{"paper_id":"8ab9276d-8fcc-4cf5-933c-43a5e5a55032","body_text":"In 2021, there were an estimated 21,410 new cases of ovarian cancer leading\nto 13,770 deaths 1 . The current\nstandard of care involves aggressive cytoreductive surgery and chemotherapy, which\ninitially elicits a response in more than 80% of cases 2 , but the majority of patients ultimately\nrelapse and develop chemotherapy-resistant disease. The estimated 5-year survival is\n49.1% in all cases; the majority of patients are diagnosed at an advanced stage, in\nwhich case the 5-year survival drops to 30.1% 1 . There is thus an urgent need for novel therapeutics in this\narena. While immunotherapies such as immune checkpoint blockade are changing the\ntrajectory of various cancers such as melanoma, the results in ovarian cancer have\nbeen disappointing. Interestingly, tumor-infiltrating B lymphocytes are present in\nmost ovarian cancers and have been associated with improved patient\noutcomes 3 – 5 . However, limited research has focused on\nhumoral response in endometriosis-associated ovarian cancers or endometriosis.\nEndometriosis is a condition in which endometrial tissue is present outside\nof the uterine cavity, which occurs in approximately 10% of women. Endometriosis is\nassociated with a two- to three-fold increase in a woman’s risk of developing\nclear cell or endometrioid ovarian cancers, and ovarian endometriosis has been\nassociated with as high as a ten-fold increased risk of clear cell and five-fold\nrisk of endometrioid ovarian cancer 6 ,\n 7 . Recent studies have concluded\nthat endometriosis is a precursor lesion to these endometriosis-associated ovarian\ncancers (EAOC), with corresponding somatic mutations identified in both 8 ,  9 .\nTherefore, we hypothesized that antitumor humoral responses generated by\ntumor-infiltrating B lymphocytes in ovarian cancer and B lymphocytes in the\nmicroenvironment of endometriosis target specific antigens that can be used to\nidentify novel, targetable antigen domains that can exert a protective effect on\nprogression of established ovarian carcinomas.\n\nHuman ovarian carcinoma tissues were procured under protocols approved\nby the Committee for the Protection of Human Subjects at Dartmouth-Hitchcock\nMedical Center (#17702), by the Institutional Review Board at Christiana Care\nHealth System (#32214), and by Advarra Institutional Review Board (#00000971)\nand H. Lee Moffitt Cancer Center Scientific Review Committee (MCC#18974). Human\nendometrioma tissues were procured under a protocol approved by the\nInstitutional Review Board at Ponce Research Institute (#1903009574). Informed\nconsent was obtained from all subjects.\nHuman ovarian cancer cell lines including OVCAR3 (RRID: CVCL_0465),\nSKOV3 (RRID: CVCL_0532), and human endometrial stromal cells (HESC), highly\ninvasive and immortalized with human telomerase reverse transcriptase (hTert),\nwere obtained from ATCC. TOV21G (RRID: CVCL_3613), RMG-I (RRID: CVCL_1662),\nCaov3 (RRID: CVCL_0201), A2780 (RRID: CVCL_0134), OVCAR4 (RRID: CVCL_1627),\nOVCAR5 (RRID: CVCL_1628), OVCAR8 (RRID: CVCL_1629), Kuramochi (RRID: CVCL_1345)\nand BRCA OVCAR were obtained as a gift from Dr. Rugang Zhang at The Wistar\nInstitute. Human endometriotic epithelial cells (12Z, RRID: CVCL_0Q73) were\nobtained as part of a collaboration with Dr. Asgerally Fazleabas and Dr. Anna\nStarzinski-Powitz 10 .\nAll cell lines except RMG-I, HESC and 12Z were cultured in RPMI 1640 medium\n(Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), penicillin\n(100 IU/mL), streptomycin (100IU/mL), L-glutamine (2mM), and sodium pyruvate\n(0.5mM). RMG-I was cultured in Ham’s F12 medium (Fisher Scientific)\nsupplemented with 10% fetal bovine serum (FBS), penicillin (100 IU/mL),\nstreptomycin (100 IU/mL), L-glutamine (2mM), and sodium pyruvate (0.5mM). 12Z\nwas cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/F12\nsupplemented with 10% FBS. HESC was cultured in phenol-free DMEM supplemented\nwith charcoal-treated 10% FBS and 1% Insulin-Transferrin-Selenium (ITS). All\ncell lines were routinely tested for Mycoplasma by PCR. Cells were used within\n20 passages from thaw for  in vitro  experiments and 10 passages\nfrom thaw for  in vivo  experiments.\nCryopreserved single-cell suspensions of two stage IIIC ovarian clear\ncell ovarian carcinomas, two stage IIIC ovarian endometrioid carcinomas, and two\nendometriomas were thawed and prepared, and CD19 +  B cells isolated,\nactivated, and immortalized, as previously described 5 . The conditioned medium from each was\ncollected and concentrated using centrifugal filter units (Millipore Sigma\nAmicon, UFC900324). From the concentrated medium, human IgA and IgG were\npurified using immunoglobulin purification kits (LigaTrap, LT-146KIT and\nLT-095KIT) according to the manufacturer’s protocols. To characterize the\nspecificities of these antibodies, they were analyzed for reactivity against a\nproteome microarray that includes greater than 80% of the human proteome\n(HuProt ™ , CDI Laboratories, Mayagüez, Puerto\nRico).\nIn order to determine which peptide would be best targeted by an\nantibody, the amino acid sequence for SDCBP was run through two epitope\nprediction tools (Bepipred Linear Epitope Prediction 2.0 and ABCPred) to\ndetermine predicted epitopes. Predicted targetable extracellular domains were\nchosen as the peptides for tetramer analysis. We tetramerized two biotinylated\npeptides (GenScript) contained in the extracellular domain of syndecan binding\nprotein (SDCBP) using PE-labelled fluorescent streptavidin (BioLegend, 405203)\nand used flow cytometry to determine the percent of specific antigen-reactive\nimmortalized B cells which are secreting antibodies against the target molecule\nfrom each of the six immortalized B cell lines, using the protocol previously\ndescribed. 5  We then\nused fluorescence-activated cell sorting with EEEIRANVAVVSGAPL peptide to sort\nendometrioid carcinoma-derived immortalized B cells specific for SDCBP from the\npool of immortalized B cells.\nSingle-cell V(D)J B-cell receptor sequencing was performed by the\nMoffitt Cancer Center Molecular Genomics Core using the 10XGenomics Chromium\nsystem. 56 cells were encapsulated and sequenced as previously\ndescribed 5 . BCR reads\nsequenced by V(D)J assay were aligned to GRCh38 reference transcriptome using\nCell Ranger VDJ (v.3.1.0, 10X Genomics). BCR heavy and light chains were\nassembled and annotated using Cell Ranger VDJ to determine clonotypes.\nRecombinant IgG4 antibodies were produced by Genscript. In brief, corresponding\nDNA sequences for the immunoglobulin heavy chain and light chain were\nsynthesized and the complete sequence was subcloned into a pcDNA3.4 vector and\nexpressed in HD 293F cells. IgG4 antibodies were eluted from cell culture\nsupernatants. Molecular weight and purity were analyzed by SDSPAGE and\nhigh-performance liquid chromatography.\nRNA was extracted from ovarian clear cell carcinoma, endometrioid\ncarcinoma, and high-grade serous carcinoma tissues and cell lines using the\nRNEasy Plus Mini Kit (Qiagen) to quantify SDCBP expression. Total RNA were\nreverse transcribed using a high-capacity cDNA reverse transcription kit with\nRNAse inhibitor (ThermoFisher, 4374966). Quantification of SDCBP was performed\non the 7900HT Real-Time PCR system (Thermo Fisher Scientific) using SYBR Select\nMaster Mix (Applied Biosystems, with forward primer:\n5’-TCTCGAAGACTTGAAGGTAGACA-3’, and reverse primer:\n5’-CGGCCACATTTGCACGTATT-3’). Expression was normalized to levels\nof the endogenous reference control gene GAPDH (forward primer:\n5’-CCTGCACCACCAACTGCTTA-3’; and reverse primer:\n5’-AGTGATGGCATGGACTGTGGT-3’).\nProteins were extracted from ovarian clear cell carcinoma, endometrioid\ncarcinoma, and high grade serous carcinoma tissues and cell lines, as well as an\nendometriosis cell line, and quantified as previously described 11 . Proteins were loaded onto a\n10% Bis-Tris polyacrylamide gel. Membranes were incubated with recombinant\nanti-SDCBP IgG4 antibodies described above (Genscript) or rabbit anti-human\nSDCBP (Sigma). After washing with TBST, the membranes were incubated with\nhorseradish peroxidase-conjugated rabbit anti-human IgG (1:5000, Cat. Ab6759,\nAbcam, RRID:AB_955434). Horseradish peroxidase-conjugated anti-β-actin\nantibody (1:5000, Cat. 5125S, Cell Signaling Technology, RRID: AB_1903890) was\nused as a loading control. Images were captured using the BioRad ChemiDoc\nimaging system and GE Healthcare Amersham ECL Prime Western Blotting Detection\nReagents (cat. 12316992, Fisher Scientific).\nAll  in vivo  protocols were approved by the University\nof South Florida’s Institutional Animal Care and Use Committee. Female\nNOD-SCID-gamma (NSG) mice, originally obtained from Jackson Laboratory, were\nmaintained by the animal facility of H. Lee Moffitt Cancer Center and Research\nInstitute. Mice were injected subcutaneously with 5×10 6  RMG-I,\nTOV21G, or OVCAR3 cells in the right flank. Once tumor uptake was demonstrated,\nseven to nine days post-injection, mice were randomly divided into two treatment\ngroups of five mice each: irrelevant IgG4 control and anti-SDCBP IgG4 treatment.\nTwice weekly, tumor volume was measured and IgG4 control and anti-SDCBP IgG4\ntreatment were administered by intratumoral or peritumoral injection\n(100μg/100μL). Tumor volume was calculated as (L ×\nW 2 )/2, in which L is length and W is width. Once any group became\nmoribund, all mice were euthanized and tumor weight was recorded. Tumor\nspecimens were formalin-fixed and paraffin-embedded or mechanically dissociated\ninto single-cell suspensions.\nAll experiments were repeated at least twice with similar results. The\nShapiro–Wilk test was applied to determine whether data were normally\ndistributed. Wilcoxon matched-pairs signed rank tests and unpaired t tests were\napplied as indicated. Two-tailed analyses were performed. Error bars represent\nstandard error of the mean. A p value less than 0.05 was considered\nstatistically significant. Statistical analyses were performed using GraphPad\nPrism 9.0 (GraphPad Prism, RRID:SCR_002798).\n\nTo identify antibodies produced by B cells recognizing possible shared\ntargets in clear cell ovarian carcinoma, endometrioid ovarian carcinoma, and\nendometriosis, we analyzed viable single-cell suspensions from two cryopreserved\nsamples of each type. B cells were isolated, activated, and immortalized using\nEpstein-Barr virus. These six immortalized B cell pools were found to secrete\nIgG and IgA at titers in the 0.7–37 mg/mL range. Using\nHuProt ™  proteome arrays containing >80% of the\nhuman proteome 5 , IgG and IgA\ntumor reactivities were decoded ( Figure\n1A ). Greater than 200 targets were identified for each sample, for both\nIgA and IgG antibodies in independent analyses.\nThere were nine molecules that met the following criteria: 1) They were\neither secreted or included an extracellular domain, and were therefore\naccessible to antibodies in live cells; 2) they were recognized by both IgA and\nIgG; and 3) reactivity was shared in every endometrioma and carcinoma sample\n( Figure 1B ). Among these, we focused on\nSDCBP, a transmembrane molecule that links syndecan-mediated signaling to the\ncytoskeleton. SDCBP regulates TGF-β1-induced Smad activation and EMT by\ninhibiting caveolin-mediated TGF-β type I receptor internalization, and\nhas been demonstrated to drive proliferation, migration, invasion, and\nangiogenesis 12 – 14 . SDCBP has been associated\nwith unfavorable prognosis in multiple solid malignancies, including breast and\ncolorectal cancer 15 – 16 . Furthermore, SDCBP has been\nreported as a therapeutic target for cancer metastases 17 , as well as cancer stemness and\nchemoresistance 18 .\nBecause antibodies targeting SDCBP are being spontaneously produced in\nthe endometriosis and ovarian cancer microenvironment, we tetramerized two\ndifferent biotinylated 16–20mer peptides contained in the extracellular\ndomain of SDCBP using fluorescent streptavidin and used flow cytometry to\ndetermine the percent of specific antigen-reactive immortalized B cells which\nare secreting antibodies against SDCBP from each of the six immortalized B cell\nlines. Based on these results, we used fluorescent activated cell sorting to\nsort endometrioid ovarian cancer-derived B cells specific for SDCBP and\nperformed single-cell B cell receptor sequencing on the sorted population of B\ncells ( Figure 2 ). Bioinformatic analysis of\nthe B cell receptor sequencing determined the sequence of the heavy chain and\nlight chain of the most common B cell receptor, identified in 96% of these cells\n( Figure 2 ). We then produced a\nrecombinant antibody targeting SDCBP using these heavy chain and light chain\nsequences on an IgG4 backbone. IgG4 was specifically selected to avoid\nantibody-dependent killing of normal cells that also express SDCBP via\nantibody-dependent cell-mediated cytotoxicity or antibody-dependent cellular\nphagocytosis.\nThe Cancer Genome Atlas RNA sequencing data were queried, demonstrating\nSDCBP mRNA expression in all tested cancer types, supporting its potential as a\ntherapeutic target for patients with a diverse range of histologies ( Figure 3A ,  Supplementary Figure 1 ) 19 . RT-qPCR was performed to\nassess the baseline prevalence of SDCBP mRNA expression in ovarian cancer cell\nlines and tumor tissues. 26/29 (90%) of the cell lines and tissue samples\nexpress SDCBP mRNA – and all of the clear cell (5/5) and endometrioid\n(7/7) cell lines and tissue samples express SDCBP ( Figure 3B ). Tumor-derived, recombinant anti-SDCBP IgG4 recognizes\nrecombinant SDCBP in Western blot analysis. Western blot demonstrated SDCBP\nprotein expression in ovarian cancer cell lines and tumor samples tested,\nincluding clear cell, endometrioid, and high grade serous histologies ( Figure 3C ,  Supplementary Figure 2 ), as well as\nexpression in endometriosis ( Figure\n3D ).\nTo investigate the antitumor effects of a recombinant anti-SDCBP IgG4\nantibody  in vivo , we created a high grade serous ovarian\ncarcinoma mouse model using subcutaneous injection of OVCAR3 cells ( Figure 4A ). Mice treated with the anti-SDCBP\nIgG4 antibody demonstrated significantly reduced tumor growth and substantially\nreduced tumor weight compared to those treated with the control irrelevant IgG4\nantibody (p=0.004 and p=0.05, respectively,  Figure\n4B – D ). To determine\nwhether this could be extrapolated to other ovarian cancer histologies,\nspecifically clear cell carcinoma – one of the endometriosis-associated\novarian cancer types, we next evaluated the anti-SDCBP antibody in an RMG-I\ntumor model. In this model, we also observed significantly reduced tumor growth\nand lower tumor weight in the treatment arm compared to the control arm\n(p<0.05 and p=0.34, respectively,  Figure\n5A – B ). To confirm these\nfindings, we performed the same experiments in a second clear cell line, TOV21G,\nwhich yielded similar results ( Figure\n5C – D ).\n\nA novel anti-SDCBP IgG4 antibody has demonstrated preclinical anti-tumor\nefficacy in HGSOC and CCC, with the possibility of use in EC and other tumor types\ngiven the broad expression of SDCBP among tumors. As HGSOC is the most common\nhistologic type of ovarian cancer, and as CCC is relatively chemotherapy-resistant\nand associated with increased risk of poor outcomes, these two disease types\nrepresent an area of high unmet need for novel therapeutic strategies. This study\nestablishes the potential of this technique in identifying novel therapeutic targets\nfor CCC and verifies the utility of the technique for HGSOC, in which we had\npreviously identified SDCBP as a target of tumor-infiltrating, IgG-producing B cells\nin six tumor samples 5 . This study\nalso highlights the importance of antibody responses in different histologic\nsubtypes of ovarian cancer and supports that intratumoral B cells, through the\nspontaneous production of antibodies, could exert a protective role against\nmalignant progression. SDCBP has been previously described in small extracellular\nvesicles 20 ; we did not\nhave enough serum samples from the patients analyzed to perform ELISAs, but it is\ntheoretically possible that reactive antibodies could be detected in circulation.\nThe concordance of antibodies being spontaneously produced in the microenvironment\nof endometriosis and endometriosis-related ovarian cancers raises the question of\nwhether antibodies such as those targeting SDCBP could prevent or delay progression\nfrom endometriosis to EAOCs - in addition to delaying tumor progression in\nestablished EAOCs.\nIn patients with ovarian cancer, high SDCBP expression is associated with a\nslight trend towards worse OS (p=0.37). The issue, however, is that the level of\nexpression of SDCBP is very high in virtually all included ovarian cancers in this\nanalysis as seen in  Figure 3A , while there is\nmore dispersion in the level of expression of SDCBP in other gynecologic and other\ncancer types, allowing a better comparison between high- and low-expressing tumors.\nOf note, high SDCBP expression is associated with statistically significant worse OS\nin breast, cervical, and endometrial cancers, among others ( Supplementary Figure 3 ) (Cancer Gene\nPrognosis Atlas,  https://cgpa.moffitt.org/ ), indicating multiple avenues for further\nresearch in other cancer types - gynecologic and otherwise. The  in\nvivo  effects of the anti-SDCBP IgG4 antibody support further\npreclinical research investigating the underlying mechanism of action, as well as\nthe continued  in vivo  study of other novel therapeutic targets\nidentified in this study, such as OLFML2B which is the target of ongoing study.\nFuture studies should also explore the effect of the anti-SDCBP IgG4 antibody in\nendometriosis models.\n\nAn α-SDCBP IgG4 has demonstrated anti-tumor efficacy in\nSDCBP +  CCC and HGSOC, and SDCBP-targeted therapy for endometriosis\nand associated malignant conditions, as well as HGSOC, warrants further\ninvestigation.","source_license":"CC0","license_restricted":false}