invention generally comprises at least one transmembrane polypeptide comprising at least one extracellular ligand binding domain and one transmembrane polypeptide comprising at least one intracellular signaling domain, such that the polypeptides assemble together to form a chimeric antigen receptor.

[0160] As used herein, the term "extracellular ligand-binding domain" is defined as an oligo- or polypeptide capable of binding to a ligand. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.

[0161] In particular, the extracellular ligand-binding domain can comprise an antigen-binding domain derived from an antibody directed against a target antigen.

[0162] In a preferred embodiment, the extracellular ligand-binding domain is a single-chain antibody fragment (scFv) comprising the light chain (VL) and heavy chain (VH) variable fragments of a target antigen-specific monoclonal antibody joined by a flexible linker.

[0163] As non-limiting examples, binding domains other than scFvs, such as camelid single domain antibody fragments or receptor ligands, antibody binding domains, antibody hypervariable loops, or CDRs, can also be used for predefined targeting of lymphocytes.

[0164] In a preferred embodiment, the transmembrane domain further comprises a stalk region between the extracellular ligand-binding domain and the transmembrane domain. As used herein, the term "stalk region" generally refers to any oligo- or polypeptide that functions to link a transmembrane domain to an extracellular ligand-binding domain. In particular, the stalk region is used to provide more flexibility and accessibility to the extracellular ligand-binding domain. The stalk region may contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids. The stalk region may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, or CD28, or all or part of an antibody constant region. Alternatively, the stalk region may be a synthetic sequence corresponding to a naturally occurring stalk sequence or may be a completely synthetic stalk sequence. In a preferred embodiment, the stalk region is a portion of the human CD8 alpha chain.

[0165] The signal transduction domain or intracellular signal transduction domain of the CAR of the present invention is involved in intracellular signal transduction after the extracellular ligand binding domain binds to a target, resulting in the activation of immune cells and immune responses. In other words, the signal transduction domain is involved in the activation of at least one of the normal effector functions of the immune cells in which the CAR is expressed. For example, the effector function of T cells can be cytolytic activity or helper activity, including the secretion of cytokines. Thus, the term "signal transduction domain" refers to the part of a protein that transmits effector signal function signals and induces cells to perform specific functions.

[0166] Signaling domains include two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation and those that act antigen-independently to provide secondary or costimulatory signals. Primary cytoplasmic signaling sequences can contain signaling motifs known as ITAMs (immunoreceptor tyrosine-based activation motifs). ITAMs are well-defined signaling motifs found in the cytoplasmic tails of various receptors that function as binding sites for the syk / zap70 class of tyrosine kinases. Examples of ITAMs used in the present invention can include, but are not limited to, those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the signaling domain of the CAR can comprise the CD3 zeta signaling domain or the cytoplasmic domain of the Fc epsilon RI beta or gamma chain. In another preferred embodiment, signaling is provided by CD3 zeta, with costimulation provided by CD28 and a tumor necrosis factor receptor (TNFr), such as, for example, 4-1BB or OX40.

[0167] In certain embodiments, the intracellular signaling domain of the CAR of the present invention comprises a costimulatory signal molecule. In some embodiments, the intracellular signaling domain contains two, three, four, or more costimulatory molecules in tandem. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.

[0168] "Costimulatory ligand" refers to a molecule on an antigen-presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing signals that mediate T cell responses, including, but not limited to, proliferation, activation, differentiation, etc., in addition to the primary signal provided by, for example, engagement of the TCR / CD3 complex with a peptide-loaded MHC molecule. Costimulatory ligands include CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, Toll ligand receptor, and in particular, a ligand that specifically binds B7-H3. Costimulatory ligands can include, but are not limited to, agonists or antibodies that bind to costimulatory molecules present on T cells, such as, but not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, and CD83.

[0169] A "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds to CD83.

[0170] In another specific embodiment, the signaling domain is a TNFR-associated factor 2 (TRAF2)-binding motif, which is the intracellular tail of the costimulatory TNFR family of members. The cytoplasmic tails of costimulatory TNFR family members contain a TRAF2-binding motif consisting of a major conserved motif (P / S / A)X(Q / E)E) or a minor motif (PXQXXD), where X is any amino acid. TRAF proteins are recruited to the intracellular tails of many TNFRs in response to receptor trimerization.

[0171] The distinctive features of suitable transmembrane polypeptides include their ability to be expressed on the surface of immune cells, particularly lymphocytes or natural killer (NK) cells, and to interact with each other to induce a cellular response of immune cells against predefined target cells. The different transmembrane polypeptides of the CAR of the present invention, including extracellular ligand binding domains and / or signaling domains, interact together to participate in signal transduction after binding to the target ligand and induce an immune response. The transmembrane domain can be derived from either natural or synthetic origin. The transmembrane domain can be derived from any membrane-bound or transmembrane protein.

[0172] The term "portion" as used herein refers to any subset of a molecule, i.e., a shorter peptide. Alternatively, functional variants of the amino acid sequence of a polypeptide can be prepared by mutation of the DNA encoding the polypeptide. Such variants or functional variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired activity, particularly specific anti-target cellular immune activity. The functionality of the CAR of the present invention in host cells can be detected by assays suitable for demonstrating the signal transduction ability of the CAR upon binding of a specific target. Such assays are available to those skilled in the art. For example, these assays allow for the detection of signal transduction pathways induced upon target binding, such as assays involving measurement of increased calcium ion release, intracellular tyrosine phosphorylation, inositol phosphate turnover, or the resulting production of interleukin (IL) 2, interferon gamma, GM-CSF, IL-3, and IL-4.

[0173] Aspects of the present invention are also directed to methods and embodiments involving CAR T cells that target two or more antigens. This can be achieved by different approaches: (a) generating two or more cell populations expressing different CARs and administering them to a subject together or sequentially (co-administration), (b) using a bicistronic vector encoding two different CARs on the same cell, (c) simultaneously engineering T cells with two different CAR constructs (co-transduction), which can generate three CAR-T subsets consisting of dual- and single-CAR-expressing cells, or (d) using a single vector to encode two CARs on the same chimeric protein (i.e., bispecific or tandem CARs).

[0174] In embodiments, dual-targeting CAR T cells can target MUC1 and one or more additional antigens. In embodiments, the one or more additional antigens can include a target on tumor cells, such as mesothelin, or a target on non-tumor cells, such as Tregs. For example, dual-targeting CAR T cells can target MUC1 on tumor cells and CCR4 on Tregs recruited to the tumor microenvironment. Such dual-targeting CAR T cells are sometimes referred to as "dual-target cell bispecific CARs."

[0175] The antigen recognition domain of the CAR can be an antibody, including an antibody fragment, as described herein. An "antibody fragment" can be a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0176] The antigen recognition domain can be directed to any antigen target of interest. In embodiments, the antigen target of interest is on the surface of a cell, such as the surface of a cancer cell. Non-limiting examples of antigen targets include Mucl and / or mesothelin.

[0177] Antigen recognition domains useful for constructing CAR-Ts, such as scFVs directed against Mucl and / or mesothelin, can be synthesized, engineered, and / or produced using nucleic acids (e.g., DNA). The DNA encoding the antigen recognition domain can be cloned in-frame with DNA encoding the necessary CAR-T elements, such as, but not limited to, the CD8 hinge region, transmembrane domain, costimulatory domain of a molecule of immunological interest, such as, but not limited to, CD28 and 41BB and CD3-zeta intracellular signaling domains.

[0178] Chimeric antigen receptors fuse an antigen recognition domain to a signaling domain (also called a stimulatory domain) that regulates (i.e., stimulates) cell signaling. Non-limiting examples of such stimulatory domains include those of CD28, 41BB, and / or CD3-zeta intracellular signaling domains.

[0179] The DNA constructs described herein, which may also be referred to as "DNA vectors," can be cloned into vectors that will be used to transduce and generate chimeric antigen receptor T cells, including those that secrete the polypeptides and / or fragments thereof. In one embodiment, the DNA constructs can be cloned into lentiviral vectors for the production of lentiviruses that will be used to transduce and generate chimeric antigen receptor T cells, including those that secrete monospecific, bispecific, or trispecific immunomodulatory antibodies / minibodies and / or antibody fusion proteins at tumor sites.

[0180] As used herein, the term "engineered" or "recombinant" cell can refer to a cell into which a recombinant gene, such as a gene encoding a chimeric antigen receptor, has been introduced. Engineered cells are therefore distinguishable from naturally occurring cells that do not contain a recombinantly introduced gene. Engineered cells are thus cells that have a gene that has been manually introduced. Recombinantly introduced genes may be in the form of either cDNA genes (i.e., they will not contain introns), copies of genomic genes, or may contain genes located adjacent to a promoter that is not naturally associated with the particular introduced gene.

[0181] In embodiments, it may be more convenient to use a cDNA version of a gene as the recombinant gene, since the size of the gene is generally much smaller than that of a genomic gene, which is typically up to an order of magnitude larger than a cDNA gene, and the use of a cDNA version offers the advantage that it is more easily used for transfection of targeted cells. However, this does not exclude the possibility of using a genomic version of a particular gene, if desired.

[0182] In embodiments, the antigen recognition domain can be linked to a signaling domain to form a CAR on the surface of any type of cell, including immune cells capable of expressing antibody fragments for cancer therapy, or cells, such as bacterial cells, that harbor an expression vector encoding a CAR. As used herein, the terms "cell," "cell line," and "cell culture" can be used interchangeably. All of these terms include their progeny, which are any and all subsequent generations. Without being bound by theory, all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing heterologous nucleic acid sequences, a "host cell" refers to a eukaryotic cell that can replicate a vector and / or express a heterologous gene encoded by the vector. Host cells can and have been used as recipients of vectors. Host cells may also be "transfected" or "transformed," which refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. Transformed cells include the primary subject cell and its progeny. As used herein, the terms "engineered" and "recombinant" or host cell can refer to a cell into which an exogenous nucleic acid sequence, such as a vector, has been introduced. Recombinant cells are therefore distinguishable from naturally occurring cells that do not contain recombinantly introduced nucleic acid.

[0183] The cells can be autologous, syngeneic, allogeneic, or in some cases xenogeneic.

[0184] In embodiments of the invention, the host cells are T cells, CD4+ T cells, NK cells, and NKT cells, including (but not limited to) cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells, or killer T cells).

[0185] For example, chimeric antigen receptor (CAR) T cell therapy redirects patient's T cells to kill tumor cells by exogenous expression of CAR.CAR is a transmembrane fusion protein that connects the antigen recognition domain of antibody or fragment with the intracellular signaling domain of T cell receptor and co-receptor.For example, chimeric antigen receptor fuses antigen-specific antibody fragment with T cell costimulatory domain and CD3 zeta intracellular signaling domain, allowing T cells to be redirected to target cells, such as the antigen presented on tumor cells.

[0186] An emerging mechanism associated with tumor progression is the immune checkpoint pathway, which consists of cellular interactions that prevent excessive T cell activation under normal conditions and allow T cell function in a self-limiting manner. As an evasion mechanism, many tumors can stimulate the expression of immune checkpoint molecules, resulting in an anergic phenotype of T cells that cannot suppress tumor progression. Emerging clinical data highlight the importance of one inhibitory ligand-receptor pair as an immune checkpoint: programmed death ligand 1 (PD-L1, B7-H1, and CD274) and programmed death receptor 1 (PD-1, CD279) in preventing cancer cell killing by cytotoxic T lymphocytes. The PD1 receptor is expressed by many cell types, including T cells, B cells, natural killer cells (NK), and host tissues. PD-L1-expressing tumors and antigen-presenting cells (APCs) can block T cell receptor (TCR) signaling of cytotoxic T lymphocytes through binding to the receptor PD-1, reducing cytokine production and T cell proliferation. PD-L1 overexpression can be found in many tumor types and may mediate immunosuppressive functions through its interactions with other proteins, including CD80 (B7.1), and block its ability to activate T cells through binding to CD28.

[0187] Genetic engineering of human lymphocytes to express tumor-specific chimeric antigen receptors (CARs) can produce anti-tumor effector cells that circumvent tumor immune evasion mechanisms due to abnormalities in protein-antigen processing and presentation. Furthermore, these transgenic receptors are directed against tumor-associated antigens that are not derived from proteins. In certain embodiments of the present invention, lymphocytes (CARTs) are modified to contain at least a CAR, and in certain embodiments of the present invention, a single CAR targets two or more antigens. In preferred embodiments, the CART is further modified to express and secrete one or more polypeptides, such as antibodies or cytokines. Such CARTs are referred to herein as armed CARTs. Armed CARTs enable simultaneous secretion of polypeptides locally at the targeting site (i.e., tumor site). For example, an anti-MUC1 antibody can be the targeting moiety of an engineered CART cell, and an anti-CCR4 antibody can be the payload of an engineered CAR T cell. However, those skilled in the art will recognize that any of several antibodies can be utilized as the payload, and this exemplary embodiment is not limiting.

[0188] The polypeptide can be, for example, an antibody or fragment thereof as described herein.For example, the second expression construct, which can be in the same DNA vector (e.g., antigen recognition domain) as that encoding CAR or in a second separate vector, can be used to encode a minibody (scFv-Fc) or antibody or fragment thereof, directed against a single or multiple antigens of interest, and can be cloned after an internal ribosome entry site (IRES).For example, the second expression cassette contains either a fluorescent molecule or an immunomodulatory minibody.

[0189] In embodiments, the engineered cells can secrete monospecific, bispecific, or trispecific minibodies, antibodies, or minibody / antibody fusion proteins at the tumor site to provide additional benefit by modifying (i.e., modulating) the immunosuppressive tumor microenvironment. For example, the secreted antibody can be an anti-CCR4 antibody.

[0190] In cancer, normal cell-cell interactions in tissues are disrupted, and a tumor microenvironment evolves to accommodate the growing tumor. The tumor microenvironment (TME) refers to the cellular environment in which a tumor resides, including components such as surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM). The tumor microenvironment is complex and is strongly influenced by the immune system.

[0191] Aspects of the present invention are further drawn to antibody-drug conjugates (or ADCs). ADCs, which may also be called immunoconjugates, combine the targeting capabilities of antibodies or antigen-binding fragments, such as those described herein, with the cancer-killing capabilities of cytotoxic drugs. Thus, ADCs are targeted therapies for the treatment of cancer patients. Unlike chemotherapy, ADCs are intended to target and kill only cancer cells, sparing healthy cells. For example, see FIG. 18, for example, a 3D1-MMAE antibody conjugate regresses tumor growth in MUC1-C+ tumors but not in MUC1-C- tumors. Monomethyl auristatin E (or MMAE) is a potent and highly toxic anti-microtubule agent. Due to its high toxicity, MMAE, which inhibits cell division by blocking tubulin polymerization, cannot be used as a single-agent chemotherapy drug. Those skilled in the art will recognize that MMAE can be replaced with one or more of a variety of chemicals known to kill tumor cells, such as MUC1+ tumor cells. For example, the antibody-drug conjugates of the invention can include, in addition to MMAE, various chemicals (ie, chemotherapy) known to those skilled in the art to kill MUC1+ tumor cells.

[0192] ADCs are complex molecules composed of antibodies linked to a biologically active cytotoxic (anti-cancer) payload or drug. Antibody-drug conjugates may also be called bioconjugates or immunoconjugates. In the development of antibody-drug conjugates, an anti-cancer drug is attached to an antibody that specifically targets a particular tumor marker (e.g., a protein ideally found only in or on tumor cells). In embodiments, the tumor marker includes, for example, MUC1. Optionally, the tumor marker can further include mesothelin. The antibody tracks these proteins throughout the body and attaches to the surface of cancer cells. A biochemical reaction between the antibody and the target protein induces a signal in the tumor cell, which then absorbs or internalizes the antibody along with a cytotoxin. After the ADC is internalized, the cytotoxic drug is released, killing the cancer. Due to this targeting, the drug ideally has fewer side effects than other chemotherapeutic agents and provides a wide therapeutic window.

[0193] A stable bond between the antibody and the cytotoxic (anti-cancer) agent is a key aspect of ADCs. A highly stable ADC linker will ensure that less of the cytotoxic payload is shed in the circulation, promoting an improved safety profile, and that more of the payload reaches cancer cells, promoting enhanced efficacy. In embodiments, the linkers utilized herein include those based on chemical motifs including disulfides, hydrazones, or peptides (cleavable), or thioethers (non-cleavable), which can control the distribution and delivery of the cytotoxic agent to target cells. Both cleavable and non-cleavable linkers have been proven safe in preclinical and clinical trials.

[0194] The availability of better, more stable linkers has changed the function of chemical bonds. The type of linker, cleavable or non-cleavable, confers specific properties to cytotoxic (anti-cancer) drugs. For example, a non-cleavable linker retains the drug intracellularly. As a result, the entire antibody, linker, and cytotoxic (anti-cancer) drug enters the targeted cancer cell, where the antibody is degraded to the amino acid level. The resulting complex (amino acids, linker, and cytotoxic drug) then becomes the active drug. In contrast, cleavable linkers are catalyzed by enzymes in the cancer cell, releasing the cytotoxic drug. The difference is that cytotoxic payloads delivered via cleavable linkers can escape from the targeted cell and attack neighboring cancer cells in a process called "bystander killing."

[0195] Other linkers, such as those that add an additional molecule between the cytotoxic drug and the cleavage site, allow for the development of ADCs with more flexibility without concerns about altering the cleavage rate.

[0196] Non-limiting examples of linkers are described in the literature. (See, e.g., Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984), which describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives attached to antibodies by oligopeptide linkers. Non-limiting examples of useful linkers that can be used with the antibodies of the invention include (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride), (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G)), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat. (21558G)), (iv) PEG-10 hydroxybenzoate (Pierce Chem. Co., Cat. (21558G)), (v) PEG-10 hydroxybenzoate (Pierce Chem. Co., Cat. (21558G)), (vi) PEG-10 hydroxybenzoate (Pierce Chem. Co., Cat. (21558G)), (vii ... #21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamido]hexanoate (Pierce Chem. Co., Cat. #2165-G), and (v) sulfo-NHS (-hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC.

[0197] The linkers described herein contain components with different attributes, thereby resulting in conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester-containing linkers are less soluble than sulfo-NHS esters. Furthermore, the linker SMPT contains a sterically hindered disulfide bond, allowing for the formation of conjugates with improved stability. Disulfide bonds are generally less stable than other bonds because they are cleaved in vitro, resulting in fewer available conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide bonds. Carbodiimide bonds (such as EDC) when used in combination with sulfo-NHS form esters that are more resistant to hydrolysis than the carbodiimide bond reaction alone.

[0198] Pharmaceutical Compositions The antibodies of the present invention that specifically bind to the MUC1 protein or fragments thereof can be administered for the treatment of cancer in the form of pharmaceutical compositions. Principles and considerations involved in the preparation of therapeutic compositions containing antibodies, as well as guidance on the selection of components, are provided, for example, in Remington: The Science and Practice of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa., 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0199] A therapeutically effective amount of an antibody of the present invention can generally relate to the amount necessary to achieve a therapeutic goal. As mentioned above, this can be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the function of the target. The amount that needs to be administered further depends on the binding affinity of the antibody for its specific antigen and also on the rate at which the administered antibody is depleted from the free volume of the subject to which it is administered. A typical range for therapeutically effective administration of an antibody or antibody fragment of the present invention can be, by way of non-limiting example, about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical administration frequencies can range, for example, from twice daily to once weekly.

[0200] When an antibody fragment is used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation can also contain two or more active compounds necessary for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can contain an agent that enhances its function, such as, for example, a cytotoxic agent, a cytokine (e.g., IL-15), a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are suitably present in combination in amounts effective for the intended purpose.

[0201] The active ingredient can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, respectively.

[0202] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0203] Sustained-release preparations can be prepared.Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles, such as films or microcapsules.Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for more than 100 days, while certain hydrogels release proteins for shorter periods of time.

[0204] The antibodies or agents of the present invention (also referred to herein as "active compounds"), as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the antibody or agent and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as liposomes and fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0205] The pharmaceutical composition of the present invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may contain the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate, and an agent for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0206] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In embodiments, the composition is sterile and fluid to the extent that easy syringability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, etc. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0207] Sterile injectable solution can be prepared by incorporating the required amount of active compound into the appropriate solvent with one or combination of the ingredients listed above as needed, and then sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other necessary ingredients listed above.For the preparation of sterile injectable solution, the method of preparation is vacuum drying and freeze-drying, which produces powder of active compound and any additional desired ingredients from the solution that has been previously sterile filtered.

[0208] Oral compositions generally contain an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which the compound in the fluid carrier is orally applied, swirled in the mouth, and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvants can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, tragacanth gum, or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, primogel, or corn starch; a lubricant such as magnesium stearate or sterol; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavor.

[0209] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0210] Systemic administration can also be by transmucosal or transdermal means.

[0211] For transmucosal or transdermal administration, penetrant suitable for the barrier to be permeated is used in formulation.Such penetrant is generally known in the art, and for example, for transmucosal administration, includes detergent, bile salt and fusidic acid derivative.Transmucosal administration can be achieved through the use of nasal spray or suppository.For transdermal administration, active compound is formulated into ointment, salve, gel or cream generally known in the art.

[0212] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0213] In one embodiment, the active compound is prepared with a carrier that will protect the compound against rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0214] For ease of administration and uniformity of dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form.Dosage unit form as used herein refers to a physically separate unit that is suitable as a single dose for the subject to be treated, and each unit contains a predetermined amount of active compound that is calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined by and directly depends on the specific characteristics of active compound and the specific therapeutic effect that should be achieved, and the inherent limitation of the technology that formulates this active compound for individual treatment.

[0215] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0216] Treatment method Antibodies or fragments that specifically bind to the MUC1 protein or its fragments, such as MUC1-SEA, can be administered to treat MUC1-related diseases or disorders. "MUC1-related diseases or disorders" include disease states and / or symptoms associated with disease states in which increased levels of MUC1 gene expression or protein levels, such as on the surface of cancer cells, and / or activation of cell signaling pathways involving MUC1 are observed. MUC1-related diseases and disorders can also be characterized by diseases in which MUC1 is abnormally glycosylated. See, e.g., Nath, S., & Mukherjee, P. (2014). MUC1: a multifaceted oncoprotein with a key role in cancer progression. Trends in molecular medicine, 20(6), 332-342, and Horm, T. M., & Schroeder, J. A. (2013). MUC1 and metastatic cancer: expression, function and therapeutic targeting. Cell adhesion & migration, 7(2), 187-198, each of which is incorporated by reference in its entirety. Exemplary MUC1-associated diseases or disorders include, but are not limited to, cancers, such as epithelial cancers.

[0217] MUC1 overexpression and aberrant glycosylation are associated with many cancers, including most human epithelial cancers. As used herein, "epithelial cancer" can refer to any cancer arising from epithelial cells, including, but not limited to, breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, oral cancer, esophageal cancer, small intestine and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer and skin cancer, such as squamous cell and basal cell carcinoma, prostate cancer, renal cell carcinoma, and other known cancers that affect epithelial cells throughout the body.

[0218] Known risk factors for epithelial cancer include, but are not limited to, family history, genetic predisposition (i.e., mutations in BRCA1 and BRCA2, BRIP1, MSH6, RAD15C), personal history of epithelial cancer, physical inactivity, or obesity.

[0219] Epithelial cancers can be diagnosed by methods known in the art, including testing for tumor markers (such as CA125), imaging via CT scan, MRI, or TVU, or fine needle biopsy.

[0220] Epithelial cancers may be treated with debulking surgery (such as removal of both the ovaries and fallopian tubes), omental biopsy of the uterus, peritoneum (lining of the abdominal cavity), and chemotherapy (such as platinum- and taxane-based chemotherapy).

[0221] With reference to the Examples, embodiments of the present invention are particularly useful in the treatment of ovarian and colon cancer.

[0222] Ovarian cancer is a cause of significant morbidity and mortality in populations worldwide. Ovarian cancer is a type of cancer that begins in the ovaries. The female reproductive system contains two ovaries, one on each side of the uterus. Each ovary is about the size of an almond and produces eggs (egg cells) and the hormones estrogen and progesterone. Ovarian cancer often goes undetected until it has spread within the pelvis and abdomen. At this later stage, ovarian cancer is more difficult to treat. Early-stage ovarian cancer, in which the disease is limited to the ovaries, is more likely to be treated successfully. Surgery and chemotherapy are commonly used to treat ovarian cancer.

[0223] Early-stage ovarian cancer rarely causes any symptoms. Advanced-stage ovarian cancer may cause a few nonspecific symptoms that are often mistaken for more common benign conditions. Signs and symptoms of ovarian cancer may include abdominal bloating or distention, feeling full quickly after eating, weight loss, discomfort in the pelvic area, changes in bowel habits such as constipation, and frequent urination.

[0224] Tests and procedures used to diagnose ovarian cancer include a pelvic exam, imaging tests (such as an ultrasound or CT scan), blood tests (such as for organ function and tumor markers), surgery, and / or biopsy.

[0225] Once ovarian cancer is diagnosed, doctors will use information from such tests and procedures to assign a stage to the cancer. Ovarian cancer stages are designated using Roman numerals ranging from I to IV, with the lowest stage indicating that the cancer is confined to the ovaries. By stage IV, the cancer has spread to distant areas of the body.

[0226] Current treatment for ovarian cancer usually involves a combination of surgery and chemotherapy.

[0227] Surgery to remove ovarian cancer may include surgery to remove one ovary, surgery to remove both ovaries and / or fallopian tubes, surgery to remove both ovaries and the uterus, or, if the cancer is advanced, chemotherapy followed by surgery to remove as much of the cancer as possible.

[0228] Chemotherapy can refer to drug treatments that use chemicals to kill rapidly growing cells in the body, including cancer cells. Chemotherapy drugs can be injected into a vein or taken orally. Sometimes drugs are injected directly into the abdomen (intraperitoneal chemotherapy). Chemotherapy is often used after surgery to kill any cancer cells that may remain. It can also be used before surgery.

[0229] Colon cancer is a type of cancer that develops in the large intestine (colon). The colon is the final segment of the digestive tract. Colon cancer typically affects older adults, but can occur at any age. It usually begins as small, noncancerous (benign) masses of cells called polyps that form on the lining of the colon. Over time, some of these polyps can develop into colon cancer. Polyps can be small and cause few, if any, symptoms. For this reason, doctors recommend regular screening tests to help prevent colon cancer by identifying and removing polyps before they become cancerous. Once colon cancer develops, many treatments are available to help control it, including surgery, radiation therapy, and drug treatments such as chemotherapy, targeted therapy, and immunotherapy. Colon cancer can also be called colorectal cancer, a combined term for colon cancer and rectal cancer, which originates in the rectum.

[0230] Signs and symptoms of colon cancer include persistent changes in bowel habits, including diarrhea or constipation or changes in stool consistency; rectal bleeding or blood in the stool; persistent abdominal discomfort such as cramps, gas, or pain; a feeling that the bowels do not empty completely; weakness or fatigue; and unexplained weight loss.

[0231] Many colon cancer patients do not experience symptoms in the early stages of the disease. If symptoms do occur, they will likely vary depending on the size of the cancer and its location in the large intestine. Doctors recommend specific screening tests for healthy individuals without signs or symptoms to look for signs of colon cancer or noncancerous colon polyps. Detecting colon cancer at its earliest stage provides the greatest opportunity for a cure. Screening has been shown to reduce the risk of dying from colon cancer.

[0232] Several screening options exist, such as blood tests or colonoscopies.

[0233] Colon cancer stages are designated by Roman numerals ranging from 0 to IV, with the lowest stage indicating cancer that is confined to the inner surface of the colon. By stage IV, the cancer is considered advanced and has spread (metastasized) to other areas of the body.

[0234] Treatment for colon cancer usually involves surgery to remove the cancer. Other treatments, such as radiation therapy and chemotherapy, may also be recommended.

[0235] An embodiment of the present invention is directed to a method for treating cancer, including epithelial cancers such as colon cancer or ovarian cancer, by administering a composition described herein to a subject suffering from cancer. The antibodies of the present invention, including fragments, bispecific, polyclonal, monoclonal, humanized, and fully human antibodies, as well as CAR-T cells, can be used as therapeutic agents. Such agents will generally be used to treat or prevent cancer in a subject, improve vaccine efficacy, or enhance natural immune responses. Antibody preparations, preferably those with high specificity and high affinity for their target antigen, are administered to a subject and generally have an effect via target binding. Administration of antibodies can neutralize, inhibit, or interfere with the activity of the MUC1 protein. Administration of antibodies can also be used to target therapeutic agents to specific cells, such as cancer cells, and / or to sensitize cancer cells to anti-cancer treatments.

[0236] The present invention provides both preventative and therapeutic methods for treating subjects at risk (or susceptible) for cancer or other cell proliferation-related diseases or disorders. Such diseases or disorders include, but are not limited to, diseases or disorders associated with aberrant expression of MUC1 and / or aberrant glycosylation of MUC1. For example, the methods are used to treat, prevent, or alleviate symptomatic cancer. Non-limiting examples of cancers that can be treated by embodiments herein include lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, or gastric cancer. In addition, the methods of the present invention can be used to treat blood cancers such as leukemia and lymphoma. Alternatively, the methods can be used to treat, prevent, or alleviate symptoms of metastatic cancer.

[0237] Thus, in one aspect, the invention provides a method for preventing, treating, or alleviating a symptomatic cancer or cell proliferative disease or disorder in a patient by administering to the patient a monoclonal antibody, an scFv antibody of the invention, or a bispecific antibody of the invention. For example, an anti-MUC1 antibody can be administered in a therapeutically effective amount.

[0238] Subjects at risk for cancer or cell proliferation-related diseases or disorders may include patients with a family history of cancer or subjects who have been exposed to agents known or suspected to cause cancer. Administration of a prophylactic agent can occur prior to the onset of cancer, such that the disease is prevented or, alternatively, its progression is delayed.

[0239] In one embodiment, tumor cell viability can be inhibited by contacting the cells with an anti-MUC1 antibody of the invention. With reference to Figure 2, for example, colon cancer cell lines expressing MUC1 exhibit reduced viability (or increased cell killing) by anti-MUC1 CAR T cells. Additionally, Figures 5 and 6 further demonstrate the tumor cell killing activity of anti-MUC1 scFv CAR T cells.

[0240] In another embodiment, tumor cell growth can be inhibited by contacting the cell with an anti-MUC1 antibody of the invention. The cell can be any cell that expresses MUC1.

[0241] The present invention also encompasses methods for increasing or enhancing the immune response to an antigen. The immune response is increased or enhanced by administering a monoclonal antibody, scFv antibody, or bispecific antibody of the present invention to a subject. The immune response is enhanced, for example, by enhancing antigen-specific T effector function. The antigen is a viral (e.g., HIV), bacterial, parasitic, or tumor antigen. The immune response is a natural immune response. A natural immune response refers to an immune response that is the result of an infectious disease. The infectious disease is a chronic infectious disease. An increase or enhancement of the immune response to an antigen can be measured by many methods known in the art. For example, the immune response can be measured by measuring any one of the following: T cell activity, T cell proliferation, T cell activation, effector cytokine production, and T cell transcriptional profile.

[0242] Alternatively, the immune response is a response induced by vaccination.

[0243] Thus, in another aspect, the present invention provides a method for increasing vaccine efficacy by administering to a subject a monoclonal antibody or scFv antibody of the present invention and a vaccine, wherein the antibody and vaccine are administered sequentially or simultaneously. The vaccine may be a tumor vaccine, a bacterial vaccine, or a viral vaccine.

[0244] In another embodiment, the present invention provides for treating cancer in a patient by administering two antibodies that bind to the same epitope of the MUC1 protein or two different epitopes of the MUC1 protein. Alternatively, cancer is treated by administering a first antibody that binds to MUC1 and a second antibody that binds to a protein other than MUC1. For example, the protein other than MUC1 includes, but is not limited to, LIGO1 and / or mesothelin. For example, the protein other than MUC1 is a tumor-associated antigen.

[0245] In some embodiments, the invention provides for the administration of anti-MUC1 antibodies, alone or with an additional antibody that recognizes another protein other than MUC1, together with cells capable of achieving or enhancing an immune response. For example, these cells can be peripheral blood mononuclear cells (PBMCs), or any cell type found in PBMCs, such as cytotoxic T cells, macrophages, and natural killer (NK) cells.

[0246] Additionally, the present invention provides for the administration of antibodies that bind to MUC1 protein and other therapeutic agents, including anti-neoplastic agents, such as small molecules, growth factors, cytokines, or biomolecules, such as peptides, peptidomimetics, peptoids, polynucleotides, lipid-derived mediators, small biogenic amines, hormones, neuropeptides, and proteases. Small molecules include, but are not limited to, inorganic molecules and small organic molecules. Suitable growth factors or cytokines include IL-2, GM-CSF, IL-12, and TNF-alpha. Small molecule libraries are known in the art. (See Lam, Anticancer Drug Des., 12:145, 1997.)

[0247] Diagnostic Assays Anti-MUC1 antibodies can be used diagnostically, e.g., as part of a clinical trial procedure to determine the effectiveness of a given therapeutic and / or prophylactic regimen, e.g., to monitor the development or progression of cancer.

[0248] In some embodiments, for diagnostic purposes, the anti-MUC1 antibodies of the invention are linked to a detectable moiety, e.g., to provide a method for detecting cancer cells in a subject at risk for or afflicted with cancer.

[0249] The detectable moiety can be conjugated directly to the antibody or fragment, or indirectly, for example, by using a fluorescent secondary antibody. Direct conjugation can be achieved, for example, by standard chemical conjugation of a fluorophore to the antibody or antibody fragment, or through genetic engineering. Chimeras, or fusion proteins containing an antibody or antibody fragment linked to a fluorescent or bioluminescent protein, can be constructed. For example, Casadei et al. describe a method for creating a vector construct capable of expressing a fusion protein of aequorin and antibody genes in mammalian cells.

[0250] As used herein, the term "labeled" with respect to a probe or antibody can encompass both direct labeling of the probe or antibody by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and biological fluids present within a subject. That is, the detection methods of the present invention can be used to detect cells expressing MUC1 in biological samples in vitro and in vivo. For example, in vitro techniques for detecting MUC1 include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. Additionally, in vivo techniques for detecting MUC1 involve introducing a labeled anti-MUC1 antibody into a subject. For example, an antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. For "targeted" conjugates, i.e., conjugates containing a targeting moiety, which is a molecule or feature designed to localize the conjugate within a subject or animal at a specific site(s), localization can refer to a state when equilibrium between the bound "localized" entity and the unbound "free" entity within the subject is essentially achieved. The rate at which such equilibrium is achieved depends on the route of administration. For example, a conjugate administered by intravenous injection can achieve localization within minutes of injection, whereas an orally administered conjugate may take several hours to achieve localization. Alternatively, localization can simply refer to the location of an entity within a subject or animal at a selected period of time after administration. As another example, localization is achieved when the moiety becomes distributed after administration.

[0251] It is understood that a reasonable estimate of the time required to achieve localization can be made by one skilled in the art. Furthermore, the state of localization as a function of time can be tracked by imaging a detectable moiety (e.g., a luminescent conjugate) according to the method of the present invention, such as with a photodetector device. The "photodetector device" used should be sensitive enough to allow imaging of weak light from within a mammal in a reasonable time and to use the signal from such a device to construct an image.

[0252] If it is possible to use extremely bright light-generating moieties and / or detect light-generating fusion proteins localized near the surface of the object or animal being imaged, "night vision" goggles or standard highly sensitive video cameras such as Silicon Intensified Tube (SIT) cameras (e.g., from Hammamatsu Photonic Systems, Bridgewater, NJ) can be used. More typically, however, more sensitive light detection methods are required.

[0253] At extremely low light levels, the photon flux per unit area becomes so low that the scene being imaged no longer appears continuous. Instead, it is represented by individual photons that differ from each other both temporally and spatially. When viewed on a monitor, such an image appears as sparkling points of light, each representing a single detected photon. By accumulating these detected photons over time in a digital image processor, an image can be acquired and constructed. In contrast to conventional cameras, where the signal at each image point is assigned an intensity value, in photon-counting imaging, the amplitude of the signal is immaterial. The objective is simply to detect the presence of a signal (photon) and count its occurrence relative to its location over time.

[0254] At least two types of photodetector devices, described below, can detect individual photons and generate a signal that can be analyzed by an image processor. Noise-reducing photodetection devices achieve sensitivity by reducing the background noise of the photon detector rather than amplifying the photon signal. Noise is primarily reduced by cooling the detector array. Devices include charge-coupled device (CCD) cameras called "back-thinned" cooled CCD cameras. In more sensitive instruments, cooling is achieved using, for example, liquid nitrogen, which brings the temperature of the CCD array to approximately -120°C. "Back-thinned" refers to an ultra-thin backplate that reduces the path length photons must travel before detection, thereby increasing quantum efficiency. A particularly sensitive back-thinned cryogenic CCD camera is the "TECH 512" Series 200 camera, available from Photometries, Ltd. (Tucson, Arizona). "Photon amplification devices" amplify photons before they hit the detection screen. This class includes CCD cameras equipped with intensifier tubes, such as microchannel intensifier tubes. Microchannel intensifier tubes typically contain a metal array of channels perpendicular to and coextensive with the camera's detection screen. The microchannel array is positioned between the sample, subject, or animal being imaged and the camera. Most photons entering a channel of the array contact the side of the channel before exiting. A voltage applied across the array results in the ejection of many electrons from each photon collision. Electrons from such collisions exit their channel of origin in a "shotgun" pattern and are detected by the camera.

[0255] Even greater sensitivity can be achieved by arranging intensifying microchannel arrays in series, so that electrons generated in the first stage in turn result in an amplified signal of electrons in the second stage. However, the increased sensitivity is achieved at the expense of spatial resolution, which decreases with each additional stage of amplification. An exemplary microchannel intensifier-tube-based single-photon detection device is the C2400 series available from Hamamatsu.

[0256] Image processors process signals generated by photon-counting photodetector devices to construct images that can be displayed on a monitor or printed on a video printer, for example. Such image processors are typically sold as part of systems that include the highly sensitive photon-counting cameras described above, and are therefore available from the same sources. Image processors are usually connected to personal computers, such as IBM-compatible PCs or Apple Macintosh computers (Apple Computer, Cupertino, Calif.), which may or may not be included as part of a purchased imaging system. Once images are in the form of digital files, they can be manipulated and printed using a variety of image processing programs (e.g., Adobe Photoshop, Adobe Systems, Adobe Systems, Mt. View, Calif.).

[0257] In one embodiment, the biological sample contains protein molecules from the test subject.Exemplary biological samples include a peripheral blood leukocyte sample isolated from the subject by conventional means, or a sample containing one or more cancer cells isolated from the subject by conventional means.Those skilled in the art will recognize that any biological sample can be used in such embodiments, and non-limiting examples include ascites, pleural effusion, urine, saliva, bronchoalveolar lavage, etc.

[0258] The present invention also encompasses kits for detecting the presence of MUC1 or MUC1-expressing cells in a biological sample. For example, the kit can include a labeled compound or agent capable of detecting cancer or tumor cells (e.g., an anti-MUC1 scFv or monoclonal antibody) in a biological sample, a means for determining the amount of MUC1 in the sample, and a means for comparing the amount of MUC1 in the sample with a standard. In some embodiments, the standard is a non-cancerous cell or a cellular extract thereof. The compound or agent can be packaged in a suitable container. The kit can further include instructions for using the kit to detect cancer in a sample.

[0259] The antibodies according to the present invention can be used as agents for detecting the presence of MUC1 (or a protein or protein fragment thereof) in a sample. Preferably, the antibody contains a detectable label. The antibody can be polyclonal or monoclonal. The intact antibody, or a fragment thereof (e.g., F a b, scFv, or F( a b)2) can be used. With respect to a probe or antibody, the term "labeled" can encompass both direct labeling of the probe or antibody by conjugating (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling a DNA probe with biotin for detection with fluorescently labeled streptavidin. The term "biological sample" can include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and biological fluids present within a subject. Thus, the use of the term "biological sample" includes blood and fractions or components of blood, including serum, plasma, or lymph. That is, the detection methods of the present invention can be used to detect analyte mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations.

[0260] Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J.R.Crowther (Ed.) Human Press, Totowa, NJ, 1995, "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996, and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Additionally, in vivo techniques for detecting an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0261] Antibodies to the MUC1 protein (or fragments thereof) can be used in methods known in the art related to localizing and / or quantitating the MUC1 protein (e.g., for use in measuring levels of MUC1 protein in an appropriate physiological sample, for use in diagnostic methods, for use in protein imaging, etc.) In certain embodiments, antibodies specific for the MUC1 protein, or derivatives, fragments, analogs, or homologs thereof, that contain an antigen-binding domain derived from the antibody, are utilized as pharmacologically active compounds (hereinafter referred to as "therapeutic agents").

[0262] Antibodies specific for the MUC1 protein of the invention can be used to isolate MUC1 polypeptides by standard techniques, such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies to the MUC1 protein (or fragments thereof) can be used diagnostically to monitor protein levels in tissues as part of a clinical testing procedure, e.g., to determine the effectiveness of a given therapeutic regimen.

[0263] Detection can be facilitated by conjugating (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 Contains H.

[0264] Other embodiments While the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0265] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0266] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, which are for illustrative purposes only, as alternative methods may be used to obtain similar results.

[0267] Example 1 Discovery of a human anti-MUC1 antibody targeting the MUC1-SEA domain We report here the discovery of a human single-chain variable fragment (scFv), termed T4E3, that recognizes human MUC1-SEA. In the scFv-Fc format, T4E3 binds to MUC1+ cells with six-fold higher affinity than the anti-MUC1-C antibody 3D1 (Figure 1). When T4E3 is utilized as the targeting moiety of CAR T cells, T4E3 CAR T cells preferentially kill MUC1+ tumor cells and not MUC1- cells. CAR T cells that recognize activated T cells and CXCR4 from the same human leukocyte donor kill neither MUC1+ nor MUC1- tumor cell lines that lack CXCR4 (Figure 2).

[0268] Example 2 Observations from CDR3 analysis G1-1-A1 V H Even if they have different VGene assignments, V H It has more similarity to T4E3 and G2-2-F8 than to G1-3-A3 and G1-2-B10. G1-3-A3 is a completely different V L V that has genes and is different from T4E3 H It has a gene that disables the binding. ·V H - The GMDV at the end of CDR3 appears to be important for binding to MUC1 (T4E3, G2-2-F8, and G1-1-A1, the highest affinity binders, share this motif). 17-18 amino acids have the highest binding V H The CDR3 lengths of G1-3-A3 and G1-2-B10 are 20 and 15, respectively. T4E3 V L CDR3 has a 3' insertion that does not have any of the low affinity hits. In addition, two consecutive serines are present in CDR3-V Lare mutated from the germline to arginine and tyrosine, respectively, and the 3' histidine in the germline is mutated to serine. None of the low affinity hits retain these mutations.

[0269] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific substances and procedures specifically described herein which equivalents are considered to be within the scope of this invention and covered by the appended claims.

Claims

1. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to the MUC1-SEA domain or a peptide corresponding to an epitope on said domain, (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence of GFTFDDYA (SEQ ID NO: 2), a CDR2 having the amino acid sequence of ISWNSGSI (SEQ ID NO: 96), and a CDR3 having the amino acid sequence of AKDIGSGSYYNYYYGMDV (SEQ ID NO: 104); (b) a light chain variable region comprising a CDR1 having the amino acid sequence of SLRSYY (SEQ ID NO: 7), a CDR2 having the amino acid sequence of GKN, and a CDR3 having the amino acid sequence of NSRDRYGNSL (SEQ ID NO: 118); An antibody or antigen-binding fragment thereof comprising:

2. V corresponding to the amino acid sequence of SEQ ID NO: 12 Ｈ and V corresponding to the amino acid sequence of SEQ ID NO: 17 Ｌ The antibody or antigen-binding fragment thereof of claim 1, comprising:

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, which is humanized.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is monospecific or bispecific.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a single-chain antibody.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, comprising a Fab fragment antibody.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, having a binding affinity in the range of 1 pM to 1 μM.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, linked to a therapeutic agent.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the therapeutic agent is a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine.

10. The antibody or antigen-binding fragment thereof of claim 8, wherein the therapeutic agent is MMAE.

11. A cell that produces the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10 and a pharmaceutically acceptable excipient.

13. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

14. A nucleic acid encoding an isolated monoclonal antibody or antigen-binding fragment thereof that binds to the MUC1-SEA domain or a peptide corresponding to an epitope on said domain, wherein said antibody or antigen-binding fragment thereof (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence of GFTFDDYA (SEQ ID NO: 2), a CDR2 having the amino acid sequence of ISWNSGSI (SEQ ID NO: 96), and a CDR3 having the amino acid sequence of AKDIGSGSYYNYYYGMDV (SEQ ID NO: 104); (b) a light chain variable region comprising a CDR1 having the amino acid sequence of SLRSYY (SEQ ID NO: 7), a CDR2 having the amino acid sequence of GKN, and a CDR3 having the amino acid sequence of NSRDRYGNSL (SEQ ID NO: 118); A nucleic acid comprising:

15. 15. The nucleic acid of claim 14, comprising the nucleotide sequences set forth in SEQ ID NO: 23 and SEQ ID NO:

28.

16. A vector comprising the nucleic acid of claim 14 or 15.

17. A cell comprising the vector of claim 16.

18. A pharmaceutical composition comprising the cells of claim 17.

19. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10.

20. 20. The CAR of claim 19, wherein the antigen-binding fragment comprises an scFv or a Fab.

21. 21. The CAR of claim 19 or 20, comprising a bispecific or dual-targeting CAR.

22. A cell comprising the CAR according to any one of claims 19 to 21.

23. 23. The cell of claim 22, comprising a T cell.

24. 24. The cell of claim 22 or 23, which further secretes an antibody or a fragment thereof.

25. 25. The cell of claim 24, wherein the secreted antibody comprises a monoclonal antibody.

26. 25. The cell of claim 24, wherein the secreted antibody comprises an immune checkpoint blockade antibody.

27. 25. The cell of claim 24, wherein the secreted antibody modulates the immune system of a subject.

28. A pharmaceutical composition comprising the cells of any one of claims 22 to 27 and a pharmaceutically acceptable excipient.

29. A nucleic acid encoding the CAR according to any one of claims 19 to 21.

30. An engineered T cell comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor is specific for a MUC1-SEA domain, and the antigen recognition domain of the CAR is a monoclonal antibody or an antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof is (a) a heavy chain variable region comprising: a CDR1 having the amino acid sequence of GFTFDDYA (SEQ ID NO: 2), a CDR2 having the amino acid sequence of ISWNSGSI (SEQ ID NO: 96), and a CDR3 having the amino acid sequence of AKDIGSGSYYNYYYGMDV (SEQ ID NO: 104); (b) a light chain variable region comprising a CDR1 having the amino acid sequence of SLRSYY (SEQ ID NO: 7), a CDR2 having the amino acid sequence of GKN, and a CDR3 having the amino acid sequence of NSRDRYGNSL (SEQ ID NO: 118); 1. An engineered T cell comprising:

31. 31. The engineered T cell of claim 30, wherein the CAR comprises an scFv or a Fab.

32. 32. The engineered T cell of claim 30 or 31, wherein the CAR comprises a bispecific CAR.

33. The nucleic acid A polypeptide, including an antibody or fragment thereof, capable of being secreted from said engineered cells.

33. The engineered T cell of any one of claims 30 to 32, further encoding:

34. 34. A pharmaceutical composition comprising the engineered T cells of any one of claims 30 to 33 and a pharmaceutically acceptable excipient.

35. 34. A pharmaceutical composition for treating a subject suffering from cancer, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or a cell according to any one of claims 30 to 33.

36. 36. The pharmaceutical composition of claim 35, wherein the cancer expresses MUC1, mesothelin, and / or other tumor-associated antigens.

37. 37. The pharmaceutical composition of claim 35 or 36, wherein the cancer comprises an epithelial cancer.

38. 38. The pharmaceutical composition of claim 37, wherein the epithelial cancer comprises breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, mouth cancer, esophageal cancer, small intestine and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer and skin cancer, squamous cell and basal cell carcinoma, prostate cancer, renal cell carcinoma, and other known cancers that affect epithelial cells throughout the body.

39. The pharmaceutical composition according to any one of claims 35 to 38, which is used in combination with a chemotherapeutic agent.

40. The pharmaceutical composition of any one of claims 35 to 39, wherein the subject is suffering from a MUC1-expressing cancer.

41. The pharmaceutical composition of claim 40, wherein the antibody or antigen-binding fragment thereof or the cell induces apoptosis of MUC1-expressing cancer cells.

42. A pharmaceutical composition for inducing apoptosis of cancer cells, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, or the CAR according to any one of claims 19 to 21; A pharmaceutical composition, wherein apoptosis of the cancer cells is induced by contacting the cancer cells with the antibody or antigen-binding fragment thereof, or the CAR.

43. The pharmaceutical composition of claim 42, wherein the cancer cells contain MUC1-SEA on their surface.

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