Target For Monoclonal Antibody Therapy Research Articles

The Role of Antibody-Based Therapies in Neuro-Oncology.

This review explores the evolving landscape of antibody-based therapies in neuro-oncology, in particular, immune checkpoint inhibitors and immunomodulatory antibodies. We discuss their mechanisms of action, blood-brain barrier (BBB) penetration, and experience in neuro-oncological conditions. Evidence from recent trials indicates that while these therapies can modulate the tumor immune microenvironment, their clinical benefits remain uncertain, largely due to challenges with BBB penetration and tumor-derived immunosuppression. This review also examines emerging targets such as TIGIT and LAG3, the potential of antibodies in modulating the myeloid compartment, and tumor-specific targets for monoclonal antibody therapy. We further delve into advanced strategies such as antibody-drug conjugates and bispecific T cell engagers. Lastly, we explore innovative techniques being investigated to enhance antibody delivery, including CAR T cell therapy. Despite current limitations, these therapies hold significant therapeutic potential for neuro-oncology. Future research should focus on optimizing antibody delivery to the CNS, identifying novel biological targets, and discovering combination therapies to address the hostile tumor microenvironment.

Recognition of mutant forms of the sodium-dependent phosphate transporter NaPi2b by ­monoclonal antibodies in ovarian cancer cells

Background. The sodium-dependent phosphate transporter NaPi2b is expressed in a number of tumors and serves as a target for monoclonal antibody therapy. The NaPi2b transporter has a large extracellular domain containing 4 cysteines and an MX35 epitope recognized by the corresponding monoclonal antibodies.
 Aim. To study the recognition of the MX35 epitope in cysteine mutants C303 and C350 of the sodium-dependent phosphate transporter NaPi2b by monoclonal antibodies in ovarian cancer cells.
 Material and methods. Ovarian cancer cells of the OVCAR-8 line were transfected with plasmids encoding mutant forms of the NaPi2b transporter. The recognition of these forms by monoclonal antibodies was studied by Western blot and flow cytometry. Statistical analysis of flow cytometry data was performed using Pearsons Chi-squared test with Yates correction.
 Results. Western blot analysis of lysates of OVCAR-8 cells expressing the wild-type NaPi2b transporter demonstrated the presence of a specific signal only in samples without the addition of dithiothreitol, while in all samples with NaPi2b cysteine mutants, even without the addition of dithiothreitol, no specific signal from monoclonal antibodies was detected. The flow cytometry results showed that proportion of OVCAR-8 cells with positive staining of the MX35 epitope with antibodies is less when expressing the NaPi2b mutants compared to the wild type.
 Conclusion. Cysteines C303 and C350 are involved in disulfide bonds formation, which is of key importance for the formation of the conformation of the extracellular domain of the sodium-dependent phosphate transporter NaPi2b, which ensures the MX35 epitope recognition by monoclonal antibodies.

RECOGNITION OF SODIUM-DEPENDENT PHOSPHATE TRANSPORTER NAPI2B BY MONOCLONAL ANTIBODIES IN BACTERIAL AND EUKARYOTIC CELLS

The membrane sodium-dependent phosphate transporter NaPi2b is attractive target for monoclonal antibody therapy. Recognition of the large extracellular domain (ECD) of NaPi2b by monoclonal antibodies L2(20/3) in Western blot analysis depends on the addition of DTT to lysates of eukaryotic cells, but not bacterial cells.

Inhibition of the CD47–SIRPα axis by TTI-621 to treat relapsed or refractory mycosis fungoides or Sézary syndrome

In a large, retrospective follow-up trial from the Cutaneous Lymphoma International Consortium, it was shown that among both US and non-US centres, there was a large treatment heterogeneity in advanced mycosis fungoides and Sézary syndrome.1 The differences in treatment were not associated with a difference in survival between US and non-US centres; however, the data showed that chemotherapy as first treatment was associated with a higher risk of death or change in therapy. Therefore, other therapeutic options should preferably serve as the first treatment approach,1 and it is agreed worldwide that new relevant targets for monoclonal antibody therapy are needed.

Photosensitizer With Illumination Enhances In Vivo Antitumor Effect of Anti-ROBO1 Immunotoxin on Maxillary Sinus Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common types of cancer worldwide. Our study focused on the axon guidance receptor roundabout guidance receptor 1 (ROBO1) as a target for monoclonal antibody therapy of HNSCC. We previously showed that saporin-conjugated anti-ROBO1 (B5209B) immunotoxin (IT-ROBO1) enhanced cytotoxic effects on HNSCC cells in combination with the photosensitizer aluminum phthalocyanine disulphonate (AlPcS2a) and illumination. We examined the effects of this combination therapy in a mouse xenograft model. IT-ROBO1 was intraperitoneally administered to HSQ-89 (derived from Japanese maxillary sinus squamous carcinoma, RCB0789; RIKEN, Tsukuba, Japan) xenografted mice. After 3 days, AlPcS2a was injected subcutaneously around the tumor and the area was illuminated at 650 nm for 30 min. The growth of the tumor was evaluated and the effects on the tumor were examined. Pronounced anti-tumor effects were elicited by the administration of IT-ROBO1 and AlPcS2a with light illumination on tumor size and pathological characteristics. The results showed that photosensitizer treatment with illumination robustly enhanced the antitumor effect of the IT-ROBO1 immunotoxin.

Membranous Expression of pan CD66, CD66a, CD66b, and CD66c and their Clinical Impact in Acute Leukemia: Cross Sectional Longitudinal Cohort Study in Saudi Arabia

CD66 and its isoforms modulate several physiologic processes and have a role in aggressiveness of malignancies. We aimed at investigating pan CD66, CD66 a, b, and c expression and their clinical implication in acute leukemia. This study included 85 cases, 50 AML, 33 ALL and 2 mixed lineage leukemia from King Abdullah Medical City, Saudi Arabia. Pan CD66, CD66a, CD66b and CD66c were detected by flow cytometry at diagnosis and pan CD66 was reanalyzed at day28. Pan CD66 and CD66c expression rate was 51.8% in B-ALL and significantly correlated with BCR/ABL gene, P-value 0.037. CD66a was detected in 11.1% and significantly associated with shorter overall survival (OS), P-value 0.045. In AML, the expression rates were 40%, 28% and 32% for pan CD66, CD66b and CD66c respectively. CD66b was significantly correlated with favorable cytogenetic and prolonged OS, P-value 0.001 and 0.025 respectively. CD66c was correlated with CD25 positivity, P-value 0.003. The expression of pan CD66 at diagnosis and day 28, were significantly correlated, P-value <0.0001. Accordingly, pan CD66 could be added to the panel for MRD. Our data were encouraging to our center to follow other centers that already included CD66c in their panel for MRD detection. CD66c may be tried as a target for monoclonal antibody therapy in CD66c positive acute leukemia. Large-scale studies are needed to verify the association of CD66b expression with cytogenetics and survival in AML

FAT1: a potential target for monoclonal antibody therapy in colon cancer

Background:Colorectal cancer (CRC) is one of the major causes of cancer-associated mortality worldwide. The currently approved therapeutic agents have limited efficacy.Methods:The atypical cadherin FAT1 was discovered as a novel CRC-associated protein by using a monoclonal antibody (mAb198.3). FAT1 expression was assessed in CRC cells by immunohistochemistry (IHC), immunoblots, flow cytometry and confocal microscopy. In addition, in vitro and in vivo tumour models were done to assess FAT1 potential value for therapeutic applications.Results:The study shows that FAT1 is broadly expressed in primary and metastatic CRC stages and detected by mAb198.3, regardless of KRAS and BRAF mutations. FAT1 mainly accumulates at the plasma membrane of cancer cells, whereas it is only marginally detected in normal human samples. Moreover, the study shows that FAT1 has an important role in cell invasiveness while it does not significantly influence apoptosis. mAb198.3 specifically recognises FAT1 on the surface of colon cancer cells and is efficiently internalised. Furthermore, it reduces cancer growth in a colon cancer xenograft model.Conclusions:This study provides evidence that FAT1 and mAb198.3 may offer new therapeutic opportunities for CRC including the tumours resistant to current EGFR-targeted therapies.

Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy.

IntroductionPodocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression.MethodsWe silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice.ResultsAlthough deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis.ConclusionsWe show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0562-7) contains supplementary material, which is available to authorized users.

Expression of enterotoxin-coding genes in methicillin-resistant Staphylococcus aureus strains isolated from Mexican haemodialysis patients.

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) causes severe catheter-related infections in haemodialysis patients ranging from local-site infections and septic thrombophlebitis to bacteraemia but the associated virulence factors and exotoxins remain unclear.FindingsWe employed an in vitro infection model using reconstituted human epithelium (RHE) to analyse the expression profiles of 4 virulence genes and 12 exotoxin-coding virulence genes in 21 MRSA strains isolated from catheter-related infections in 21 Mexican patients undergoing haemodialysis.All 21 strains (100%) expressed the seg, seh, sei, eta, etb, or hla genes coding staphylococcal toxins. Eleven MRSA strains (52.3%) expressed the sea gene coding staphylococcal enterotoxin A, and two strains (9.5%) expressed the v8 gene coding serine protease. The tst, chp, and arcA genes coding toxic shock syndrome toxin 1, chemotaxis inhibitory protein, and arginine deiminase, respectively, were expressed in separate single strains (4.7%). The most frequent expression profile (42.8% of the strains) comprised seg, seh, sei, eta, etb, and hla.ConclusionIt is likely that the SEG, SEH, SEI, ETA, ETB, and Hla toxins may play a role in MRSA catheter-related infections. Consideration of these toxins in the development of a vaccine or as targets for monoclonal antibody therapy could provide an improved therapeutic strategy for the treatment of catheter-related infections in haemodialysis patients.

Abstract 4622: Discovery of a novel target for monoclonal antibody therapy of breast and ovary cancers

Abstract In our recent research activities, we identified a panel of novel candidate markers for prevalent cancers by a systematic immune-histochemical screening with a large collection of polyclonal antibodies (approximately 1600) raised against membrane-associated and secreted human proteins currently marginally characterized. Of utmost interest were those proteins over-expressed detected in one or more tumors showing surface exposure on cancer cells. Among them, here we describe the molecular characterization of a novel surface-associated protein (EXN36) belonging to the lectin-binding protein family for which very little is presently known. We found that the protein is mainly over-expressed in breast and ovary cancers (frequency of approximately 30-40% of tested cases, based on analysis of 50 patients) and it is involved in cell proliferation, migration and invasiveness, as demonstrated by gene silencing. With the intent of exploiting EXN36 as therapeutic target for monoclonal antibody (mAb) therapy, highly specific murine mAbs were generated and proved to recognize the protein on the surface of selected breast and ovary cell lines. Of particular interest are two mAbs, which, upon binding, are efficiently internalized by breast and ovary cancer cells. These two mAbs influence the viability of breast and ovary cells in vitro. One of them shows antibody-dependent cell-mediated cytotoxicity in vitro in breast cancer cells. An analysis of their biodistribution in normal human tissues showed that these mAbs have limited cytoplasmic reactivity in some tissues. Overall, the functional properties of the anti-EXN36 mAbs make them amenable for the generation of antibody-drug conjugates (ADCs). Moreover, they could be exploited for immune-nanoparticles targeted delivery systems. In vivo efficacy in breast and ovary xenograft cancer models are ongoing to evaluate their therapeutic activity either as naked antibodies or ADCs. Overall, data indicate that EXN36 and the specific mAbs represent innovative and promising tools for a targeted mAb therapy of breast and colon cancer, either alone or in combinatorial therapeutic strategies. Citation Format: Alberto Grandi, Susanna Campagnoli, Matteo Parri, Elisa De Camilli, Boquan Jin, Paolo Sarmientos, Guido Grandi, Luigi Terracciano, Giuseppe Viale, Piero Pileri, Renata Maria Grifantini. Discovery of a novel target for monoclonal antibody therapy of breast and ovary cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4622. doi:10.1158/1538-7445.AM2014-4622

Novel IgM-derived antibody-drug conjugate selectively kills chronic lymphocytic leukemia cells through binding of Fcμ-receptor leading to rapid internalization and intracellular cytotoxic payload delivery (VAC3P.953)

Abstract The IgM Fc receptor (FcμR) is a previously unexploited target for monoclonal antibody therapy and antibody drug conjugate (ADC) therapy of B-cell chronic lymphocytic leukemia (CLL). While anti-FcμR IgG binding does not cause FcμR internalization, IgM bound to FcμR are rapidly internalized (&amp;gt;80% internalized within 5 minutes). FcμR is overexpressed in CLL with only limited expression on normal B, T, and NK cells. In our study, 29 out of 30 CLL patient samples (97%) evaluated by flow cytometry had high FcμR expression by CLL cells (mean(SD)=89.1(7.1)%) and the remaining patient had moderate CLL FcμR expression (57.9%). In contrast, FcμR expression by T cells (mean(SD)=8.9(5.2)%) was significantly lower (p&amp;lt;0.0001). We developed a FcμR targeting ADC consisting of the CH2-CH3-CH4 IgM regions conjugated to monomethyl auristatin F. Flow cytometric analyses of CLL patient samples showed potent ADC mediated killing of CLL cells (mean(SD)=78.8(10.1)%, p&amp;lt; 0.0001 versus scaffold treated control) but not T cells (mean(SD)=8.4(3.8)%, p=0.3210 versus scaffold treated control). The ADC also greatly reduced tumor burden in NOD/SCID/IL-2Rγnull mice xenografted with 4 CLL patient samples (mean reduction(SD)=74(20)% in blood and 65(19)% in spleen, n=30). In summary, FcμR is a promising new target for therapy of CLL and possibly other malignancies. Our preclinical results provide translational evidence for the clinical utility of IgM-derived protein scaffolds to target FcμR expressing cells.

New strategies for treatment of inflammatory bowel disease.

The etiology of inflammatory bowel disease (IBD), of which ulcerative colitis (UC) and Crohn’s disease (CD) are the two most prevailing entities, is unknown. However, IBD is characterized by an imbalanced synthesis of pro-inflammatory mediators of the inflamed intestine, and for more than a decade tumor necrosis factor-(TNF) α has been a major target for monoclonal antibody therapy. However, TNF inhibitors are not useful for one third of all patients (i.e. “primary failures”), and further one third lose effect over time (“secondary failures”). Therefore, other strategies have in later years been developed including monoclonal antibodies targeting the interleukin (IL)-6 family of receptors (the p40 subunit of IL-12/IL-23) as well as monoclonal antibodies inhibiting adhesion molecules (the α4β7 heterodimers), which direct leukocytes to the intestinal mucosa. Recently, small molecules, which are inhibitors of Janus kinases (JAKs), hold promise with a tolerable safety profile and efficacy in UC, and the field of nanomedicine is emerging with siRNAs loaded into polyactide nanoparticles that may silence gene transcripts at sites of intestinal inflammation. Thus, drug development for IBD holds great promise, and patients as well as their treating physicians can be hopeful for the future.

Expression of enterotoxin-coding genes in methicillin-resistant Staphylococcus aureus strains isolated from Mexican haemodialysis patients

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) causes severe catheter-related infections in haemodialysis patients ranging from local-site infections and septic thrombophlebitis to bacteraemia but the associated virulence factors and exotoxins remain unclear.FindingsWe employed an in vitro infection model using reconstituted human epithelium (RHE) to analyse the expression profiles of 4 virulence genes and 12 exotoxin-coding virulence genes in 21 MRSA strains isolated from catheter-related infections in 21 Mexican patients undergoing haemodialysis.All 21 strains (100%) expressed the seg, seh, sei, eta, etb, or hla genes coding staphylococcal toxins. Eleven MRSA strains (52.3%) expressed the sea gene coding staphylococcal enterotoxin A, and two strains (9.5%) expressed the v8 gene coding serine protease. The tst, chp, and arcA genes coding toxic shock syndrome toxin 1, chemotaxis inhibitory protein, and arginine deiminase, respectively, were expressed in separate single strains (4.7%). The most frequent expression profile (42.8% of the strains) comprised seg, seh, sei, eta, etb, and hla.ConclusionIt is likely that the SEG, SEH, SEI, ETA, ETB, and Hla toxins may play a role in MRSA catheter-related infections. Consideration of these toxins in the development of a vaccine or as targets for monoclonal antibody therapy could provide an improved therapeutic strategy for the treatment of catheter-related infections in haemodialysis patients.

Abstract C165: Discovery of a lectin-like receptor family protein as a novel target for monoclonal antibody therapy.

Abstract In our recent research activities, we identified a panel of novel candidate markers for prevalent cancers by a systematic tissue microarray analysis (TMA) of a large collection of polyclonal antibodies (approximately 1600) raised against membrane-associated and secreted human proteins currently marginally characterized. Of major interest were those proteins specifically detected in one or more of the five tumors with significant frequencies (data from 50 patients per each of the five cancers) and showing surface exposure on cancer cells. Among them, here we describe the molecular characterization of novel surface-associated protein belonging to the lectin-binding family proteins (involved in the innate immune response) for which very little knowledge exists. The protein was mainly over-expressed in breast, lung and ovary cancers (approximately 30-40% of tested cases) and it is involved in cell proliferation, migration and and invasiveness, as demonstrated by gene silencing using specific siRNA technology. Highly specific murine monoclonal antibodies (mAbs) were generated toward it are able to recognize the protein on the surface of breast and ovary cell lines. Of particular interest are two mAbs, which, upon binding, are efficiently internalized by breast and ovary cancer cells, indicating that they are amenable for the generation of antibody-drug conjugates. These two mAbs influence viability of breast and ovary cells in vitro. One of them shows antibody-dependent cell-mediated cytotoxicity in vitro on breast cancer cells. In vivo efficacy studies are ongoing to evaluate their therapeutic activity. Overall, data indicate that the lectin-binding protein and the specific mAbs here described could be developed as a novel tool for a targeted therapy alone or in combination with other treatments. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C165. Citation Format: Susanna Campagnoli, Matteo Parri, Alberto Grandi, Elisa De Camilli, Luigi Terracciano, Boquan Jin, Giuseppe Viale, Paolo Sarmientos, Guido Grandi, Piero Pileri, Renata Maria Grifantini. Discovery of a lectin-like receptor family protein as a novel target for monoclonal antibody therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C165.

Abstract 5533: Novel targets and monoclonal antibodies for cancer therapy.

Abstract Cancer therapeutic targets is an extremely active research field in both academia and pharmaceutical companies. In our recent research activities we identified by a high-throughput immunohistochemistry screening of Tissue microarray (TMA), a panel of 89 novel candidate tumor markers for prevalent cancers representing breast, lung colon ovary and prostate carcinomas. The novel marker candidates were found over-expressed in one or more of the five tumors under analysis, with significant frequency. Three of them seems to be promising therapeutic targets for monoclonal antibody therapy, being exposed on the surface of cancer cells. They include: 1) a lectin binding protein, over-expressed in breast, lung and ovary cancer; 2) a protein involved in iron homoeostasis and over-expressed in breast, colon, lung and ovary cancers, 3) a cadherin homologous protein detected in colon, lung and ovary cancers. Gene silencing using siRNA technology and/or over-expression experiments using marker-encoding plasmids significantly alter cell proliferation, migration, invasiveness and clonal growth in vitro, indicating that expression of the three proteins confers cell phenotypes relevant for tumor progression. Highly specific murine monoclonal antibodies (mAbs) were generated against the three proteins and proved to bind the surface of cancer cells lines. Of particular interest is a murine mAb targeting the cadherin-like protein that, upon binding to the cell surface, is efficiently internalized by cancer cells, suggesting that it is amenable to the development of antibody-drug conjugates. This mAb did not show any relevant IHC cross-reactivity in the 35 human tissues requested by FDA to demonstrate antibody specificity. Furthermore this mAb significantly inhibited tumor growth in athymic nude mice bearing HCT15 and HT29 colon cancer xenograft models. Finally, IHC analysis of approximately 300 colon cancer clinical samples indicates that the antibody stains a large fraction of colon cancer samples, and gives intense membranous staining in specific patients’ group. Overall, data indicate that this antibody could be developed as a novel tool for a targeted therapy of colo-rectal cancer, alone or in combination with other treatments Citation Format: Alberto Grandi, Matteo Parri, Susanna Campagnoli, Renzo Nogarotto, Elisa De Camilli, Ilaria Naldi, Caterina Cinti, Paolo Sarmientos, Guido Grandi, Luigi Terracciano, Giuseppe Viale, Piero Pileri, Renata Maria Grifantini. Novel targets and monoclonal antibodies for cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5533. doi:10.1158/1538-7445.AM2013-5533

The Role of Mesothelin in Tumor Progression and Targeted Therapy

Mesothelin, a glycosylphosphatidylinositol (GPI) anchored cell surface protein, is a potential target for antibody-based cancer therapy due to its high expression in mesothelioma, ovarian cancer, pancreatic cancer, cholangiocarcinoma and other cancers. The SS1P immunotoxin and MORAb-009 (amatuximab), a chimeric monoclonal antibody, are currently being evaluated in clinical trials. In this review, we discuss the role of mesothelin in cancer progression and provide new insights into mesothelin-targeted cancer therapy. Recent studies highlight three mechanisms by which mesothelin plays a role in cancer progression. First, mesothelin may aid in the peritoneal implantation and metastasis of tumors through its interaction with mucin MUC16 (also known as CA125). Second, mesothelin may promote cancer cell survival and proliferation via the NF-κB signaling pathway. Finally, mesothelin expression promotes resistance to certain chemotherapy drugs such as TNF-α, paclitaxel, and a combination of platinum and cyclophosphamide. However, its cancerspecific expression makes mesothelin a potential target for monoclonal antibody therapy. New human monoclonal antibodies targeting mesothelin have been isolated by phage display technology and may provide opportunities for novel cancer therapy.

547 Novel Targets for Monoclonal Antibody Therapy

547 Novel Targets for Monoclonal Antibody Therapy

Dlk-1, a cell surface antigen on foetal hepatic stem/progenitor cells, is expressed in hepatocellular, colon, pancreas and breast carcinomas at a high frequency

Delta-like 1 protein (Dlk-1), also known as preadipocyte factor 1 (Pref-1), is a transmembrane and secreted protein with epidermal growth factor (EGF)-like repeats. Dlk-1 is known to be expressed in foetal liver, but absent in neonatal and adult liver in mice and rats. Dlk-1 is also expressed in a subpopulation of hepatic oval cells, which are considered as stem/progenitor cells in rat adult liver. In this study, we generated monoclonal antibodies against human Dlk-1 (hDlk-1) and investigated hDlk-1 expression in human liver and hepatocellular carcinoma (HCC). Like rodent livers, hDlk-1 was detected in foetal liver, but not in adult liver. In HCC, hDlk-1 was positive for 20.5% of the cases examined and was localized in both cytoplasm and cell membrane, whereas hDlk-1 was undetected in viral hepatitis, nodular cirrhosis. Interestingly, hDlk-1 positive HCC was found more frequently in younger patients and its expression was correlated with alpha-fetoprotein expression. Furthermore, hDlk-1 was also detected frequently in colon adenocarcinomas (58%), pancreatic islet carcinoma (50%), and small cell lung carcinoma (50%). Thus, hDlk-1 is a cell surface protein expressed in many carcinomas including HCC and may be a potential target for monoclonal antibody therapy for carcinomas.

A CD20 tandem-epitope immunogen elicits antibody in mice that binds murine cell surface CD20 and depletes splenic B cells in vivo

CD20 is an important target for monoclonal antibody therapy of B-cell malignancies and for some autoimmune disorders. Though there is interest in evaluating the induction of active immunity to CD20 in the mouse model, the CD20 extracellular domain (ECD) has significant secondary and tertiary structure which make it difficult to target using peptide immunogens. We constructed, expressed, and purified a recombinant immunogen, CD20ECD-6, which displays six tandemly repeated copies of the C-terminus of the murine CD20 ECD covalently linked to maltose-binding protein. Analysis of the purified protein suggested a complex conformation as the protein migrated in significantly retarded fashion by SDS-PAGE analysis. Immunization of mice and rabbits with the CD20ECD-6 led to the induction of antibodies reactive with the C-terminal ECD peptide sequence by ELISA and more importantly, with native cell surface CD20 on the murine B-cell lymphomas, 38C13 and A20. Immunoprecipitation using the rabbit antisera and non-denaturing detergent confirmed the identity of the bound cell surface protein as murine CD20 and suggested that the cell-binding antibodies were specific for the native, folded conformation. Finally, immunization of mice with the CD20ECD-6 using Freund's or QS-21 adjuvants was shown to exert significant biological effects in vivo with the pronounced depletion of splenic B cells. The tandem-epitope immunogen represents a promising tool for eliciting and studying active autoimmunity to CD20, as a basis for potential development of new immunotherapeutics for cancer and autoimmune diseases.

CD32B Expression Reflects Phenotypic and Functional Heterogeneity in Multiple Myeloma (MM)

Intraclonal heterogeneity (IH) in MM may provide a basis for the selection of resistance to therapy that occurs over time. Model systems to study IH in MM have not been defined so we focused on the expression of CD32B, the low-affinity inhibitory Fcγ-receptor that is a key participant in normal plasma cell apoptosis and a potential target for monoclonal antibody therapy. At diagnosis, CD138+ MM marrow cells are largely CD32B+ (n=72; median 94%, range 1–100) while at relapse the CD32B+ MM subpopulation is significantly smaller in both bone marrow (n=25, median 83%, (10–100), P = 0.02) and extramedullary sites (n=8, median 17% (0–78); P << 0.01). Among human MM cell lines only two of ten tested expressed cell-surface CD32B. We hypothesize that the CD32B phenotype of MM subpopulations contributes to the functional heterogeneity that is critical to the selection of resistance, and sought a model system to study CD32B-related IH. The KMS-12BM cell line (BM), a human MM line from marrow, has CD32B+ and CD32B− subpopulations as MM marrows do, while the KMS-12PE cell line (PE), from the pleural fluid of the same patient, is completely CD32B negative, as is often the case in extramedullary MM. Both BM and PE cells share t(11;14), del 17p and other cytogenetic abnormalities, and both contain intact CD32B genes (FCGR2B). We used 3 populations, BM CD32B+, BM CD32B− and PE (CD32B−), as well as sorted patient MM cells, to study the IH associated with CD32B. RNAi studies demonstrated that the expression of FCGR2B in BM cells is dependent on the Ets family transcription factor PU.1, while PE cells do not express PU.1. RT-PCR of patient MM cells sorted into paired CD32B+ and CD32B− fractions showed PU.1 and FCGR2B (CD32B) expression only in CD138+/CD32B+ cells. Cell-tracking studies with CFSE showed that BM CD32B+ cells gave rise to BM CD32B− cells but that the reverse did not occur. Cell-culture studies showed that BM CD32B+ cells have significantly lower apoptotic rates than BM CD32B− cells, while the latter display greater dependence on insulin for survival and on IGF-1 and MIP-1α for proliferation. In order to better understand the differences between CD32B+ and CD32B− populations, we performed transcript profiling. BM CD32B+ cells have significantly higher expression of the activin inhibin, while BM CD32B− cells overexpress insulin-response genes such as the kinase Akt2. Compared to BM cells, PE cells overexpress germ-cell transcription factors and inflammasome-related genes, while BM cells overexpress tetraspanins (CD9, CD37, CD53) and insulin-response genes. Of interest, there are also significant differences in the expression of BMP and Wnt-related genes (but not Hh or Notch genes). BM cells overexpress BMPR1A, FZD2 and Wnt5A while PE cells overexpress CRIM1, FZD1 and LEF1. These data indicate that functional IH exists in MM, that the KMS-12 cell lines provide a useful model for its study, and that MM may originate in a CD32B+ cell that begets CD32B− heirs. We continue to study PU.1 and the generation of CD32B− cells by CD32B+ cells; to validate transcript data using sorted patient specimens; and to assess with RNAi the contributions of BMP, Wnt and insulin-response genes to the survival and proliferation of CD32B+ and CD32B− cells, seeking novel therapeutic targets on which selection of resistance may depend.