HERV-K antibody therapeutic drug

Abstract

The present invention provides therapeutic humanized anti-HERV-K antibodies, CARs, or bispecific T cell engagers (BiTEs) against CD3 and CDS, DNA-encoded BiTEs (DBiTEs), or fusions thereof consisting of antibody-drug conjugates (ADCs). The present invention also relates to peptides, proteins, nucleic acids, and cells for use in immunotherapy. In particular, the present invention relates to immunotherapy using cancer peptides bound to MHC molecules or peptides that can also be targets for antibodies and other binding molecules.

Description

[Technical Field]

[0001] The present invention relates generally to cancer antigens.

[0002] REFERENCE TO RELATED APPLICATIONS This patent matter is related to and claims priority to U.S. Provisional Patent Application No. 63 / 080,009, filed September 17, 2020. [Background technology]

[0003] Human endogenous retroviruses (HERVs) are known as genomic repeats that harbor numerous copies in the genome. As a result, approximately 8% of the human genome is of retroviral origin. See scientific reference 1 below. HERVs arose from thousands of ancient integration events that incorporated retroviral DNA into germline cells. 2 Normally, retroviruses lose their infectious potential due to the accumulation of mutations. Therefore, these genes are largely silent and not expressed in normal adult human tissues, except in pathological conditions such as cancer. Most biologically active HERVs are members of the HERV-K family. HERV-K possesses the complete sequence required for the expression of all elements required for replication-competent retroviruses (scientific references 3 and 4), yet remains silent in normal cells. However, in some situations, such as tumors, the inventors have reported that HERV-K expression is activated and its envelope (Env) protein can be detected at levels significantly higher in several different types of tumors than in normal tissues. See scientific references 5-23. This indicates that it is an excellent tumor-associated antigen and may be an ideal target for cancer immunotherapy because it is expressed in tumors and absent from normal tissues, minimizing off-target effects.

[0004] An important consideration in the development of cancer therapeutics is the expression profile of tumor-associated antigens. HERV-K is transcriptionally active in germ cell tumors (Scientific Reference 24), melanoma (Scientific Reference 25), breast cancer cell lines (T47D) (Scientific References 26-28), breast cancer tissues (Scientific References 15, 29), and ovarian cancer (Scientific Reference 13). The inventors specifically identified HERV proteins and sequences in cancer cell lines and patient tumors. The inventors observed expression of HERVs, particularly HERV-K sequences, in breast, lung, prostate, ovarian, colon, pancreatic, and other solid tumors. See Scientific References 11, 12, 16, 17, 20, 30-34. They also found that expression of HERV-K env transcripts in breast cancer is specifically associated with basal breast cancer, a particularly aggressive subtype. (20)

[0005] Several diagnostic agents can be used as companion diagnostics for patient selection. One strategy targets endogenous viral antigens found only on cancer cells and not on normal tissues. Viral RNA is released from these tumors, and both HERV-K RNA (env or gag) and anti-HERV-K antibodies have been found by our group to appear in the circulation of cancer patients. See scientific references 31-33, 35. These proteins of non-human origin can be utilized as ideal targets for cancer therapy and as companion diagnostics for therapeutic antibodies targeting HERV-K.

[0006] The significantly increased number of clinical trials of immunotherapies in multiple cancer types has fueled the search for effective immunotherapies for breast cancer. Improved understanding of the tumor microenvironment in breast cancer is crucial for the design of rational, efficient therapies. One problem that has limited the success of therapies against solid tumors is the lack of tumor antigens that are highly expressed in tumor cells but not in normal cells.

[0007] In our previous study, we demonstrated that HERV-K Env protein is commonly expressed on the surface of breast cancer cells. 30 Epithelial-mesenchymal transition (EMT) reduces CD4 or CD8 T cell infiltration in some tumors. 36 HERV-K expression has been demonstrated to induce EMT and increase cell motility. 37 Both of these factors favor tumor dissemination. Scientific publications 10, 33, and 37 provide strong evidence that HERV-K overexpression leads to cancer development and contributes to cancer progression. A chimeric antigen receptor (CAR) (K-CAR) specific for HERV-K Env protein was generated from an anti-HERV-K monoclonal antibody (mAb) (designated 6H5), and the anti-metastatic tumor efficacy of K-CAR therapy was demonstrated in breast cancer and melanoma. 33, 35 Importantly, downregulation of HERV-K and Ras expression was demonstrated in cancer cells treated with either K-CART cells or shRNAenv. See scientific references 10, 33, 38. Summary of the Invention [Means for solving the problem]

[0008] We found that checkpoint molecule levels in serum and tumor-infiltrating lymphocytes (TILs) were highly correlated with HERV-K antibody titers, particularly in patients with high-grade breast cancer (patients with invasive ductal carcinoma (IDC) or invasive breast cancer (IMC)). The phenotypic and functional characteristics of TILs in breast cancer are associated with HERV-K status, suggesting that the combination of checkpoint blockade and HERV-K antibody therapy may result in better killing efficacy.

[0009] The present invention provides therapeutic humanized anti-HERV-K antibodies or their fusions consisting of bispecific T cell engagers (BiTEs) against CD3 and CD8, DNA-encoded BiTEs (DBiTEs) or antibody-drug conjugates (ADCs).

[0010] In a first embodiment, the present invention provides cancer cells that overexpress HERV-K, which may be particularly good targets and models for the anti-HERV-K humanized antibodies and ADCs of the present invention, since more antibody can be bound per cell.

[0011] In a second embodiment, the present invention provides two humanized antibody clones (HUM1 and HUM2) produced from bacteria and a humanized antibody (hu6H5) produced from mammalian cells. Both clones can bind to antigens produced from recombinant HERV-K Env surface fusion protein (KSU) and lysates from MDA-MB-231 breast cancer cells. The mammalian cell-produced hu6H5 was compared with other forms of our anti-HERV-K antibody. hu6H5 has binding affinity for the HERV-K antigen similar to that of the murine antibody (m6H5), chimeric antibody (cAb), or humanized antibody (HUM1). The hu6H5 antibody induces cancer cells to undergo apoptosis, inhibits cancer cell proliferation, and kills cancer cells expressing the HERV-K antigen. Importantly, the hu6H5 antibody was demonstrated to reduce tumor survival in mouse MDA-MB-231 xenografts, and was particularly able to reduce cancer cell metastasis to the lungs and lymph nodes. Mice bearing human breast cancer tumors treated with these humanized antibodies had longer survival times compared to control mice that did not receive antibody treatment.

[0012] In a third embodiment, the present invention provides a HERV-K env gene produced from a breast cancer patient as an oncogene capable of inducing cancer cell proliferation, tumor growth, and metastasis to the lungs and lymph nodes. Cells expressing HERV-K showed reduced expression of genes associated with tumor suppression, including caspases 3 and 9, pRB, SIRT-1, and CIDEA, and increased expression of genes associated with tumorigenesis, including Ras, p-ERK, PP-38, and beta-catenin.

[0013] In a fourth embodiment, the present invention provides BiTEs directed against T cell CD3 or CD8 and the tumor-associated antigen HERV-K. The inventors generated BiTEs consisting of either CD3 or CD8 and an antibody targeting HERV-K (VL-VH 6H5scFv---VH-VLhuCD3 or CD8+c-myc+FLAG) or (VL-VH hu6H5scFv---VH-VLhuCD3 or huCD8+c-myc+FLAG). The FLAG-tag (DYKDDDDK) (SEQ ID NO: 39), a peptide recognized by the antibody, and the Myc-tag (EQKLISEEDL) (SEQ ID NO: 40), a short peptide recognized by the antibody.

[0014] In a fifth embodiment, the present invention provides T cells expressing a lentiviral CAR expression vector carrying a humanized or fully human HERV-K scFv.

[0015] [ka]

[0016] These T cells effectively lyse and kill tumor cells from several different cancers. Humanized K-CARs expressed from lentiviral vectors are pan-cancer CAR-Ts.

[0017] In a sixth embodiment, the present invention provides a humanized single-chain variable fragment (scFv) antibody. This antibody can bind to an antigen generated from a recombinant HERV-K Env surface fusion protein (KSU) and a lysate from MDA-MB-231 breast cancer cells. A CAR generated from this humanized scFv can be cloned into a lentiviral vector. This recombinant vector can be used in combination with therapies including, but not limited to, K-CAR T cells and checkpoint inhibitors, pro-inflammatory cytokines such as interleukin (IL)-12 and IL-18, oncolytic viruses, and kinase inhibitors. Kinase inhibitors include, but are not limited to, p-RSK and p-ERK.

[0018] In a seventh embodiment, the present invention provides HERV-K staining, which often overlaps with that of the serum tumor marker CK. HERV-K can be a CTC marker as well as a target for HERV-K antibody therapy.

[0019] In an eighth embodiment, the present invention provides HERV-K as a stem cell marker. Targeting HERV-K can slow down or prevent the growth of cancer stem cells, thereby blocking tumor progression. Targeting HERV-K with circulating therapeutic antibodies or other therapies can also kill CTCs and prevent the metastasis of these circulating cells to distant sites.

[0020] In a ninth embodiment, the present invention provides that forced overexpression of HERV-K using agents that induce expression of HERV-K through the innate immune response (e.g., poly I:C treatment) or LTR hypomethylation (such as with 5-Aza) induces cancer cells to increase production of targets that may make them more sensitive to targeted therapies, including targeted immunotherapy.

[0021] In a tenth embodiment, the present invention improves the in vivo enrichment technique (IVE: approximately 20-fold enhancement) in SCID / beige mice, enabling rapid expansion and B cell activation. This improved technique can produce a large number of antigen-specific plasmablasts. For cancer-bearing donors with higher antibody titers, the improved technique uses a protocol that uses humanized mice (HM) or human tumor mice (HTM) instead of SCID / beige mice. For normal donors without cancer and without memory B cells, the improved technique uses a modified protocol: mice are treated with a cytokine cocktail (days 1, 7, and 14) and boosted with antigen on days 14 and 21. Serum is collected from the mice and binding affinity is tested by ELISA. After an increase in antibody titer is detected, spleens are harvested, analyzed, and used to generate hybridomas. Higher antibody titers were detected in mice using the IVE protocol.

[0022] In an eleventh embodiment, the present invention provides a method for determining cells that not only produce antibodies but also bind to antigens and kill cancer cells, which can efficiently stimulate and expand CD40- B cells to high numbers with high purity (>90%) and induce their antibody secretion.

[0023] In a twelfth embodiment, the present invention provides a method in which after incubation of treated B cells, glass coverslips are washed, tagged with fluorescent anti-human IgG antibodies, and read using microengraving technology that reveals distinct spots corresponding to the secretion of antigen-specific antibodies by single B cells.

[0024] In a thirteenth embodiment, the present invention provides for the development of a platform for determining the binding kinetics and cell-cell interactions of all cells in a microwell slab.

[0025] In a fourteenth embodiment, the present invention remarkably provides significantly enhanced expression of six circulating immune checkpoint proteins in the plasma of breast cancer patients. The present invention also provides a significant decrease in immune checkpoint protein levels 6 or 18 months after surgery compared to before surgery. Importantly, a positive correlation is observed between soluble immune checkpoint protein molecule levels and HERV-K antibody titers induced by HERV-K expression in tumors. HERV-K antibody titers can affect immune checkpoint protein levels in breast cancer. Therefore, HERV-K expression can control the immune response of breast cancer patients.

[0026] In another aspect, these findings collectively indicate that the immunosuppressive domain (ISD) of HERV-K is an as yet unrecognized immune checkpoint on cancer cells, similar to the PD-L1 immune checkpoint. In a fifteenth embodiment, the present invention provides that blockade of the ISD of HERV-K with immune checkpoint inhibitors, including but not limited to monoclonal antibodies and drugs that target the ISD of HERV-K, is a cancer immunomodulator therapy that will allow T cells to continue operating, unrestricting the immune response to cancer as well as enhancing existing responses and promoting the elimination of cancer cells.

[0027] In a sixteenth embodiment, the present invention provides humanized and fully human (hTab) antibodies targeting HERV-K. These antibodies enhance the efficacy of checkpoint blockade antibody treatment. Effective combination cancer therapies include, but are not limited to, (a) HERV-K humanized or hTAb (1.5 mg / kg), (b) K-CAR, (c) K-BiTE, (d) HERV-K shRNA or CRISPR / Cas9 genome editing technology to knock down HERV-K gene expression, or (e) preventative or therapeutic HERV-K vaccines containing full-length and truncated HERV-K Env proteins and HERV-K Env peptides. Effective combination cancer therapies include, but are not limited to, full-length and truncated HERV-K Env proteins and HERV-K Env peptides in combination with (a) anti-ICP antibodies, (b) cancer chemotherapy, (c) 5-azacytidine, 5-aza-2'-deoxycytidine or other epigenetic modulating agents, such as DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi), (d) EMT inhibitors, (e) inhibitors of cell migration or invasion, (f) induction of S or G2 phase cell cycle arrest, (g) inhibitors of the PI3K / AKT / mTOR or MAPK / ERK signaling pathways, or (f) factors that induce signaling to HIF1α.

[0028] In a seventeenth embodiment, the present invention provides a humanized antibody targeting HERV-K that can be used for ADCs to deliver drugs into cancer cells and tumors.

[0029] In an eighteenth embodiment, the present invention provides an antibody targeting HERV-K that can be used for tumor imaging.

[0030] In a nineteenth embodiment, the present invention provides a novel CAR that uses the hu6H5 scFv.

[0031] In a twentieth embodiment, the present invention provides novel BITEs using hu6H5 scFv, including CD3 and CD8 BiTEs. [Brief explanation of the drawings]

[0032] [Figure 1] Figure 1 shows a Western blot used to detect the VH and VL chains of a humanized anti-HERV-K antibody on an SDS-PAGE gel under reducing conditions. The VH chain had a molecular weight of 49 KDa and the VL chain had a molecular weight of 23 KDa.

[0033] [Figure 2] In Figure 2, size exclusion chromatography (SEC) separation by size and / or molecular weight was further used to determine protein expression. Only two peaks were detected, with the concentration of peak 2 being greater than 99% of the combined total size of peaks 1 and 2.

[0034] [Figure 3] In Figure 3, ELISA was used to compare the binding of chimeric 6H5, HUM1 (produced from bacteria), and the novel hu6H5 (a novel humanized anti-HERV-K antibody produced from mammalian cells) to the HERV-K env target at the following dilutions: 1000, 1:1000; 2000, 1:2000; 4000, 1:4000; and 8000, 1:8000.

[0035] [Figure 4] In Figure 4, an apoptosis assay was used to determine the cytotoxicity of murine and humanized anti-HERV-K antibodies directed against cancer cells. Cancer cells containing MDA-MB-231-pLVXK(231K) (a breast cancer cell line transduced with a pLVX vector expressing the HERV-K env protein) or MDA-MB-231-pLVXC(231C) (the same breast cancer cell line transduced with a pLVX empty vector) were treated with either m6H5 or hu6H5 (1 or 10 µg per ml) for 4 or 16 hours. Annexin V and 7AAD were used to determine the percentage of apoptotic cells. Results after 4 hours of antibody treatment are shown here.

[0036] [Figure 5] In Figure 5, a live / dead cell viability assay was used to evaluate the induction of cell death after anti-HERV-K antibody treatment. MDA-MB-231 cells were seeded overnight in 24-well plates. Cells were treated with various antibodies (10 μg / ml) and incubated for 16 hours at 37°C in a cell culture incubator. Calcein Am (4 μl / 10 ml medium) and Eth-D1 (20 μl / 10 ml medium) were then added at 200 μl per well, and the cells were incubated for 30 minutes at room temperature. Eth-D1 penetrates cells with membrane damage and binds to nucleic acids in dead cells, producing red fluorescence. A co-stained live / dead viability assay was used to identify live cells (green; calcein Am) and presumed dead cells (red; Eth-D1). Human IgG or mouse IgG was used as a control. Cells lacking red fluorescence were observed after treatment with control human or mouse IgG. However, red fluorescent cells were observed in cells treated with either the humanized or mouse 6H5 anti-HERV-K antibody.

[0037] [Figure 6]In Figure 6, an MTS assay was used to determine the inhibition of proliferation of cells treated with hu6H5. Significantly reduced cell proliferation was observed in cells treated with either 6H5 antibody (human or mouse). The inhibition was more pronounced in 231K cells, which express high levels of HERV-K, than in 231C cells, which do not express higher levels of HERV-K.

[0038] [Figure 7] ADCC was used to determine the mechanism of antibody-induced cell killing in Figure 7. Greater ADCC lysis of cancer cells was observed with increasing percentages of PBMCs in cells treated with hu6H5 than with m6H5.

[0039] [Figure 8-1] In Figure 8, mice were inoculated with 231K or 231C cells (2 million cells by subcutaneous injection). The mice were then treated with the hu6H5 antibody (n=3; 4 mg / kg, twice a week). Tumor growth was monitored and measured three times a week, and mouse survival was examined. Treatment of mice with hu6H5 led to longer survival than treatment of a separate cohort of mice (n=4) with a control antibody. Shorter survival was observed in mice inoculated with 231K cells than in mice inoculated with 231C cells, indicating that overexpression of HERV-K in breast cancer cells shortens tumor-related survival. [Figure 8-2] Same as above.

[0040] [Figure 9-1]In Figure 9, tissues from the hu6H5 and no antibody treatment groups were stained with H&E, and the staining results are shown here at 2X, 4X, and 10X magnification. A reduction in tumor viability was demonstrated in mice inoculated with 231C cells treated with hu6H5 (20%; B4) compared with the same cells without antibody treatment (60%; B14; Figure 9A). A reduction in tumor viability was also demonstrated in mice inoculated with 231K cells treated with hu6H5 (45%; B1; Figure 9B) compared with the same cells without antibody treatment (using anti-Ki67 and anti-HERV-K mAb (6H5) (Figure 9C). A reduction in tumor viability was also demonstrated in mice incubated with 231C cells treated with hu6H5 (20%; lower panel) compared with the control (60%; upper panel). [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above.

[0041] [Figure 10-1] In Figure 10, metastasis to the lungs and lymph nodes was observed in mice inoculated with 231K cells. Metastasis to the lungs (Figures 10A and 10B) or lymph nodes (Figure 10C) was observed only in mice inoculated with 231K cells. Decreased tumor viability and increased tumor necrosis were detected in the lungs of mice inoculated with 231K cells and treated with hu6H5 (Figure 10B). Visible enlargement of lymph nodes was observed in mice inoculated with 231K but not in mice inoculated with 231C cells. Decreased tumor viability and increased tumor necrosis were detected in lymph nodes obtained from mice inoculated with 231K cells and treated with hu6H5 (KAB) (Figure 10C; B18; 40%, lower panel) compared with 231K cells without added antibody (KCON; upper panel) (B26>95%). These results indicate that HERV-K expression is a causative factor in tumor development, particularly metastasis to distant organ sites. Importantly, our humanized anti-HERV-K antibody can reduce tumor viability, increase tumor necrosis, and reduce metastasis to the lungs and lymph nodes. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0042] [Figure 11] Figure 11 shows CD3BiTE-mediated secretion of IFN-gamma from normal donor PBMCs in the presence of MDA-MB-231 luc cells. 5 x 10-3 cells / well were seeded in 96-well plates. PBMCs from ND number 230341 (positive control) and four normal donors were used as effector cells. The effector / tumor cell ratio was 10 / 1. 140 μg / ml of CD3BiTE was used. 72 hours after plate setup, supernatants were collected for IFN-gamma assay.

[0043] [Figure 12] Figure 12A shows images of viability (green) or death (red) of MCF-7 cells treated with PBMCs and K3Bi (0 ng / ml; upper panel) or K3Bi (100 ng / ml; lower panel) for 72 hours. Figure 12B shows significantly increased cancer cell death in the supernatants of effector:tumor cells (10:1) treated with 0 ng / ml or 100 ng / ml K3Bi + PBMCs for 72 hours, as demonstrated by LDH release assay. Figure 12C shows significantly increased IFN-γ secretion in three breast cancer cell lines treated with K3Bi (100 ng / ml) for 72 hours. Untreated cells, PBMCs alone, or BiTE alone were used as controls.

[0044] [Figure 13] In Figure 13, NOD / SCID / IL-2Rγ null (NSG) mice were inoculated with MDA-MB-231 HERV-K positive breast cancer cells on day 0 and administered PBMCs (red arrows) or BiTEs (black arrows) on the indicated days. Tumor volume was calculated throughout the study by measuring tumor volume using calipers.

[0045] [Figure 14]In Figure 14, the percentage of CAR-A / CAR-B transduced CD4 positive PBMCs stained with K10-labeled AF488 protein is higher than the percentage of naive T cells stained with K10-labeled AF488 protein.

[0046] [Figure 15] Figure 15 illustrates the microengraving process. In Figure 15A, enriched B cells were mixed with tumor cells and co-cultured for 2 to 16 hours in wells covered with HERV-K antigen-coated glass slides for immunoassay (top right). B cells capable of producing antibodies and killing tumor cells were recovered using CellCelector for RT-PCR (bottom right) and recloned to generate antibodies (bottom left). In Figure 15B, mammospheres (days 7 and 14) generated from tumor tissue were used as target cells. Autologous PBMCs were stimulated with the cocktail for 4 days to enrich for antibody-producing B cells. The B cells were then co-cultured with tumor target cells. A cover slide coated with HERV-K Env protein was incubated with the co-cultured cells. A co-stained live / dead viability assay was used to identify putative dead cells (red). EthD-1 penetrates cells with membrane damage and binds to nucleic acids in dead cells, producing red fluorescence. An ELISA assay on a cover slide in the same location in the same well was used to detect B cells producing antibodies capable of binding to the HERV-K Env protein (red square). In Figure 15C, B cells (red circle; left) that were HERV-K+ (green) and IgG+ (red) were picked by CellCelector. Cells are shown before (top right) and after (bottom right) picking.

[0047] [Figure 16]In Figure 16A, ELISPOT was used to detect IFN-γ-secreting splenocytes in mice immunized with HERV-K transmembrane (TM) proteins (mouse M1-M4) or PBS (M5-M6). See Figures 16B and 16C. ELISA was used to detect anti-HERV-K antibody titers in mice. Higher antibody titers were detected in mice treated with KSU Env protein, regardless of CpG (Figure 16B) or CDN (Figure 16C) status. In Figure 16D, anti-HERV-K antibody titers were detected by ELISA using anti-human IgG mAb in HTM models vaccinated with MDA-MB-231 (HTM1) or MDA-MB-468 (HTM2) and in HMs (1-2) immunized with HERV-K SU Env protein.

[0048] [Figure 17]Figure 17 shows a scheme of in vivo HERV-K-driven plasmablast differentiation in human-SCID chimeras. On day 0, 50 million PBMCs obtained from subjects premixed in vitro with HERV-K protein (100 μg) were intrasplenic injected into humanized mice. A cytokine cocktail (BAFF: 50 μg, IL-2: 50 ng, IL-6: 50 ng, and IL-21: 50 ng) was injected intraperitoneally on days 1, 4, and 7. HERV-K Env protein (100 μg) was boosted intraperitoneally on day 2. IgG+, CD38+, and HERV-K+ cells were selected by flow cytometry or microengraving platform for subsequent analysis. Half of the splenocytes were used to generate hybridomas with MFP-2 fusion partners. An ELISA assay was used to detect anti-ZIKV Env antibodies from hybridoma clones. Supernatant (100 μl) from each hybridoma clone was added and incubated for 1 hour. Goat anti-human IgG / A / M-HRP antibody was then added (1:4,000 dilution), followed by an additional 1 hour of incubation. High antibody titers were demonstrated in several hybridoma clones from donor 322336. Supernatant from an anti-flavivirus 4G2 mAb was used as a positive control (D1-4G2-4-15; ATCC HB-112).

[0049] [Figure 18]In Figure 18A, the percentages of CD33, CD3, and CD19 cells were quantified in huCD45+ cells obtained 4 weeks after inoculation of TNBC PDX cells and in MDA-MB-231 HTM models co-transplanted with CD34+ hematopoietic stem cells 7 weeks after inoculation. Figure 18B shows flow data from splenocytes 7 weeks after inoculation of MDA-MB-231 cells. In Figure 18C, immunofluorescence staining was used to detect HERV-K expression in MDA-MB-231 tumors obtained from HTM using anti-HERV-K mAb 6H5 (green). F-actin (red) was used as a control (two left panels). huCD3+ cells (green) were also detected in tumor tissue (two right panels). In Figure 18D, anti-human IgG mAb was used to detect anti-HERV-K antibody titers by ELISA in HTM models vaccinated with MDA-MB-231 (HTM1) or MDA-MB-468 (HTM2), and in HM1 and HM2 immunized with HERV-K SU Env protein.

[0050] [Figure 19]Figure 19 illustrates baseline immune status in relation to HERV-K status in breast cancer patients: combined HERV-K and immune checkpoint assays. Expression of soluble immune checkpoint proteins was examined by Luminex assay in breast cancer patients, including DCIS and high-grade breast cancer, compared with normal donors. Figure 19A shows a comparison of the expression of six ICPs in DCIS, high-grade breast cancer (aBC), and normal female donors. A notable finding was the significantly enhanced expression of six circulating ICPs in the plasma of breast cancer patients (Figure 19A). Figure 19B(a-c): An additional finding was a significant decrease in immune checkpoint protein levels in patients 6 months (B; time point 2) or 18 months (data not shown) after surgery compared with pre-surgery (time point 1). Importantly, a positive correlation was observed between the soluble ICP molecule levels in tumors and the HERV-K antibody titers induced by HERV-K expression (Figure 19C), suggesting that HERV-K antibody titers may affect ICP levels in breast cancer. Therefore, HERV-K expression can regulate the immune response in breast cancer patients.

[0051] [Figure 20-1]In Figure 20A, 6H5 conjugation with r-Gel was demonstrated using an immunoblot assay. In Figure 20B, delivery of recombinant gelonin toxin (r-Gel) was observed in HERV-K-positive cancer cells using an anti-HERV-K 6H5-rGel ADC. Surface and cytoplasmic expression of HERV-K was detected in DOV13 ovarian cancer cells using an anti-HERV-K 6H5 mAb. Furthermore, r-Gel expression was detected in DOV13 cells 4 hours after treatment using an anti-rGel antibody. In Figure 20C, HERV-K env protein (6H5; red) or rGel signal (green) was detected in SKBr3, MCF-7, and MDA-MB-231 breast cancer cells 1 hour after internalization. Yellow-orange coloring indicates colocalization of HERV-K env protein and rGel toxin in the target cytoplasm (right panel). The antitumor effects were compared in mice inoculated with MDA-MB-231 cells treated with 6H5 (p=0.0052) and 6H5-r-Gel (p<0.0001) compared to mice treated with control IgG (Figure 20D). [Figure 20-2] Same as above.

[0052] [Figure 21-1] In Figure 21, gold nanoparticle (GNP) delivery was demonstrated in various HERV-K-positive breast cancer cell lines in vitro and in vivo. GNPs (dark dots) were detected in MDAMB231 cells in vitro using TEM after 2 hours of incubation with naked GNPs (Figure 21A) or 6H5-GNPs (Figure 21B). GNPs were detected in MDAMB231 tumors (Figure 21C) or SKBr3 tumors (Figure 21D) 24 hours after intravenous injection of 6H5-GNPs or 6H5scFv-GNPs in the tail vein of mice using a silver-enhanced assay. (E / F) GNPs (white arrows) were detected in MDAMB231 cells in tumors isolated from mice by TEM 24 hours after intravenous injection of 6H5-GNPs. HERV viral particles (green arrows) were observed adjacent to tumor cells. [Figure 21-2] Same as above.

[0053] [Figure 22] In FIG. 22, in vivo imaging using the Nuance system detected higher densities of 6H5 mAb in tumor nodules from mice 24 hours after intravenous injection with 6H5-Alexa647 (red).

[0054] [Figure 23] In Figure 23, mice were immunized with five MAPS identified from BC patient serum samples, and the affinity of antibodies generated in three mice to various HERV viral proteins, including HERV-K SU envelope protein (K10G15), ERV3 (E3G4), Rec, Np9, and HERV-K TM envelope protein, was examined. Anti-HERV-KSU antibodies were demonstrated in the mouse serum, and the antibody sequences were generated from hybridoma cells generated from mouse number 2. DETAILED DESCRIPTION OF THE INVENTION

[0055] usefulness The present specification provides a method for producing a humanized anti-HERV-K antibody. The anti-tumor effect of hu6H5 was demonstrated in vitro and in vivo.

[0056] The present invention provides methods for treating patients suffering from cancer. In a twentieth embodiment, the present invention provides a method for treating cancer, comprising administering a therapeutic humanized anti-HERV-K antibody or a fusion thereof consisting of a CAR, BiTE, or ADC, or a cancer vaccine, optionally combined with one or more immune checkpoint blockade agents. Each of these therapeutic agents individually targets the immune system. In a twenty-first embodiment, the method of the present invention inhibits metastasis. In a twenty-second embodiment, the method of the present invention reduces tumor size. In a twenty-third embodiment, the method of the present invention inhibits tumor cell growth. In a twenty-fourth embodiment, the method of the present invention detects cancer and cancer metastasis.

[0057] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are listed below. Unless otherwise stated or implied from the context, these terms and phrases have the following meanings. These definitions are intended to aid in the description of particular embodiments and are not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by those skilled in the art of molecular biology. In the event of any apparent discrepancy between the meaning of a term and the definition provided herein, the meaning provided herein shall prevail.

[0058] "5-Aza" has the biotechnology art-recognized meaning of 5-azacytidine.

[0059] "6H5" has the biotechnology art-recognized meaning of a murine anti-HERV-K monoclonal antibody developed in the laboratory of the present inventors.

[0060] "About" is understood by those skilled in the art of molecular biology and will vary to some extent depending on the context in which it is used. If there are uses of the term that are not clear to those skilled in the art of molecular biology given the context in which it is used, "about" will mean up to plus or minus 10% of the value.

[0061] "Antibody-drug conjugate (ADC)" has the art-recognized meaning in biotechnology of a highly potent biological drug constructed by attaching a small molecule anticancer drug or another therapeutic agent to an antibody using either a permanent or labile linker. The antibody targets a specific antigen found only on target cells.

[0062] The term "B7 family" has the technically recognized meaning of an inhibitory ligand with an undefined receptor in biotechnology. The B7 family includes B7-H3 and B7-H4, both of which are upregulated in tumor cells and tumor-infiltrating cells. The complete hB7-H3 and hB7-H4 sequences can be found under GenBank accession numbers Q5ZPR3 and AAZ17406, respectively.

[0063] "BiTE" has its art-recognized meaning in biotechnology of a bispecific T cell engager. BiTE refers to a recombinant bispecific protein with two linked scFvs derived from two different antibodies, one targeting a cell surface molecule on a T cell (e.g., CD3ε) and the other targeting an antigen on the surface of a malignant cell. The two scFvs are linked together by a short flexible linker. The term DNA-encoded BiTE (DBiTE) includes any DNA plasmid encoding a BiTE that can be expressed in vivo.

[0064] "Cancer antigen" or "tumor antigen" has the art-recognized meaning in biotechnology of (i) a tumor-specific antigen, (ii) a tumor-associated antigen, (iii) a cell expressing a tumor-specific antigen, (iv) a cell expressing a tumor-associated antigen, (v) an embryonic antigen on a tumor, (vi) an autologous tumor cell, (vii) a tumor-specific membrane antigen, (viii) a tumor-associated membrane antigen, (ix) a growth factor receptor, (x) a growth factor ligand, and (xi) any other type of antigen or antigen-presenting cell or material associated with cancer.

[0065] "Combination therapy" encompasses the administration of each agent or therapy in a sequential manner in a regimen that provides the beneficial effect of the combination, as well as the co-administration of these agents or therapies in a substantially simultaneous manner, e.g., in a single capsule having a fixed ratio of the active agents or in multiple, separate capsules for each agent. Combination therapy also includes combinations in which the individual elements can be administered at separate times and / or by different routes, but which act in combination to provide a beneficial effect through the simultaneous action or pharmacokinetic and pharmacodynamic effects of each agent or tumor treatment approach of the combination therapy.

[0066] "CTL" has the art-recognized meaning of cytolytic or cytotoxic T cell biotechnology.

[0067] "Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4)" is a T-cell surface molecule and a member of the immunoglobulin superfamily. This protein downregulates the immune system by binding to CD80 and CD86. As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4 and analogs that share at least one common epitope with hCTLA-4. The complete hCTLA-4 sequence can be found under GenBank accession number P16410.

[0068] "Derived from" a designated polypeptide or protein has its art-recognized meaning in biotechnology of the polypeptide's origin. Preferably, a polypeptide or amino acid sequence derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, where the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or is otherwise identifiable as having its origin in the sequence by one skilled in the art of molecular biology. A polypeptide derived from another peptide may have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues substituted with another amino acid residue, or one or more amino acid residues inserted or deleted. A polypeptide may include an amino acid sequence that does not occur in nature. Such variants necessarily have less than 100% sequence identity or similarity to the starting molecule. In some embodiments, the peptide is encoded by a nucleotide sequence. The nucleotide sequences of the present invention may be useful for several applications, including cloning, gene therapy, protein expression and purification, mutagenesis, DNA vaccination of a host in need thereof, antibody generation for passive immunization, PCR, primer and probe generation, and the like.

[0069] "Effector cell" has the art-recognized meaning in biotechnology of an immune cell involved in the effector aspect of an immune response, as opposed to the cognitive and activation aspects of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells (CTLs)), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express specific Fc receptors (FcRs) and perform specific immune functions.

[0070] "Epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents. Epitopes can include amino acid residues directly involved in binding (also called immunodominant components of the epitope) and other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked by a specific antigen-binding peptide (in other words, amino acid residues within the footprint of the specific antigen-binding peptide).

[0071] "FACS" refers to fluorescence activated cell sorter.

[0072] "HERV" has its biotechnology art-recognized meaning of human endogenous retrovirus, and "HERV-K" has its biotechnology art-recognized meaning of the HERV-K family of endogenous retroviruses. "Human endogenous retroviruses" (HERVs) are retroviruses that exist in the form of proviral DNA integrated into the genome of all normal cells and are transmitted by Mendelian inheritance patterns. "HERV-X" (where "X" is an English letter) has its biotechnology art-recognized meaning of other families of HERVs. "Env" has its biotechnology art-recognized meaning of viral envelope protein. "KSU" has its biotechnology art-recognized meaning of HERV-K envelope surface fusion protein, and "KTM" has its biotechnology art-recognized meaning of HERV-K Env transmembrane protein. "env" has its biotechnology art-recognized meaning of viral envelope RNA. pLVXK has its biotechnology art-recognized meaning of HERV-K expression vector. The term MDA-MB-231 pLVXK or 231-K refers to MDA-MB-231 cells transduced with pLVXK. pLVXC has its art-recognized meaning in biotechnology for the control expression vector only. The term MDA-MB-231 pLVXC or 231-C refers to MDA-MB-231 cells transduced with pLVXC.HERV-K is expressed in numerous tumor types, including, but not limited to, melanoma (Muster et al., 2003; Buscher et al., 2005; Li et al., 2010; Reiche et al., 2010; Serafino et al., 2009), breast cancer (Patience et al., 1996; Wang-Johanning et al., 2003; Seifarth et al., 1995), ovarian cancer (Wang-Johanning et al., 2007), lymphoma (Contreras-Galindo et al., 2008), and teratocarcinoma (Bieda et al., 2001; Lower et al., 1993). Additionally, infected cells, including those infected by HIV (Jones et al., 2012), also express HERV-K. This offers an attractive opportunity to treat a variety of cancers and infectious diseases using a single CAR design that targets HERV-K.

[0073] "HM" has its art-recognized meaning in biotechnology of humanized mice, and "HTM" has its art-recognized meaning in biotechnology of human tumor mice.

[0074] "hTAb" has the biotechnology art-recognized meaning of fully human tumor antibody.

[0075] "Human endogenous retrovirus-K," "HERV-K," "HERV," "human endogenous retrovirus," "endogenous retrovirus," and "ERV" include any variant, isoform, and species homolog of an endogenous retrovirus that is naturally expressed by a cell or expressed in a cell transfected with the genes of the endogenous retrovirus.

[0076] "ICP" has its art-recognized meaning of immune checkpoint biotechnology.

[0077] "IHC" has its art-recognized meaning in the biotechnology field of immunohistochemistry.

[0078] "ILC" has its biotechnology art-recognized meaning of invasive lobular carcinoma. "DCIS" has its biotechnology art-recognized meaning of ductal carcinoma in situ. "IDC" has its biotechnology art-recognized meaning of invasive ductal carcinoma.

[0079] "Immune cells" are cells of hematopoietic origin that play a role in the immune response. Immune cells include lymphocytes (e.g., B cells and T cells), natural killer cells, and myeloid cells (e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes).

[0080] "Immune checkpoint blocker" has its art-recognized meaning in biotechnology of a molecule that fully or partially reduces, inhibits, interferes with, or modulates one or more checkpoint proteins. In some embodiments, an immune checkpoint blocker interferes with inhibitory signals associated with an immune checkpoint. In some embodiments, an immune checkpoint blocker is an antibody or fragment thereof that disrupts inhibitory signaling associated with an immune checkpoint. In some embodiments, an immune checkpoint blocker is a small molecule that disrupts inhibitory signaling. In some embodiments, an immune checkpoint blocker is an antibody, fragment thereof, or antibody mimetic that interferes with the interaction between checkpoint blocker proteins, e.g., an antibody or fragment thereof that interferes with the interaction between PD-1 and PD-L1. In some embodiments, an immune checkpoint blocker is an antibody or fragment thereof that interferes with the interaction between CTLA-4 and CD80 or CD86. In some embodiments, an immune checkpoint blocker is an antibody or fragment thereof that interferes with the interaction between LAG3 and its ligand or between TIM-3 and its ligand. The checkpoint blocker may also be in the form of a soluble form of the molecule (or a variant thereof) itself, for example, soluble PD-L1 or a PD-L1 fusion.

[0081] "Immune checkpoint" has its biotechnology art-recognized meaning of costimulatory and inhibitory signals that regulate the amplitude and quality of T cell receptor recognition of antigen. In some embodiments, the immune checkpoint is an inhibitory signal. In some embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1. In some embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86, which displaces CD28 binding. In some embodiments, the inhibitory signal is the interaction between LAG3 and an MHC class II molecule. In some embodiments, the inhibitory signal is the interaction between TIM3 and galectin-9.

[0082] "In vivo" has its art-recognized meaning in biotechnology of a process occurring in a living organism. The terms "mammal" or "subject" or "patient," as used herein, include both humans and non-humans, including, but not limited to, humans, non-human primates, dogs, cats, rodents, cattle, horses, and pigs.

[0083] "Inhibiting growth" (e.g., with reference to cells such as tumor cells) is intended to include any measurable decrease in cell growth, e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% inhibition of growth of a cell culture when contacted with a HERV-K specific therapeutic agent, compared to the growth of the same cells not contacted with the HERV-K specific therapeutic agent. Such a decrease in cell growth can occur through a variety of mechanisms exerted by the anti-HERV-K agent either individually or in combination, e.g., apoptosis.

[0084] "ISD" has the biotechnology art-recognized meaning of immunosuppressive domain.

[0085] The terms "K-CAR" or "HERV-Kenv CAR" have their biotechnology art-recognized meaning of a HERV-K envelope gene (surface or transmembrane) chimeric antigen receptor (CAR) genetic construct. The term "HERV-Kenv CAR-T cell" or "K-CAR-T cell" have their biotechnology art-recognized meaning of a T cell transduced with a K-CAR or HERV-Kenv CAR lentiviral or Sleeping Beauty expression system.

[0086] "KD" normally has its biotechnology art-recognized meaning of knockdown by shRNA.

[0087] "Linked," "fused," or "fusion" are used interchangeably. These terms refer to joining two or more elements or components or domains together by any means, including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional cross-linkers) are known in the art.

[0088] "Linker" or "linker domain" has its biotechnology art-recognized meaning of a sequence that connects two or more domains in a linear sequence (e.g., a humanized antibody targeting HERV-K and an antibody targeting a T-cell protein). Constructs suitable for use in the methods disclosed herein can use one or more "linker domains," e.g., polypeptide linkers. "Polypeptide linker" has its biotechnology art-recognized meaning of a peptide or polypeptide sequence (e.g., a synthetic peptide or polypeptide sequence) that connects two or more domains in the amino acid sequence of a linear polypeptide chain. Such polypeptide linkers can provide flexibility to the polypeptide molecule. Polypeptide linkers can be used to connect (e.g., genetically fuse) one or more Fc domains and / or drugs.

[0089] "Lymphocyte activation gene-3 (LAG3)" is an inhibitory receptor associated with the inhibition of lymphocyte activity by binding to MHC class II molecules. This receptor enhances Treg cell function and inhibits CD8+ effector T cell function. "LAG3," as used herein, includes human LAG3 (hLAG3), variants, isoforms, and species homologs of hLAG3 and analogs sharing at least one common epitope. The complete hLAG3 sequence can be found under GenBank accession number P18627.

[0090] "Mammosphere" has its art-recognized meaning in biotechnology of breast or mammary gland cells cultured under non-adherent, non-differentiating conditions to form discrete clusters of cells.

[0091] "Nucleic acid" has the art-recognized meaning of deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, it encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. See Batzer et al., Nucleic Acid Res., 19, 5081 (1991); Ohtsuka et al., Biol. Chem., 260, 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes, 8, 91-98 (1994). For arginine and leucine, modifications at the second base can also be conservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by gene.

[0092] "PBMC" has its art-recognized meaning in biotechnology of peripheral blood mononuclear cells.

[0093] "PDX" has its art-recognized meaning in biotechnology of a patient-derived xenograft. PDXs are typically generated by implanting human tumor cells or tumor tissue into immunocompromised mouse models of human cancer.

[0094] "Percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have a specified percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN, or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, "percent identity" can be over a region of the sequences being compared, such as a functional domain, or over the entire length of the two sequences to be compared. For sequence comparison, one sequence usually serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, and sequence algorithm program parameters are designated, if necessary. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math., 2, 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol., 48, 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci., USA, 85, 2444 (1988), by computerized implementations of these algorithms (GAP, BESHERV-KIT, FASTA and HERV-KASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin, USA), or by visual inspection.One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215, 403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.

[0095] "Pharmaceutically acceptable" generally means compounds, materials and compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs and / or body fluids of human beings and animals without excessive toxicity, irritation, allergic response or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0096] "Programmed death-ligand-1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1 and analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank accession number Q9NZQ7.

[0097] The "programmed death-1 (PD-1)" receptor has the biotechnology art-recognized meaning of an immunoinhibitory receptor belonging to the CD28 family. PD-1 is expressed primarily on pre-activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. "PD-1," as used herein, includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1 and analogs that share at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank accession number AAC51773.

[0098] A "recombinant host cell" (or simply "host cell") has the art-recognized meaning in biotechnology of a cell into which an expression vector has been introduced. Such term refers not only to the particular subject cell but also to the progeny of such a cell. Because some modifications may occur in subsequent generations, either due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of "host cell" as used herein. Recombinant host cells include, for example, transfectomas, e.g., CHO cells, HEK293 cells, NS / 0 cells, and lymphoid cells.

[0099] "scFv" has the biotechnology art-recognized meaning of single-chain variable fragment.

[0100] "SU" has its art-recognized meaning in biotechnology of a HERV-K surface protein.

[0101] By "amount sufficient" or "an amount sufficient to" is meant an amount sufficient to produce a desired effect, for example, an amount sufficient to reduce the size of a tumor.

[0102] "Synergism" or "synergistic," in reference to the effect produced by two or more individual components, has its recognized meaning in the art of biotechnology, the phenomenon whereby the total effect produced by those components, when utilized in combination, is greater than the sum of the individual effects of each component acting alone.

[0103] "T-cell membrane protein-3 (TIM3)" is an inhibitory receptor involved in inhibiting lymphocyte activity by inhibiting TH1 cell responses. Its ligand is galectin 9, which is upregulated in various types of cancer. As used herein, "TIM3" includes human TIM3 (hTIM3), variants, isoforms, and species homologs of hTIM3 and analogs that share at least one common epitope. The complete hTIM3 sequence can be found under GenBank accession number Q8TDQo.

[0104] "T cell" has its art-recognized meaning of CD4+ T cell or CD8+ T cell biotechnology. The term T cell encompasses TH1 cells, TH2 cells, and TH17 cells.

[0105] A "therapeutically effective amount" is an amount that is effective in ameliorating symptoms of a disease. A therapeutically effective amount can be a "prophylactically effective amount" since prevention can be a therapy being considered.

[0106] "TM" has its art-recognized meaning in biotechnology for HERV-K transmembrane protein.

[0107] "TNBC" has its art-recognized meaning of triple-negative breast cancer biotechnology.

[0108] "Transgenic non-human animal" has its art-recognized meaning in biotechnology of a non-human animal having a genome that contains one or more human heavy and / or light chain transgenes or transchromosomes (integrated or not integrated into the animal's native genomic DNA) and is capable of expressing fully human antibodies. For example, a transgenic mouse may have a human light chain transgene and either a human heavy chain transgene or a human heavy chain transchromosome, such that the mouse produces human anti-HERV-K antibodies upon immunization with HERV-K antigen and / or cells expressing HERV-K. The human heavy chain transgene may be integrated into the chromosomal DNA of the mouse, as in the case of a transgenic mouse, e.g., a HuMAb mouse, or the human heavy chain transgene may be maintained extrachromosomally, as in the case of a transchromosomal KM mouse as described in WO 02 / 43478. Such transgenic and transchromosomal mice (collectively referred to herein as "transgenic mice") can generate multiple isotypes of human mAbs (such as IgG, IgA, IgM, IgD, or IgE) against a given antigen by undergoing VDJ recombination and isotype switching. Transgenic non-human animals can also be used for the production of antibodies against specific antigens by introducing genes encoding such specific antibodies, e.g., by operably linking them to genes that are expressed in the animal's milk.

[0109] "Treatment" means the administration of an effective amount of a therapeutically active compound of the present invention to relieve, ameliorate, arrest, or eradicate (cure) the symptoms or disease state.

[0110] The term "vector" has its biotechnology-recognized meaning of a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which has its biotechnology-recognized meaning of a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Upon introduction into a host cell, other vectors (such as non-episomal mammalian vectors) can be integrated into the genome of the host cell, thereby being replicated along with the host genome. Additionally, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Because the plasmid is the most common form of vector, the terms "plasmid" and "vector" are used interchangeably herein. However, the invention is intended to include such other forms of expression vectors, such as viral vectors, including replication defective retroviruses, adenoviruses and adeno-associated viruses, which serve equivalent functions.

[0111] Over the past 15 years, the development of cancer therapeutic antibodies, such as Herceptin® (trastuzumab), Avastin® (bevacizumab), Erbitux® (cetuximab), and others, has saved tens of thousands of lives worldwide. In particular, treatment of HER2-positive metastatic breast or ovarian cancer with trastuzumab has dramatically altered patient outcomes. 40 Antibody therapeutics offer distinct advances over small molecule drugs: (i) a defined mechanism of action, (ii) greater specificity and fewer off-target effects, and (iii) a predictable safety and toxicological profile. 41, 42 Currently, >200 antibody therapeutics are in clinical trials in the United States. As extensive studies using anti-Her2 and anti-EGFR monoclonals demonstrate, only a few of the thousands of antibodies identified based on their ability to bind to their molecular targets with high affinity exhibit the properties necessary for clinically effective cancer cell killing. 41 The efficacy of therapeutic antibodies is primarily due to their ability to induce potent tumor cytotoxicity by direct induction of apoptosis in target cells or through effector-mediated functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0112] The primary methodologies for antibody isolation are (i) in vitro screening of libraries from immunized animals or synthetic libraries using phage or microbial display43-45, and (ii) isolation of antibodies after B cell immortalization or cloning46-48. These methodologies suffer from one or both of the following drawbacks, severely limiting the number of unique antibodies that can be isolated: (i) the need for extensive screening to isolate even a few high-affinity antibodies and (ii) the immune response to these antibodies when injected into humans. Therefore, regardless of the methodology used for screening / isolation of therapeutic monoclonal antibodies (mAbs), the speed of transition from discovery to the clinic is inefficient and laborious47,49.

[0113] One advancement that has accelerated the approval of therapeutic mAbs has been the generation of humanized antibodies by complementarity-determining region (CDR) grafting technology. See scientific reference 10. In CDR grafting, non-human antibody CDR sequences are translated into human framework sequences to maintain target specificity.

[0114] Humanized antibodies and antibody drug conjugate (ADC) pharmaceutical compositions Cancer cells that overexpress HERV-K may be particularly good targets for the anti-HERV-K humanized antibodies and ADCs of the present invention because more antibody may be bound per cell. Thus, in a 25th embodiment, cancer patients to be treated with the anti-HERV-K humanized antibodies or ADCs of the present invention are patients diagnosed with HERV-K overexpression in their tumor cells, such as breast cancer, ovarian cancer, pancreatic cancer, lung cancer, or colorectal cancer patients.

[0115] Upon purification of the anti-HERV-K humanized antibodies or ADCs, they can be formulated into pharmaceutical compositions using well-known pharmaceutical carriers or excipients.

[0116] Pharmaceutical compositions can be formulated using pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed. (Mack Publishing Co., Easton, Pa., 1995).

[0117] Pharmaceutically acceptable carriers or diluents, as well as any other known adjuvants and excipients, must be suitable for the humanized antibody or ADC of the present invention and the selected mode of administration. The suitability of carriers and other components of pharmaceutical compositions is determined based on the lack of a significant negative effect on the desired biological properties of the selected compound of the present invention or pharmaceutical composition upon antigen binding (e.g., less than a substantial effect (10% or less relative inhibition, 5% or less relative inhibition, etc.)).

[0118] Pharmaceutical compositions of the present invention may also include diluents, fillers, salts, buffers, surfactants (e.g., non-ionic surfactants such as Tween-20 or Tween-80), stabilizers (e.g., sugars or non-protein amino acids), preservatives, tissue fixatives, solubilizing agents and / or other materials suitable for inclusion in a pharmaceutical composition.

[0119] The actual dosage level of the humanized antibody or ADC in the pharmaceutical compositions of the invention can be varied to obtain an amount of the humanized antibody or ADC that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration of the particular compound being used, the time of administration, rate of excretion, duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0120] Pharmaceutical compositions can be administered by any suitable route and mode. Suitable routes for administering the humanized antibodies or ADCs of the present invention are well known in the art and can be selected by those skilled in the art of molecular biology.

[0121] In a twenty-sixth embodiment, the pharmaceutical composition of the invention is administered parenterally.

[0122] The phrases "parenteral administration" and "parenterally administered" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include epithelial, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion.

[0123] In a twenty-seventh embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0124] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, absorption delaying agents, and the like that are physiologically compatible with the humanized antibodies or ADCs of the invention.

[0125] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solution, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical arts.

[0126] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is well known in the art.Except where any conventional media or agent is incompatible with the anti-HERV-K humanized antibody or ADC of the present invention, it is contemplated to use it in the pharmaceutical compositions of the present invention.

[0127] Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size of the dispersion, and by the use of surfactants.

[0128] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, e.g., ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, e.g., ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelators, e.g., citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0129] The pharmaceutical compositions of the present invention may also include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride in the composition.

[0130] The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersing agents, preservatives, or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The anti-HERV-K humanized antibodies or ADCs of the present invention can be prepared using carriers that will protect the compound from rapid release, for example, sustained-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, alone or in combination with waxes or other materials well known in the art of molecular biology. Methods for preparing such formulations are generally known to those skilled in the art of molecular biology. See, for example, *Sustained and Controlled Release Drug Delivery Systems*, J.R. Robinson, ed. (Marcel Dekker, Inc., New York, 1978).

[0131] In a twenty-eighth embodiment, the anti-HERV-K humanized antibody or ADC of the present invention can be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. 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 pharmaceutical compositions of the present invention is contemplated. Supplementary active compounds can also be incorporated into the composition.

[0132] Pharmaceutical compositions for injections are usually sterile and must be stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be an aqueous or non-aqueous solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained by using a coating such as lecithin to maintain the required particle size of the dispersion, and by using surfactants. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition agents that delay absorption, such as monostearate salts and gelatin. Sterile injectable solutions can be prepared by incorporating the required amount of anti-HERV-K humanized antibody or ADC into an appropriate solvent with, for example, one or a combination of ingredients as listed above, followed by sterile microfiltration. Generally, dispersions are prepared by incorporating the anti-HERV-K humanized antibody or ADC into a sterile vehicle containing a basic dispersion medium and the required other ingredients, for example, from those listed above. Examples of preparation methods for sterile powders for the preparation of sterile injectable solutions include vacuum drying and freeze-drying (lyophilization), which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0133] Sterile injectable solutions can be prepared by incorporating the required amount of anti-HERV-K humanized antibody or ADC into an appropriate solvent with one or a combination of ingredients as listed above, followed by sterile microfiltration, as needed. Generally, dispersions are prepared by incorporating the anti-HERV-K humanized antibody or ADC into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those listed above. Examples of preparation methods for sterile powders for the preparation of sterile injectable solutions include vacuum drying and freeze-drying (lyophilization), which yield a powder of the anti-HERV-K humanized antibody or ADC and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0134] The pharmaceutical compositions of the invention may contain an anti-HERV-K humanized antibody or ADC of the invention or a combination of anti-HERV-K humanized antibodies or ADCs of the invention.

[0135] The effective dosage and administration regimen of an anti-HERV-K humanized antibody or ADC varies depending on the disease or condition to be treated and can be determined by one skilled in the art of molecular biology. An exemplary, non-limiting range of a therapeutically effective amount of a compound of the present invention is about 0.1 to 100 mg / kg, for example, about 0.1 to 50 mg / kg, for example, about 0.1 to 20 mg / kg, for example, about 0.1 to 10 mg / kg, for example, about 0.5 to 5 mg / kg, for example, about 5 mg / kg, for example, about 4 mg / kg or about 3 mg / kg or about 2 mg / kg or about 1 mg / kg or about 0.5 mg / kg or about 0.3 mg / kg. An exemplary non-limiting range of a therapeutically effective amount of an anti-HERV-K humanized antibody or ADC of the invention is about 0.02 to 30 mg / kg, e.g., about 0.1 to 20 mg / kg or about 0.5 to 10 mg / kg or about 0.5 to 5 mg / kg, e.g., about 1 to 2 mg / kg, particularly of antibody 011, 098, 114 or 111 as disclosed herein.

[0136] A physician with ordinary skill in the art of molecular biology can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, a physician can start the dosage of the anti-HERV-K humanized antibody or ADC used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. A suitable daily dose of the composition of the present invention will be the amount of compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally vary depending on the factors described above. Administration can be intravenous, intramuscular, intraperitoneal, or subcutaneous, for example, administered proximal to the target site. If necessary, an effective daily dose of the pharmaceutical composition can be administered as two, three, four, five, six, or more subdoses administered separately at appropriate intervals throughout the day in unit dosage form, as appropriate. While the anti-HERV-K humanized antibody or ADC of the present invention can be administered alone, it is preferable to administer the anti-HERV-K humanized antibody or ADC as a pharmaceutical composition as described above.

[0137] In a 29th embodiment, the anti-HERV-K humanized antibody or ADC may be administered by infusion at a weekly dose of 10 to 1500 mg / m, e.g., 30 to 1500 mg / m, or e.g., 50 to 1000 mg / m, or e.g., 10 to 500 mg / m, or e.g., 100 to 300 mg / m. Such administration may be repeated, e.g., 1 to 8 times, e.g., 3 to 5 times. Administration may be by continuous infusion over a period of 2 to 24 hours, e.g., 2 to 12 hours.

[0138] In a thirtieth embodiment, the anti-HERV-K humanized antibody or ADC may be administered by infusion every three weeks at a dose of 30 to 1500 mg / m², e.g., 50 to 1000 mg / m² or 100 to 300 mg / m². Such administration may be repeated, for example, one to eight times, e.g., three to five times. Administration may be by continuous infusion over a period of 2 to 24 hours, e.g., 2 to 12 hours.

[0139] In a thirty-first embodiment, the anti-HERV-K humanized antibody or ADC may be administered by slow continuous infusion over an extended period of time, for example, greater than 24 hours, to reduce toxic side effects.

[0140] In a 32nd embodiment, the anti-HERV-K humanized antibody or ADC may be administered in a weekly dose of 50 mg to 2000 mg, e.g., 50 mg, 100 mg, 200 mg, 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg, or 2000 mg, up to 16 doses, e.g., 4 to 10 doses, e.g., 4 to 6 doses. Administration may be performed by continuous infusion over a period of 2 to 24 hours, e.g., 2 to 12 hours. Such a regimen may be repeated one or more times as needed, e.g., after 6 or 12 months. The dosage may be determined or adjusted by, for example, collecting a biological sample and measuring the amount of the anti-HERV-K humanized antibody or ADC of the present invention in the blood at the time of administration, by using an anti-idiotypic antibody targeting the antigen-binding region of the anti-HERV-K humanized antibody or ADC of the present invention.

[0141] In a thirty-third embodiment, the anti-HERV-K humanized antibody or ADC may be administered by maintenance therapy, such as once weekly for a period of six months or more.

[0142] In a thirty-fourth embodiment, the ADC may be administered by a regimen comprising one infusion of an ADC of the invention followed by an infusion of an anti-HERV-K antibody of the invention, for example, antibody 6H5hum.

[0143] Bispecific T cell engagers (BiTEs) In a thirty-fifth embodiment, provided herein is a method of treating HERV-K positive cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific antibody comprising two different antigen-binding regions, one with binding specificity for CD3 or CD8 and the other with binding specificity for HERV-K.

[0144] In a thirty-sixth embodiment, the invention relates to a bispecific antibody comprising a first single-chain human variable region that binds to HERV-K in tandem with a second single-chain human variable region that binds to the T-cell activating ligands CD3 or CD8, the first and second single-chain human variable regions being in amino to carboxy order, a linker sequence being interposed between each of said segments, and a spacer polypeptide connecting the first and second single-chain variable regions.

[0145] In a thirty-seventh embodiment of the method, the administering is intravenous or intraperitoneal.

[0146] In a thirty-eighth embodiment of the method, the bispecific binding molecule is not bound to T cells during said administering step.

[0147] In a thirty-ninth embodiment of the methods described herein, the method further comprises administering to the subject T cells. In certain embodiments, the T cells are not bound to the same molecule as the bispecific binding molecule.

[0148] In a fortieth embodiment, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of a bispecific binding molecule, a pharmaceutically acceptable carrier, and T cells. In a fortieth embodiment, T cells are bound to the bispecific binding molecule. In a fortieth embodiment, the binding of the T cells to the bispecific binding molecule is non-covalent. In a fortieth embodiment, the administering is performed in combination with T cell infusion into the subject for the treatment of HERV-K positive cancer. In a fortieth embodiment, the administering is performed after treatment of the patient with T cell infusion. In a fortieth embodiment, the T cells are autologous to the subject to whom they are administered. In a fortieth embodiment, the T cells are allogeneic to the subject to whom they are administered. In a fortieth embodiment, the T cells are human T cells.

[0149] In a 48th embodiment of the methods described herein, the subject is a human.

[0150] In a forty-ninth embodiment of the method, the bispecific binding molecule is contained in a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier.

[0151] In a 50th embodiment of the bispecific binding molecule, the bispecific binding molecule does not bind to Fc receptors in its soluble or cell-bound form. In some embodiments of the bispecific binding molecule, the heavy chain is mutated to destroy an N-linked glycosylation site. In a 51st embodiment of the bispecific binding molecule, the heavy chain has an amino acid substitution that replaces an asparagine, an N-linked glycosylation site, with an amino acid that does not function as a glycosylation site. In a 52nd embodiment of the bispecific binding molecule, the heavy chain is mutated to destroy a C1q binding site. In a 53rd embodiment, the bispecific binding molecule does not activate complement.

[0152] In a 54th embodiment of the bispecific binding molecule, the HERV-K positive cancer is breast cancer, ovarian cancer, prostate cancer, pancreatic cancer, melanoma, colorectal cancer, small cell lung cancer, non-small cell lung cancer or any other neoplastic tissue that expresses HERV-K. In a 55th embodiment, the HERV-K positive cancer is a primary tumor or a metastatic tumor, for example, a brain, bone or lung metastasis.

[0153] DNA-encoded bispecific T cell engager (DBiTE) Certain antibody therapies, including mAbs and bispecific T cell engagers (BiTEs), are important tools in cancer immunotherapy. BiTEs are a class of engineered bispecific monoclonal antibodies that have the potential to transform the landscape of cancer immunotherapy. BiTEs direct the host immune system, specifically the cytotoxic activity of T cells, against cancer cells. BiTEs have two binding domains: one domain binds to the targeted tumor (such as cells expressing HERV-K), and the other domain engages the immune system by directly binding to a molecule on T cells. This dual binding activity drives T cell activation directly at the tumor, resulting in killing and tumor destruction. DBiTEs share many of the advantages of bispecific monoclonal antibodies. Both are composed of engineered DNA sequences encoding two antibody fragments. The patient's own cells then manufacture functional BiTEs encoded by the delivered DBiTE sequence. This allows for the delivery of BiTEs and the administration of a combination of DBiTEs at once as a multi-pronged approach to treating resistant cancers. Synthetic DNA design of BiTE-like molecules involves engineering and encoding them into optimized synthetic plasmid DNA cassettes. DBiTEs are then injected locally into muscle, allowing muscle cells to convert the genetic instructions into proteins and deliver the molecules directly into the bloodstream in vivo to seek out and destroy tumors. See Perales-Puchalt et al., DNA-encoded bispecific T cell engagers and antibodies present long-term antitumor activity, JCI Insight, 4(8), e126086 (April 18, 2019). In preclinical studies, DBiTEs have demonstrated a unique profile compared to conventional BiTEs and overcome some of the technical challenges associated with their production. For further information, see PCT Patent Publications WO2016 / 054153 (The Wistar Institute of Anatomy and Biology) and WO2018 / 041827 (Psioxus Therapeutics Limited).

[0154] HERV-K CAR-T therapy Numerous formulations of CARs specific to target antigens have been developed. See, for example, International Patent Publication WO2014 / 186469 (Council, Texas System University). This specification provides a method for generating chimeric antigen receptor (CAR)-mediated T cells with the potential for long life in vivo, for example, to treat leukemia patients exhibiting minimal residual disease (MRD). Overall, this method describes a method by which soluble molecules, such as cytokines, can be fused to the cell surface to enhance therapeutic potential. The core of this method relies on co-modifying CAR cells with a human cytokine mutein of interleukin-15 (IL-15), hereafter referred to as mIL15. The mIL15 fusion protein consists of a codon-optimized cDNA sequence of IL-15 fused to the full-length IL15 receptor alpha via a flexible serine-glycine linker. This IL-15 mutein was designed in this manner to (i) restrict mIL15 expression to the surface of CAR T cells, limiting cytokine delivery to non-target in vivo environments and potentially improving their safety profile, as exogenous soluble cytokine administration led to toxicity, and (ii) present IL-15 in the context of IL-15Ra to mimic physiologically relevant qualitative signaling and to stabilize and recycle the IL15 / IL15Ra complex for longer cytokine half-life. T cells expressing mIL15 are able to sustain supportive cytokine signaling, which is important for their survival after infusion. mIL15 CAR T cells generated by non-viral Sleeping Beauty-based genetic modification and subsequent ex vivo expansion in a clinically applicable platform resulted in T cell infusion products with enhanced persistence after infusion in mouse models with high, low, or no tumor burden. Furthermore, mIL15 CAR T cells also demonstrated improved antitumor efficacy in both high and low tumor burden models. The hu6H5 scFv was used in a lentiviral vector to generate K-CAR.

[0155] Combination therapy Therapies herein rely on the binding of antibodies or fragments to surface antigens on HERV-K+ cancer cells in situ, stimulating an immune attack thereon, and can be used without modification. Alternatively, the methods can be performed using antibodies or binding fragments to which a cytotoxic agent is attached. Binding of the cytotoxic antibody or antibody-binding fragment to tumor cells inhibits cell growth or kills the cells.

[0156] Antibodies specific for the HERV-K env protein can be used in conjunction with other expressed HERV antigens. This may be particularly useful for immunotherapy and antibody treatment of diseases in which several different HERVs are expressed, such as HERV-E in prostate cancer, ERV3, HERV-E and HERV-K in ovarian cancer, and ERV3, HERV-H and HERV-W in other cancers.

[0157] Cytokines in the common gamma chain receptor family (γC) are key costimulatory molecules for T cells, critical for lymphoid function, survival, and proliferation. IL-15 possesses several desirable properties for adoptive therapy. IL-15 is a homeostatic cytokine that supports the survival of long-lived memory cytotoxic T cells, promotes the eradication of established tumors through the relief of functional suppression of tumor-resident cells, and inhibits activation-induced cell death (AICD). IL-15 is tissue-restricted and only observed under pathological conditions, with any level observed in serum or systemically. Unlike other γC cytokines that are secreted into the surrounding environment, IL-15 is transpresented to T cells by producing cells in the context of the IL-15 receptor alpha (IL-15Ra). This cytokine's unique delivery mechanism to T cells and other responsive cells is (i) highly targeted and localized, (ii) increases the stability and half-life of IL-15, and (iii) results in signaling that is qualitatively different from that achieved by soluble IL-15.

[0158] Pharmaceutical Composition This specification is also directed to pharmaceutical compositions comprising a therapy that specifically binds to a HERV-K env protein, together with a pharmaceutically acceptable carrier, excipient, or diluent. Such pharmaceutical compositions can be administered in any suitable manner, including parenterally, topically, orally, or topically (such as by aerosol or transdermally), or any combination thereof. A suitable regimen also includes an initial administration by intravenous bolus injection, followed by repeat doses at one or more intervals.

[0159] Pharmaceutical compositions of the compounds of the present disclosure are prepared for storage by mixing the peptide ligand containing the compound having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 18th ed., 1990) in the form of a lyophilized formulation or aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.

[0160] The compositions herein may also contain more than one active compound as needed for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other.Alternatively, or in addition, the compositions may contain cytotoxic agents, cytokines, growth inhibitory agents and / or cardioprotective agents.These molecules are suitably present in a combination and amount that is effective for the intended purpose.

[0161] The present invention is further illustrated by the following examples, which are not intended to limit the scope or content of the invention in any way. For example, the present disclosure provides: [Section 1] An isolated antibody that binds to human endogenous retrovirus-K (HERV-K), the isolated antibody comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR). [Section 2] The antibody of paragraph 1, comprising a humanized or human framework region. [Section 3] The antibody of item 1, which is a HERV-K antagonist. [Section 4] The antibody of paragraph 2 for use in reducing tumor growth. [Section 5] The antibody of paragraph 2 for use in reducing metastasis to the lungs, lymph nodes or other organs. [Section 6] An isolated nucleic acid comprising a nucleotide sequence encoding the HCVR, LCVR or combination thereof described in paragraph 1. [Section 7] An expression vector comprising the nucleic acid according to Item 6. [Section 8] A host cell transformed with the expression vector according to item 7. [Section 9] A method for treating cancer in a mammal, comprising administering an effective amount of the antibody of paragraph 2 to a mammal in need thereof. [Section 10] 10. The method of paragraph 9, wherein the antibody is conjugated to the cytotoxic drug, an auristatin, or a functional peptide analog or derivative thereof, via a linker. [Section 11] Item 10. The method of item 9, wherein the cancer is selected from the group consisting of melanoma, chronic lymphocytic leukemia, breast cancer, pancreatic cancer, head and neck cancer, ovarian cancer, cervical cancer, colorectal cancer, testicular cancer, gastric cancer, kidney cancer, endometrial cancer, uterine cancer, bladder cancer, prostate cancer, esophageal cancer, liver cancer and non-small cell lung cancer. [Section 12] The method of claim 9, wherein the antibody is a full-length antibody. [Section 13] The method of paragraph 9, wherein the antibody is a human monoclonal IgG1 or IgG4 antibody. [Section 14] Humanized antibodies for use in CAR T, CAR NK or BiTE assays. [Section 15] A humanized antibody for use in a CAR T, CAR NK or BiTE assay, where the assay is used to develop a CAR T, CAR NK or BiTE. [Section 16] Cancer cells that overexpress HERV-K for use as targets for the anti-HERV-K humanized antibodies and ADCs of the present invention. [Section 17] hu6H5 clones generated from bacteria (HUM1 and HUM2) or mammalian cells (FWJ1 and FWJ2). [Section 18] A BiTE directed against T cell CD3 or CD8 and a humanized scFv against the tumor-associated antigen HERV-K, the BiTE comprising an antibody that targets either CD3 or CD8 and HERV-K. [Section 19] T cells expressing a lentiviral CAR expression vector carrying a humanized or fully human HERV-K scFv. [Section 20] A humanized single-chain variable fragment (scFv) antibody capable of binding to an antigen generated from a recombinant HERV-K Env surface fusion protein (KSU) and a lysate from cancer cells expressing the HERV-K Env protein. [Section 21] A CAR generated from the humanized scFv described in paragraph 19. [Section 22] 1. An improved in vivo enrichment method for rapid expansion and activation of B cells for donors lacking memory B cells, comprising: (a) treating mice with a cytokine cocktail on days 1, 7, and 14; and (b) boosting the mice with antigen on days 14 and 21 A method comprising: [Section 23] Cells that produce antibodies that can bind to antigens and kill cancer cells. [Section 24] A method of blocking the immunosuppressive domain (ISD) of HERV-K using an immune checkpoint inhibitor. [Section 25] Item 43. The method of item 42, wherein the HERV-K immune checkpoint inhibitor is selected from the group consisting of monoclonal antibodies and drugs that target the HERV-K ISD. [Section 26] Humanized and fully human antibodies targeting HERV-K for use in enhancing checkpoint blockade antibody treatment efficacy. [Section 27] A method for generating antibodies from mice immunized with five multiple antigenic peptides (MAPS) generated from the HERV-K SU protein produced by a cancer patient. [Section 28] Methods for generating HERV-K CAR A:VH-VLhu6H5-CD8-CD28-4-1BB-CD3 Zeta. [Example]

[0162] [Example 1] [ka]

[0163] VH CAGGTGAAGCTGCAGCAGTCAGGACCTGACCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGCAAGGCGTCTGGTTACTCATTCACTGGCTACTACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGACGTGTTAATCCTAACAGTGGTGTACAAGCTA CAACCAGAAGTTCAAGGACAAGGCCATATTAACTGTAGACAAGTCATCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGAAAGGTAACTACTTCTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTC A( Sequence number 2)

[0164] >FWJ_VH QVKLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWVKQSHGKSLEWIGRVNPNSGGTSYNQKFKDKAILTVDKSSSTAYMELRSLTSEDSAVYYCARSKGNYFYAMDYWGQGTTVTVSS (SEQ ID NO: 3)

Chem.

Table 1

[0165] VL GACATCGAGCTCACTCAGTCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATATCCTGCAGAGCCAGTGAAAGTGTTGATAGTCATGGCACTAGTTTTATGCACTGGTACCAGCAGAAACCAGGACAGCCACCCAAATTCCTCATCTATCGTGCATCCAACCTAGAATCTGGGATCCCTGCCAGGTTCAGTGGCAGTGGGTCTAGGACAGACTTCACCCTCACCATTAATCCTGTGGAGACAGATGATGTTGCAATCTATTACTGTCAGCAAAGTAATGAGGATCCTCCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAA A( (SEQ ID NO: 4)

[0166] >FWJ_VL DIELTQSPASLAVSLGQRATISCRASESVDSHGTSFMHWYQQKPGQPPKFLIYRASNLESGIPARFSGSGSRTDFTLTINPVETDDVAIYYCQQSNEDPPTFGGGTKLEIK (SEQ ID NO: 5)

Table 2

Chem.

[0167] Design of humanized single-chain variable fragment (scFv) antibodies Antibody numbering scheme and CDR definitions: The antibody numbering server, part of the KabatMan database (http: / / www.bioinf.org.uk / ), was used to number all antibody sequences in this study according to the enhanced Chothia scheme. In this humanization study, we combined the enhanced Chothia numbering with the contact CDR definitions of the antibody sequences to place the CDRs of the antibody light and heavy chains at the following positions: H-CDR1 30-35, H-CDR2 47-58, H-CDR3 93-101, L-CDR1 30-36, L-CDR2 46-55, and L-CDR3 89-96.

[0168] Selection of human templates: To generate humanized scFv genes, the six complementarity-determining regions (CDRs) of mouse VH and VL were grafted onto selected human frameworks (FRs) that showed the highest amino acid sequence identity for the humanization of a given antibody. Human immunoglobulin germline sequences were used as the selected human FRs for the mouse FWJ antibody clone (Figure 1). Human immunoglobulin germline sequences showing the highest amino acid sequence similarity in the FRs between human and mouse FWJ VH and VL were independently identified using the V-quest server (http: / / www.imgt.org / IMGT_vquest) and the Ig-BLAST server (http: / / www.ncbi.nlm.nih.gov / igblast). The selected heavy chain VHIII and light KI chains were based on conserved germline sequences. Consensus human FRs were designed among the selected germline genes to graft the CDR residues of FWJ. Amino acid sequences in the FRs of mouse VH and VL that differed from the consensus human FRs were replaced with human residues, while preserving mouse residues at positions known as Vernier zone residues and chain packing residues.

[0169] >HUM1-FWJVH EVQLVESGGGLVQPGGSLRLSCKASGYSFTGYYMHWVRQAPGKGLEWIGRVNPNSGGTSYNQKFKDRATLSVDNSKNTAYLQMNSLRAEDTAVYYCARSKGNYFYAMDYWGQGTLVTVSS (SEQ ID NO: 6). The Z-score of the query sequence is 0.7.

[0170] >Hum2 FWJVH VH EVQLVESGGGLVQPGGSLKVSCKASGYSFTGYYMHWVRQASGKGLEWIGRVNPNSGGTSYNQKFKDRFTISRDKSISTLYLQMSSLRSEDTAVYYCARSKGNYFYAMDYWGQGTLVTVSS (SEQ ID NO: 7). The Z-score of the query sequence is 0.5.

[0171] >FWJ_VH QVKLQQSGPDLVKPGASVKISCKASGYSFTGYYMHWVKQSHGKSLEWIGRVNPNSGGTSYNQKFKDKAILTVDKSSSTAYMELRSLTSEDSAVYYCARSKGNYFYAMDYWGQGTTVTVSS (SEQ ID NO: 8). The Z-score of the query sequence is -1.7.

[0172] [Table 3]

[0173] [Table 4]

[0174] [Table 5]

[0175] >HUM1FWJVL DIQMTQSPSSLSASVGDRVTITCRASESVDSHGTSFMHWYQQKPGKAPKFLIYRASNLESGIPSRFSGSGSGTDFTLTISSVQPEDFAVYYCQQSNEDPPTFGGGTKVEIK (SEQ ID NO: 9). The Z-score of the query sequence is 0.1.

[0176] >HUM2FWJVL DIQMTQSPSSLSASVGDRVTISCRASESVDSHGTSFMHWYQQKPGKSPKFLIYRASNLESGIPSRFSGSGSGTDFTLTISSLQPEDFAIYYCQQSNEDPPTFGGGTKVEIK (SEQ ID NO: 10). The Z-score of the query sequence is 0.0.

[0177] >FWJ_VL DIELTQSPASLAVSLGQRATISCRASESVDSHGTSFMHWYQQKPGQPPKFLIYRASNLESGIPARFSGSGSRTDFTLTINPVETDDVAIYYCQQSNEDPPTFGGGTKLEIK (SEQ ID NO: 11). The Z-score of the query sequence is -1.0.

[0178] Final humanized version of the scFv gene Humanized scFv-1 EVQLVESGGGLVQPGGSLRLSCKASGYSFTGYYMHWVRQAPGKGLEWIGRVNPNSGGTSYNQKFKDRATLSVDNSKNTAYLQMNSLRAEDTAVYYCARSKGNYFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASESVDSHGTSFMHWYQQKPGKAPKFLIYRASNLESGIPSRFSGSGSGTDFTLTISSVQPEDFAVYYCQQSNEDPPTFGGGTKVEIK (SEQ ID NO: 12)

[0179] Humanized scFv-2 EVQLVESGGGLVQPGGSLKVSCKASGYSFTGYYMHWVRQASGKGLEWIGRVNPNSGGTSYNQKFKDRFTISRDKSISTLYLQMSSLRSEDTAVYYCARSKGNYFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTISCRASESVDSHGTSFMHWYQQKPGKSPKFLIYRASNLESGIPSRFSGSGSGTDFTLTISSLQPEDFAIYYCQQSNEDPPTFGGGTKVEIK (SEQ ID NO: 13)

[0180] Construction of scFvs and Testing of Biological Activity Against Human KV and 231 Antigens. Clones of the variable heavy and light chains of the FWJ_1 and FWJ_2 antibody genes were amplified and synthesized. The scFv-encoding genes were VH-linker-VL with a standard 20-amino acid linker (Gly4Ser)3GGGAR (SEQ ID NO: 14). The amplified genes were digested with BssHII and NheI restriction enzymes and inserted into a pET-based vector (PAB-myc) (Novagen, Madison, Wisconsin, USA) containing the pelB promoter for periplasmic protein expression, along with a C-terminal 6x histidine tag for purification by metal affinity chromatography, and transformed into the DH5α bacterial strain. The transformed clones were amplified overnight in LB with ampicillin broth. Plasmid DNA was prepared and sent for DNA sequencing. The correct sequence of the scFv plasmid was transformed into a T7 shuttle bacterial strain, and the transformed bacteria were used for soluble protein production in the periplasmic compartment.

[0181] FWJ_1 and FWJ_2_scFv_ Genes and Translated Protein Sequences: The diagram below depicts the heavy and light chains and linker arms of FWJ_1 and FWJ_2_scFv. In engineering the FWJ_1 and FWJ_2_scFv genes, two epitope tags were engineered onto the C-terminus: 1) a his tag to facilitate purification of the encoded scFv by nickel affinity chromatography; and 2) a myc tag to facilitate rapid immunochemical recognition of the expressed scFv.

[0182] Final humanized version of the scFv gene Humanized scFv-1 FWJ_humscFv-1 EVQLVESGGGLVQPGGSLRLSCKASGYSFTGYYMHWVRQAPGKGLEWIGRVNPNSGGTSYNQKFKDRATLSVDNSKNTAYLQMNSLRAEDTAVYYCARSKGNYFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASESVDSHGTSFMHWYQQKPGKAPKFLIYRASNLESGIPSRFSGSGSGTDFTLTISSVQPEDFAVYYCQQSNEDPPTFGGGTKVEIK (SEQ ID NO: 15)

[0183] [ka] [ka]

[0184] Humanized FWJ_humscFv-2 EVQLVESGGGLVQPGGSLKVSCKASGYSFTGYYMHWVRQASGKGLEWIGRVNPNSGGTSYNQKFKDRFTISRDKSISTLYLQMSSLRSEDTAVYYCARSKGNYFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTISCRASESVDSHGTSFMHWYQQKPGKSPKFLIYRASNLESGIPSRFSGSGSGTDFTLTISSLQPEDFAIYYCQQSNEDPPTFGGGTKVEIK (SEQ ID NO: 18)

[0185] [ka] [ka]

[0186] Induction of ScFv proteins in bacterial hosts: The FWJ_1 and FWJ_2 scFv clones were transformed into the T7 shuttle bacterial strain. T7 shuttle cells were grown in 1.4 L of 2xYT and ampicillin medium at 37°C to log-phage (OD600 = 0.5), induced with 0.3 mM IPTG, and grown for an additional 16 hours at 30°C. After induction, bacteria were harvested by centrifugation at 8000g for 15 minutes at 4°C, and the pellet was stored at -20°C for at least 2 hours. The frozen pellet was briefly thawed and suspended in 40 ml of lysis buffer (1 mg / ml lysozyme and EDTA-free protease inhibitor cocktail (Thermo Scientific, Waltham, MA, USA) in PBS). The lysis mixture was incubated on ice for 1 h, and then 10 mM MgCl2 and 1 μg / ml DNase I were added, and the mixture was incubated at 25°C for 20 min. The final lysis mixture was centrifuged at 12,000 g for 20 min, and the supernatant was collected. This supernatant was referred to as the periplasmic extract and was used for nickel column affinity chromatography.

[0187] Western blot analysis using FWJ_1 and FWJ_2 scFv proteins: Lysate Ag and KSU proteins were used as antigen targets in dot blot analysis. Two to five μg of Ag protein under non-reducing conditions and 1 μg of purified protein as a negative control were loaded onto a nitrocellulose membrane. The membrane was blocked with 3% nonfat milk in PBS for 3 hours at room temperature. The membrane was then incubated overnight at 4°C with periplasmic extracts of FWJ_1 and FWJ_2 scFv proteins. The membrane was washed three times with sodium phosphate-buffered saline (PBST) containing 0.05% Tween 20 buffer. The washed membrane was then incubated with anti-c-Myc mouse IgG for 1 hour at room temperature to identify the location of the antigen bound by the scFv. After washing with PBST, the membrane was incubated with goat anti-mouse IgG (H+L) HRP conjugate diluted in PBS (1:3000 v / v) for 1 h at room temperature, and specific immunoreactive bands were visualized using a mixture of TMB substrates.

[0188] We identified the anti-HERV-K mAb 6H5 heavy chain CDRs (H-CDR1 30-35, H-CDR2 47-58, H-CDR3 93-101) and light chain CDRs (L-CDR1 30-36, L-CDR2 46-55, and L-CDR3 89-96) and grafted them onto selected human frameworks (FRs) to optimize the humanization of a given antibody. Human immunoglobulin germline sequences showing the highest amino acid sequence similarity in FRs between human and mouse VH and VL were independently identified from the V-quest (http: / / www.imgt.org / IMGT_vquest) and Ig-BLAST (http: / / www.ncbi.nlm.nih.gov / igblast) servers. Amino acid sequences in the mouse VH and VL FRs that differed from the consensus human FRs were replaced with human residues, while preserving murine residues at positions known as Vernier zone residues and chain packing residues. VH and VL chain clones of candidate humanized antibody genes were amplified and synthesized. The scFv-encoding gene, comprising the VH-linker-VL with the standard 20-amino acid linker (Gly4Ser3GGGAR), was inserted into a pET-based vector (PAB-myc) (Novagen, Madison, Wisconsin, USA) containing the pelB promoter for periplasmic protein expression, along with a C-terminal 6x histidine tag for purification by metal affinity chromatography and a myc tag to facilitate rapid immunochemical recognition of the expressed scFv. The correct sequence of the scFv plasmid was used for soluble protein production in the periplasmic compartment. Two hu6H5 clones (FWJ1 and FWJ2) were selected, and their binding affinities to the antigen were determined. Both clones were able to bind antigen generated from recombinant HERV-K Env surface fusion protein (KSU) and lysates from MDA-MB-231 breast cancer cells.

[0189] HuVH or HuVL carrying human IgG1 were cloned into the pcDNA3.4 vector to generate VH-CH (human IgG1) or VL-CL (human kappa). For mammalian expression, the plasmids were transiently transfected into Expi293 cells. The ratio of H-chain to L-chain plasmids was 2:3. Expression was determined using Western blot, and the predicted MW of 49 / 23 kDa (H-chain / L-chain) was detected under reducing conditions (Figure 1).

[0190] Size-exclusion chromatography (SEC) separation by size and / or molecular weight was further used to determine protein expression (Figure 2). Finally, humanized 6H5 antibody (purity >95%) with endotoxin levels <1 EU / mg was used to determine antitumor efficacy in vitro and in vivo.

[0191] An ELISA assay was used to compare the antigen binding sensitivity and specificity of hu6H5 versus m6H5 (Figure 3). No significant differences were detected between these two parameters.

[0192] An apoptosis assay was used to compare the efficacy of hu6H5 and m6H5 in killing cancer cells. MDA-MB-231 breast cancer cells were treated with each antibody (1 or 10 μg per ml) for 4 and 24 hours (Figure 4). Cells treated without antibody or with mIgG or human IgG were used as controls. The results showed that hu6H5 was as effective as m6H5 in killing these breast cancer cells. To further evaluate the efficacy in cell killing, MDA-MB-231 cells were treated with various antibodies (10 μg / ml) for 16 hours. A co-staining live / dead viability assay was used to identify live cells (green; calcein Am) and presumed dead cells (red; EthD-1) (Figure 5). The results showed that hu6H5 was as effective as m6H5 in killing breast cancer cells. Furthermore, we confirmed that hu6H5 could inhibit cancer cell growth using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Figure 6). We used ADCC to determine the mechanism of BC cell killing, and our results supported effector cell-mediated secretion of cytotoxic molecules that lyse antibody-coated target cells (Figure 7).

[0193] Flow cytometry was used to determine whether hu6H5 could downregulate the expression of p-ERK, Ras, and SIRT-1. 231C or 231K cells were treated with 10 μg of hu6H5 per ml for 16 hours. Expression of HERV-K, SIRT-1 (Figure 8A), p-ERK, and Ras (Figure 8B) in both permissive and non-permissive cells. Downregulated expression of HERV-K, p-ERK, Ras, and SIRT-1 was demonstrated in 231K or 231C cells treated with hu6H5.

[0194] [ka]

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[0196] [ka]

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[0198] pLVXK is a HERV-K expression vector, and MDA-MB-231 pLVXK is MDA-MB-231 cells transduced with pLVXK. Similarly, pLVXC is the control expression vector alone, and MDA-MB-231 pLVXK is MDA-MB-231 cells transduced with pLVXC. NSG female mice (8 weeks old) were inoculated with MDA-MB-231 pLVXC (231-C; subcutaneous, 2 million cells) versus MDA_MB-231 pLVXK (231-K; subcutaneous, 2 million cells). On day 6, mice were treated with hu6H5 (4 mg / kg intraperitoneally, twice a week for 3 weeks). Tumor growth was monitored and measured every other day. The percentage of surviving mice at various time intervals is shown in Figure 9A. Mice bearing 231-C and 231-K cells treated with antibody demonstrated increased survival. Tumor and lung tissues were collected from each mouse. Larger lymph nodes were detected in some mice bearing 231-K cells, but not in mice bearing 231-C cells.

[0199] Hematoxylin and eosin (H&E) staining was further used to evaluate the morphological characteristics of tumor tissue (Figure 9) and tissues from other organs (lung and lymph node; Figure 10). Tumor viability and tumor necrosis were quantified by pathologists by measuring tumor area by H&E staining. Humanized antibody treatment resulted in smaller tumor volume, less tumor focality and number, less invasive borders, and reduced mitotic activity. A reduction in the percentage of tumor viability was observed in mice bearing 231-C cells (Figure 9B) or in 231-K cells treated with the antibody (Figure 10B). Reduced tumor variability was demonstrated in 231C (Figure 9B) or 231K cells treated with hu6H5 (Figure 9C) compared with their controls. Anti-Ki67 and anti-HERV-K mAbs were used (Figure 9D). Mice treated with hu6H5 demonstrated reduced tumor survival (20%; lower panel) compared with controls (60%; upper panel; Figure 9B). The antibody-treated group was more uniform in appearance, had fewer polymorphonuclei and smaller nucleoli, and tumor-infiltrating lymphocytes were significantly increased in number.

[0200] Metastatic tumor cells were also found in lung tissue obtained from mice bearing 231-K cells, but not in mice bearing 231-C cells (Figure 10A). A reduction in the percentage of tumor viability was observed in the lungs of 231-K-bearing mice treated with antibody compared to those not treated with antibody (Figure 10B). Metastatic lymph nodes were detected only in mice inoculated with 231K cells (Figure 10C). A tumor viability rate of greater than 95% was detected in the lymph nodes of mice bearing 231-K cells (Figure 10C; >95%; upper panel), and a reduction in the percentage of tumor viability was observed in the lymph nodes of mice treated with antibody (Figure 10C; 40%; lower panel) compared to mice not treated with antibody (Figure 10D). Ascites was demonstrated in mice bearing 231K or 231C tumor cells without antibody treatment.

[0201] [Example 2] Efficacy of bispecific T cell engagers (BiTEs) targeting HERV-K We generated BiTEs directed against T cell CD3 or CD8 and the tumor-associated antigen HERV-K, consisting of antibodies targeting either CD3 or CD8 and HERV-K. These BiTEs were shown to induce interferon-gamma (IFN-gamma) cytotoxic activity against MDA-MB-231 breast cancer cells expressing tumor histocompatibility class (MHC) molecules loaded with HERV-K epitopes, resulting in a 20- to 30-fold increase in IFN-gamma expression after treatment with the BiTEs (Figure 11).

[0202] BiTEs are recombinant protein constructs constructed as single-chain antibody constructs that redirect T cells to tumor cells without requiring the expansion of endogenous T cells by antigen-presenting cells. See scientific reference 50. BiTE molecules can be administered directly to patients, and BiTE-mediated T cell activation does not depend on the presence of MHC class I molecules, as does CAR. Given the success of targeting HERV-K Env as a tumor-associated antigen (TAA) and the fact that nearly all breast cancer cell lines express Kenv protein, we hypothesize that BiTEs specific for Kenv and CD3 (K3Bi) will effectively treat metastatic disease, as have K-CARs. We designed and synthesized K3Bi, which has bispecificity for Kenv and CD3. Thus, T cells are directed to target HERV-K+ tumor cells. We generated, purified, and validated K3Bi and CD8 BiTEs (K8Bi). This was performed using mAb 6H5, which was also used in CAR constructs (Scientific Reference 33), and OKT3, an antibody against human CD3 previously used in other BiTEs. These antibodies were humanized and attached with a flexible linker and two C-terminal epitope tags (MYC and FLAG) for purification and staining. A CD8 single-chain antibody (scFv) derived from OKT8 hybridoma cells was generated in our laboratory and used to generate K8Bi (VL-VH6H5 linker VH-VLCD8-MYC and FLAG). K3Bi and K8Bi were cloned into pLJM1-EGFP lenti or pGEX-6P-1 vectors for recombinant protein expression. The ability of K3Bi or K8Bi to bind to T cells and HERV-K+ breast cancer cell lines was determined by several immunoassays. We found that increasing BiTE concentration increased the number of target cells bound to the BiTE.

[0203] We also investigated the ability of K3Bi to induce T cell activation, proliferation, cytokine production, and lysis of target tumor cells. As described in scientific reference 51, bulk PBMCs (50,000 per well) obtained from healthy controls were co-cultured with K3Bi (0, 1, 10, 100, and 1,000 ng / ml) and tumor cells (5,000 per well) to achieve a 10:1 effector:target cell ratio. One result is shown in Figure 12. PBMCs + MCF-7 + K3Bi demonstrated increased cancer cell killing compared with PBMCs + MCF-7 without K3Bi (Figure 12B). As previously performed by the inventors, an LDH release assay was used to detect cell viability and cytotoxicity. See scientific reference 33. Enhanced IFN-γ production, as assayed by ELISA (Figure 12C), was observed in MDA-MB-231, MDA-MB-468, and MCF-7 cells treated with K3Bi. Untreated cells, PBMCs alone, or BiTE alone were used as controls, and no IFN-γ production was observed in these control groups.

[0204] Furthermore, treatment of immunodeficient NSG mice bearing HERV-K-positive MDA-MB-231 breast cancer cells with PBMCs and CD3 HERV-K BiTEs and IL-2 or CD8 HERV-K BiTEs and PBMCs and IL-2 resulted in a greater reduction in tumor growth (Figure 13).

[0205] [Example 3] Staining results of normal donor PBMCs transduced with CAR-A and CAR-B lentiviral vectors PBMCs from normal donors were transduced with two CAR-T lentiviral vector constructs: K-CAR-A (CAR-A) or K-CAR B (CAR-B); pWPT-GFP with psPAX2 and pMD2g; and VH-VLhu6H5-CD8-CD28-4-1BB-CD3 zeta. An alternative to the Sleeping Beauty transduction process, i.e., a protocol for generating HERV-Kenv CAR-T cells by lentiviral transduction, is as follows: 1. Thaw PBMCs (2x107) and deplete monocytes by plastic adherence (1 hour incubation at 37°C, 5% CO2). 2. Culture monocyte-depleted PBMCs in RPMI 1640 (complete medium) supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Stimulate T cells with anti-CD3 / CD28 beads at a 3:1 bead:cell ratio and 40 IU / mL IL-2 for 24 hours. 3. Transduce activated T cells with CAR-A or CAR-B lentiviral particles (CD19 CAR as a control). 4. 24 hours after transduction, T cells are cultured in complete medium containing 300 IU / mL IL-2 and γ-irradiated (100 Gy) MDA-MB-231-Kenv (Kenv is the envelope protein of HERV-K) aAPCs at an aAPC / T cell ratio of 2:1 to stimulate CAR-T cell proliferation. γ-irradiated K562-CD19 is used as a control aAPC. Remove the anti-CD3 / CD28 beads on day 5.5. Replenish the CAR-T cells with fresh medium containing IL-2 every 2-3 days. 6. Conduct further experiments using CAR-T cells if proliferation shows a decline from log phase.

[0206] CAR-A or CAR-B transduced cells were co-cultured with γ-irradiated (100 Gy) MDA MB 231 antigen-presenting cells. Soluble IL-2 cytokine (50 U / ml) was added every other day. On day 14, cells were harvested for staining. They were first stained with BV450 live and dead stain at a 1:1000 dilution for 20 minutes at 4°C. After 20 minutes, the cells were washed and stained with K10-AF 488 protein (1 μg / ml), CD4 Amcyan, CD3 PeCy7, and goat anti-human IgG Fc AF 594 antibodies for 30 minutes at 4°C according to the manufacturer's recommendations, followed by washing with PBS. Cells were fixed with 4% PFA for 15–30 minutes, washed, and then analyzed by flow cytometry. Samples were positive for GFP because they were transfected with GFP + CAR-A / CAR-B.

[0207] The percentage of CD4+ cells was determined by gating on populations negative for BV450 and positive for each color. The percentage of CD4-negative (referred to as CD8-positive cells) was gated by selecting populations negative for BV450 and negative for CD4 Amcyan color. The results show that the percentage of CAR-A / CAR-B-transduced CD4-positive PBMCs stained with K10-labeled AF488 protein was higher than the percentage of naive T cells stained with K10-labeled AF488 protein (Figure 14). This indicates that CAR-A or CAR-B-transduced T cells stain with HERV-K10 protein.

[0208] T cells expressing lentiviral CAR expression vectors harboring humanized or fully human HERV-K scFv effectively lyse and kill tumor cells from several different cancers. Humanized K-CARs expressed from lentiviral vectors are pan-cancer CAR-Ts.

[0209] [Example 4] HERV-K-specific humanized chimeric antigen receptor (K-CAR) therapy The inventors generated a humanized single-chain variable fragment (scFv) antibody (Example 1) that was capable of binding to an antigen generated from a recombinant HERV-K Env surface fusion protein (KSU) (Example 3 immediately preceding) and to a lysate from MDA-MB-231 breast cancer cells. CARs generated from this humanized scFv were cloned into lentiviral vectors and used in combination with therapies including, but not limited to, K-CART cells and checkpoint inhibitors, pro-inflammatory cytokines such as interleukin (IL)-12 and IL-18, oncolytic viruses, and kinase inhibitors (including, but not limited to, p-RSK, p-ERK).

[0210] [Example 5] Identification of an ultra-rare B cell-derived human therapeutic antibody (hTAb) that displays potent target specificity and high sensitivity Generation of fully human therapeutic antibodies from the human adaptive immune system: To directly use B cells from breast cancer patients as a source of high-affinity antibodies, we performed indirect ELISAs or immunoblots using HERV-K Env recombinant fusion proteins and used them to detect anti-HERV-K Env-specific responses in several different breast cancer patients. Patients with higher titers of anti-HERV-K antibodies were selected for single B cell experiments. PBMCs obtained from breast cancer patients were polyclonally activated: 1) using irradiated 3T3-CD40L fibroblasts for a period of 2 weeks. This method can efficiently stimulate CD40- B cells, expand them to high numbers with high purity (>90%), and induce their antibody secretion; and 2) ex vivo for 4 days using recombinant human IL-21, IL-2, soluble CD40 ligand, and anti-APO1. This second method allows for the highest percentage of B cells to secrete antibodies using the shortest culture time. IL-21 is known to promote differentiation into antibody-secreting cells. See Scientific References 53 and 54. In vitro IL-2 stimulation can induce human plasma cell differentiation, which requires appropriate T cell help to reach the induction threshold. See Scientific Reference 55. sCD40L associates with CD40 expressed on the cell surface of B cells to mimic T cell-mediated activation. See Scientific Reference 56. Because activation also induces cell death, anti-APO1 was used to rescue B cells from Fas-induced apoptosis. See Scientific Reference 57. Almost no cytotoxic B cells were detected.

[0211] Development of a platform for determining the binding kinetics and cell-to-cell interactions of all cells in a microwell slab. Details of the microengraving process, which allows for the screening and monitoring of B cell interactions and allows for single-cell cloning of antibody-producing B cells over time, are shown in Figure 15A. Nanowell arrays were fabricated in polydimethylsiloxane (PDMS), and cells obtained from mammospheres derived from patient breast tumor tissue, generated and cultured in our laboratory (Figure 15B; left panel), were used as targets to determine the efficacy of breast cancer cell killing. B cells and mammosphere cells (1:1 ratio) from the same donor were loaded into the nanowell array (one cell per well), and the cells were allowed to settle by gravity (Figure 15B, center panel). Dead tumor cells (red) and B cells are shown in the same well (Figure 15B). Anti-HERV-K antibodies produced by the B cells were detected at the same location on a glass cover slide (right panel, red square). Single B cells were then selected by CellCelector for RT-PCR (Figure 15C). Our results show that HERV-K-specific memory B cells expressed anti-HERV-K antibodies and exhibited cytotoxicity against autologous mammosphere cells.

[0212] Therapeutic antibody discovery using in vivo enrichment (IVE) applications: Our platform allows for the isolation of antibodies that can not only bind to target cancer cells but also kill them. Instead of breast cancer patient donors, normal donors without memory B cells can be used to generate hTAbs. Because B cells can produce therapeutic antibodies for treatments that are extremely rare even after ex vivo enrichment, we developed the following platform to identify extremely rare hTAbs:

[0213] Groups (N=10 / group) of wild-type Balb / c mice (female, 6 weeks old) were immunized on day 1 and boosted on weeks 3 and 5. ELISPOT was used to determine IFN-γ secretion by CD8+ T cells obtained from immunized mice (Figure 16A). ELISA assays (Figures 16B, 16C, and 16D) were used to detect anti-HERV-K IgG titers in immunized mouse sera.

[0214] Example 5.1 To generate large numbers of antigen-specific plasmablasts, we adapted the in vivo enrichment technology (IVE: approximately 20-fold enrichment) in SCID / beige mice, enabling rapid expansion and B cell activation. See Figure 11A. This platform generates fully human antibodies from B cells in as little as 8 days. As proof of principle, we developed the IVE technology to generate fully human anti-Zika antibodies in hybridoma cells generated from splenocytes on day 8 of fusion with MFP-2 partner cells (Figures 17A and 17B).

[0215] Recently, we successfully generated humanized mice (HM) and human tumor-bearing mice (HTM) by intravenous injection of CD34+ cells (1-2 × 105 cells / mouse) for HM generation and immunization with HERV-K SU or PD-L1 recombinant fusion proteins. We also co-transplanted CD34+ hematopoietic stem cells into the mammary fat pad with 5 × 104 to 3 × 106 triple-negative breast cancer patient-derived xenografts (TNBC PDX cells or MDA-MB-231 or MDA-MB-468 TNBC cells) for HTM generation. The percentage of hCD19 or hCD45 cells was higher in mice that had been inoculated with CD34 cells for a longer period (Figures 18A and 18B). Exposure to the antigen was associated with HERV-K expression in tumors, with higher antibody titers detected (HTM 2: 40 days vs. HTM 1: 30 days; Figures 18C and 18D). Importantly, this indicates that HTM can produce anti-HERV-K antibodies in mice inoculated with breast cancer cells. This finding prompted us to explore the use of HM and / or HTM to generate complete hTAbs, particularly using normal donors with no prior exposure to the antigen. NSG mice, which lack T-, B-, and NK cell activity, are contemplated as ideal candidates for establishing HM. We recently developed mice (Figure 18B) with higher engraftment rates of human CD45+ cells than those seen in earlier studies, without any significant toxicity.

[0216] Protocol 1. For cancer-bearing donors with higher antibody titers, we use a protocol similar to that in Figure 17A, using HM instead of SCID / beige mice. PBMCs (50 x 106) obtained from breast cancer patients are polyclonally activated with IL-21, IL-2, soluble CD40 ligand, and anti-APO1 and premixed with antigen (HERV-K or PD-L1; 100 μg). B cells isolated from the PBMCs by negative selection using the EasySep™ Human B Cell Enrichment Kit (Stemcell Technologies) are co-injected with CD34 cells in mice treated with busulfan (see Scientific Reference 61) (Fisher: 30 mg / kg intraperitoneally) on day 0. Mice are treated with a cytokine cocktail (days 1, 4, and 7) and boosted with antigen on day 2. This protocol can be completed relatively quickly (8 days).

[0217] Protocol 2. For normal donors without cancer and without memory B cells, we use Protocol 1 with a modification: mice are treated with a cytokine cocktail (days 1, 7, and 14) and boosted with antigen on days 14 and 21. Serum is collected from the mice and binding affinity is tested by ELISA. After an increase in antibody titer is detected, spleens are harvested, analyzed, and used to generate hybridomas. Higher antibody titers were detected in mice using IVE Protocol 2 at week 2.

[0218] Example 5.2 After IVE, half of the spleen is harvested and used for flow cytometry analysis, microengraving, and other analyses. Flow cytometry analysis of B cell surface and intracellular markers and CFSE labeling (Invitrogen CellTrace CFSE kit) is performed using the following: anti-CD19 PECy5, anti-CD27 allophycocyanin, anti-CD38 PECy7, anti-IgG FITC, or anti-IgM PE isotype control of mouse IgG1k conjugated to FITC, PE, PECy5, PECy7, Alexa 700, or allophycocyanin (all from BD Bioscience). Total CD19+ B cells from the spleen were isolated using negative magnetic immunoaffinity bead separation (Miltenyi Biotec) and stimulated with CpG2006 (10 ng / ml; Oligos, Inc.) for 72 hours in the presence of recombinant human B cell activating factor (BAFF; 75 ng / ml; GenScript), IL-2 (20 IU / ml), IL-10 (50 ng / ml), and IL-15 (10 ng / ml) (all from BD Biosciences). Our multiwell microengraving platform (up to 400,000 wells; Figure 15) was used with autologous tumor cells or HERV-K+ TNBC cells as target cells to determine tumor-killing B cells obtained directly from Protocols 1 or 2. As in Figure 15, we determined cells that not only produced antibodies but also bound antigens and killed cancer cells.

[0219] Example 5.3 We then developed human hybridoma cells to ensure long-term antibody availability. To develop fully human hybridomas, we used ClonaCell™-HY (Stemcell Technologies Inc.) according to its protocol, using MFP-2 cells along with the remaining half of the spleen as partners to generate hybridomas. Polyethylene glycol (PEG) was used to fuse human lymphocytes with MFP-2 cells, and the methylcellulose-based semi-solid medium in this kit was used for cloning and selection of hybridoma cells. Clones that grew after selection were pipetted into 96-well plates and screened for reactivity to HERV-K Env protein by ELISA. The isotype of positive clones was determined using a human IgG antibody isotyping kit from Thermo Fisher Scientific. The clones were then adapted to serum-free medium conditions and expanded. Hybridoma supernatants were collected, and antibodies were purified using Hi-Trap Protein A or Protein G columns, depending on the human antibody isotype. Protein A columns are known to have high affinity for antibodies of isotype IgG1, 2, and 4 and variable binding for antibodies of isotype IgM, while Protein G columns are known to show high binding for antibodies of isotype IgG1, 2, 3, and 4, but no binding to IgM antibodies.

[0220] Example 5.4 We will evaluate the antitumor efficacy of candidate B cells obtained from the above protocol in vitro, including effects on cell growth, proliferation, and apoptosis, as we routinely do in our laboratory. We will also perform in vivo studies to evaluate the efficacy of hTAbs in immunodeficient mouse models using breast cancer cell lines and primary tumor cells to assess efficacy and compare it with matched uninvolved control breast cells.

[0221] [Example 6] Combination therapy Our breast cancer data strongly support the feasibility of combination therapy approaches involving HERV-K. Thus, humanized and fully human antibodies targeting HERV-K enhance the efficacy of checkpoint blockade antibody therapy. Effective combination cancer therapies include, but are not limited to, (a) HERV-K hTAb (1.5 mg / kg), (b) K-CAR, (c) K-BiTE, (d) HERV-K shRNA or CRISPR / Cas9 genome editing technology to knock down HERV-K gene expression, (e) or full-length and truncated HERV-K Env proteins and HERV-K. Combinations of prophylactic or therapeutic HERV-K vaccines comprising Env peptides and (a) anti-ICP antibodies (Figure 19), (b) cancer chemotherapy, (c) 5-azacytidine, 5-aza-2'-deoxycytidine or other epigenetic modulating agents, such as DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi), (d) EMT inhibitors, (e) inhibitors of cell migration or invasion, (f) induction of S or G2 phase cell cycle arrest, (g) inhibitors of the PI3K / AKT / mTOR or MAPK / ERK signaling pathways or (h) signaling to HIF1α.

[0222] [Example 7] [ka]

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[0231] IgG domain order: VL-VH6H5---VH-VLhuCD3 or CD8 + c-myc tag + FLAG or VL-VHhu6H5---VH-VLhuCD3 or huCD8 + c-myc tag + FLAG

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[0234] Mice were immunized with five Maps, and serum was collected and tested by ELISA using various HERV fusion proteins (Figure 23). Only the HERV-K SU protein was positive. Hybridoma cells were generated from mice immunized with the five Maps, and scFvs with the following sequences were selected:

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[0236] [Example 8] A humanized antibody targeting HERV-K that can be used in ADCs to deliver drugs into cancer cells and tumors Recombinant gelonin (r-Gel) toxin was conjugated with 6H5 (Figure 20A). r-Gel was detected in OVCAR3 (Figure 20B), SKBr3, MCF-7, and MDA-MB-231 cells (Figure 20C) after 1 hour of internalization using an anti-r-Gel antibody. Furthermore, gold nanoparticles (GNPs) were detected by transmission electron microscopy (TEM) in MDA-MB-231 cells after 2 hours of incubation with naked GNPs (Figure 21A) or 6H5-GNPs (Figure 21B). GNPs were detected using a silver enhancement assay in MDA-MB-231 (Figures 21C, 21E, and 21F) or SKBr3 (Figure 21D) tumors isolated from mice 24 hours after intravenous injection with 6H5-GNPs (Figures 21C, 21E, and 21F) or 6H5scFV-GNPs (Figure 21D). When placed in a radiofrequency magnetic field, GNPs generate heat that kills targeted tumor cells.

[0237] [Example 9] In vivo imaging of anti-HERV-K antibodies in tumor nodules in mice In vivo imaging using the Nuance system detected higher densities of 6H5 in tumor nodules from mice 24 hours after intravenous injection with the anti-HERV-K-Alexa647 conjugate 6H5-Alexa647 (red) (Figure 22).

[0238] List of Embodiments Specific compositions and methods for HERV-K antibody therapeutics. The scope of the present invention is to be defined solely by the claims. Those skilled in the biomedical arts will interpret all claim terms in the broadest possible manner consistent with the context and spirit of the disclosure. The detailed descriptions herein are exemplary and are not limiting or exhaustive. The present invention is not limited to the specific methodology, protocols, and reagents described herein, which may, in fact, vary. Where the specification or claims recite ordered steps or functions, alternative embodiments may perform those functions in a different order or substantially simultaneously. Those skilled in the biomedical arts will recognize that other equivalents and modifications, in addition to those already described, are possible without departing from the inventive concepts described herein.

[0239] All patents and publications cited throughout this specification are incorporated by reference into this disclosure and describe materials and methods used with the technology described herein. Patents and publications are provided solely for their disclosure prior to the filing date of this specification. All statements regarding the disclosure and publication dates of patents and publications are to the information and belief of the inventors. The inventors make no admission as to the accuracy of the contents or dates of these documents. In the event of a conflict between the dates provided herein and the actual publication date, the actual publication date shall prevail. The inventors may have antedated such disclosure by prior invention or for other reasons. In the event of a conflict between the scientific or technical teachings of a prior patent or publication and this specification, the teachings of this specification and these claims shall prevail.

[0240] Where this specification provides a range of values, each intervening value between the upper and lower limits of that range is within the range of values ​​unless otherwise indicated.

[0241] Among the embodiments provided herein are the following:

[0242] 1. An isolated antibody that binds to human endogenous retrovirus-K (HERV-K), the isolated antibody comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR). The humanized anti-HERV-K antibody can reduce tumor growth, particularly metastasis to the lungs, lymph nodes, and other organs.

[0243] 2. The antibody according to embodiment 1, comprising humanized or human framework regions.

[0244] 3. The antibody according to embodiment 1, which is a HERV-K antagonist.

[0245] 4. An isolated nucleic acid comprising a nucleotide sequence encoding the HCVR, LCVR, or combination thereof of embodiment 1.

[0246] 5. An expression vector comprising the nucleic acid of embodiment 4.

[0247] 6. A host cell transformed with the expression vector of embodiment 5.

[0248] 7. A method for producing an antibody comprising an HCVR, an LCVR, or a combination thereof, comprising growing a host cell of embodiment 1 under conditions such that the host cell expresses an antibody comprising an HCVR, an LCVR, or a combination thereof, and isolating the antibody comprising the HCVR, the LCVR, or a combination thereof.

[0249] 10. A method for treating cancer in a mammal, comprising administering an effective amount of an antibody according to embodiment 1 to a mammal in need thereof.

[0250] 11. A method of treating cancer, comprising administering to an individual in need thereof an effective amount of an ADC comprising an antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH region comprises CDR1, CDR2, and CDR3, and the VL region comprises CDR1, CDR2, and CDR3, and wherein the antibody is conjugated via a linker to a cytotoxic drug, an auristatin, or a functional peptide analog or derivative thereof.

[0251] 12. The method of embodiment 11, wherein the ADC is administered in combination with one or more additional therapeutic agents.

[0252] 13. The method of embodiment 11, wherein the one or more additional therapeutic agents comprises a chemotherapeutic agent.

[0253] 14. The method of embodiment 11, wherein the cancer is selected from the group consisting of melanoma, chronic lymphocytic leukemia, breast cancer, pancreatic cancer, head and neck cancer, ovarian cancer, cervical cancer, colorectal cancer, testicular cancer, gastric cancer, kidney cancer, endometrial cancer, uterine cancer, bladder cancer, prostate cancer, esophageal cancer, liver cancer and non-small cell lung cancer.

[0254] Humanized antibodies developed for CAR T, CAR NK, and BiTE research.

[0255] 15. The method of embodiment 11, wherein the antibody is a full-length antibody.

[0256] 16. The method of embodiment 11, wherein the antibody is a human monoclonal IgG1 or IgG4 antibody.

[0257] 17. The method of embodiment 11, wherein the auristatin is monomethyl auristatin E (MMAE).

[0258] 18. The method of embodiment 11, wherein the auristatin is monomethyl auristatin F (MMAF).

[0259] 19. The method of embodiment 11, wherein the cytotoxic agent is emtansine (DM1).

[0260] 20. The method of embodiment 11, wherein the cytotoxic agent is ozagamicin (calicheamicin).

[0261] 21. The method of embodiment 11, wherein the cytotoxic drug is deruxtecan (DXd).

[0262] 22. The method of embodiment 11, wherein the cytotoxic agent is govitecan (SN-38).

[0263] 23. The method of embodiment 11, wherein the cytotoxic agent is mafodotin (MMAF).

[0264] 24. The method of embodiment 11, wherein the cytotoxic drug is duocarmazine (duocarmycin).

[0265] 25. The method of embodiment 11, wherein the cytotoxic agent is BAT8001 (a maytansinoid) soravtansine (DM4).

[0266] 26. The method of embodiment 11, wherein the cytotoxic drug is tesirine (PBD).

[0267] 27. The method of embodiment 11, wherein the linker is attached to a sulfhydryl residue of the antibody obtained by partial reduction of the antibody.

[0268] 28. The method of embodiment 11, wherein the linker-auristatin is vcMMAF or vcMMAE.

[0269] 29. Early detection, metastasis or HERV-K and immune checkpoint biomarkers substantially as described herein.

[0270] 30. An antibody-based therapeutic agent substantially as described herein.

[0271] 32. Cancer cells that overexpress HERV-K as targets for the anti-HERV-K humanized antibodies and ADCs of the present invention.

[0272] 33. hu6H5 clones (FWJ1 and FWJ2) generated from bacteria (HUM1 and HUM2) or mammalian cells.

[0273] 34. A BiTE directed against T cell CD3 or CD8 and a humanized scFv against the tumor-associated antigen HERV-K, the BiTE comprising an antibody targeting either CD3 or CD8 and HERV-K.

[0274] 35. T cells expressing a lentiviral CAR expression vector carrying a humanized or fully human HERV-K scFv.

[0275] 36. A humanized single-chain variable fragment (scFv) antibody capable of binding to an antigen generated from a recombinant HERV-K Env surface fusion protein (KSU) and a lysate from cancer cells expressing the HERV-K Env protein.

[0276] 37. A CAR generated from the humanized scFv of embodiment 28.

[0277] 38. A CAR generated from the humanized scFv of embodiment 28, cloned into a lentiviral vector.

[0278] 39. A CAR generated from the humanized scFv of embodiment 28, cloned into a lentiviral vector, for use in combination therapy.

[0279] 44. An improved in vivo enrichment method for rapid expansion and B cell activation for donors without memory B cells, comprising treating mice with a cytokine cocktail on days 1, 7, and 14 and boosting the mice with antigen on days 14 and 21.

[0280] 45. Cells that not only produce antibodies, but also bind to antigens and kill cancer cells, and cells that express antigens can be killed by antibodies.

[0281] 47. Significantly enhanced expression of six circulating immune checkpoint proteins in the plasma of breast cancer patients.

[0282] 50. A method of blocking the immunosuppressive domain (ISD) of HERV-K using immune checkpoint inhibitors.

[0283] 51. The method of embodiment 42, wherein the HERV-K immune checkpoint inhibitor is selected from the group consisting of monoclonal antibodies and drugs that target the ISD of HERV-K.

[0284] 52. Humanized and fully human antibodies targeting HERV-K for use in enhancing the efficacy of checkpoint blockade antibody therapy.

[0285] 53. A method for generating novel antibodies from mice immunized with five multiple antigenic peptides (MAPS) generated from the HERV-K SU protein produced by a cancer patient.

[0286] 54. Method for generating HERV-K CAR A:VH-VLhu6H5-CD8-CD28-4-1BB-CD3 Zeta.

[0287] References Those skilled in the art of molecular biology may use the following patents, patent applications and scientific references as guidance as to the predictable results in making and using the present invention:

[0288] Patent references: U.S. Patent No. 9,243,055 (Wang-Johanning) discloses and claims cancer diagnostics and therapies. Methods and compositions for detecting, preventing, and treating HERV-K+ cancers are provided. One method is to prevent or inhibit cancer cell growth by administering to a subject a cancer cell growth-blocking or -reducing amount of a HERV-K env protein-binding antibody.

[0289] International Patent Publication WO2014 / 186469 (Regent, Texas System University). This patent publication relates to methods and compositions for immunotherapy using engineered T cells containing chimeric antigen receptors (CARs). CAR-expressing T cells are generated using electroporation in conjunction with a transposon-based integration system to generate a population of CAR-expressing cells that require minimal ex vivo expansion or can be administered directly to patients for cancer treatment.

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Claims

1. A human endogenous retrovirus-K (HERV-K) binding protein comprising an antibody heavy chain variable domain and an antibody light chain variable domain comprising SEQ ID NOs: 6 and 9, or SEQ ID NOs: 7 and 10, respectively.

2. The HERV-K binding protein of claim 1, wherein the HERV-K binding protein is an antibody.

3. The HERV-K binding protein of claim 2, wherein the antibody is of the human IgG1 isotype subtype.

4. 4. The HERV-K binding protein of claim 3, wherein the antibody comprises a heavy chain and a light chain comprising SEQ ID NOs: 42 and 44, respectively.

5. The HERV-K binding protein of any of claims 2 to 4, wherein the antibody is conjugated to a cytotoxic drug.

6. The HERV-K binding protein of claim 1, wherein the HERV-K binding protein is an scFv or comprises an scFv as part thereof.

7. 7. The HERV-K binding protein of claim 6, wherein the scFv comprises SEQ ID NO: 12 or 13.

8. The HERV-K binding protein of claim 6 or 7, wherein the HERV-K binding protein is a chimeric antigen receptor (CAR).

9. The HERV-K binding protein of claim 6, wherein the HERV-K binding protein is a bispecific T cell engager (BiTE) that further comprises a CD8-binding domain or a CD3-binding domain in addition to the scFv.

10. 10. The HERV-K binding protein of claim 9, wherein the CD8 binding domain comprises SEQ ID NO: 36 or the CD3 binding domain comprises SEQ ID NO:

38.

11. One or more isolated polynucleotides encoding the HERV-K binding protein of any one of claims 1 to 10.

12. One or more vectors comprising one or more polynucleotides of claim 11.

13. A host cell comprising one or more vectors of claim 12.

14. A method for producing a HERV-K binding protein, comprising: Culturing the host cell of claim 13 under conditions that allow expression of the HERV-K binding protein; and Optionally, isolating the HERV-K binding protein from the culture; A method comprising:

15. A pharmaceutical composition comprising a HERV-K binding protein of any of claims 1 to 7, 9 and 10 or a T cell expressing the HERV-K binding protein of claim 8, for use in a method for treating a HERV-K positive cancer in a subject in need thereof.

16. A pharmaceutical composition described in claim 15 for use in combination with an immune checkpoint inhibitor.

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