Anti-MUC4β antibodies as a novel therapeutic modality for the treatment of pancreatic cancer

The deregulation of cell surface glycoproteins, such as mucins, is a hallmark of many tumors of epithelial origin. The transmembrane mucin MUC4, is differentially overexpressed in several malignancies, including pancreatic, breast, ovarian, lung, cervical, and head and neck cancers. Of all the aforementioned cancers, the role of MUC4 has been the most thoroughly in pancreatic cancer (PC), which is the 4th leading cause of cancer-related deaths in the United States. While MUC4 is undetectable in the normal or inflamed pancreas (pancreatitis), its expression progressively increases during PC progression and its higher expression correlates with poor survival. The role of MUC4 in neoplastic transformation, enhanced motility, invasiveness, and drug resistance of cancer cells in vitro, and in tumorigenicity and metastasis in vivo has been conclusively established. Due to the differential overexpression of MUC4 in cancer cells and its functional involvement in disease pathobiology, it is an attractive therapeutic target. Of specific interest is the MUC4β-subunit of the MUC4 glycoprotein. The α-domain of MUC4 can be shed from the surface of the cell thus rendering it a feckless target. Thus, we propose to target the MUC4β-domain, a growth factor-like subunit that remains attached to the cell surface. To date no MUC4-targeted therapeutics have been developed. In the first part of the dissertation research, I sought to develop monoclonal antibodies (mAb) against the MUC4β subunit. MAb-based therapeutics have emerged as a promising cancer treatment modality due to their low toxicity and high specificity. Antibodies used in the treatment of solid tumors may inhibit oncogenic signaling, block cell-cell interactions, or engage immune effector cells to attack the tumor by antibody-dependent cellular cytotoxicity (ADCC) in a target expression-specific manner. Alternatively, mAbs can serve as vectors to deliver cytotoxic cargo (drugs, radionuclides, or toxins) to cancer cells in antigen-specific manner. The development of MUC4 specific mAbs was achieved through the use of hybridoma technology. The immunization of BALB/c mice with recombinant MUC4β protein purified from E.coli allowed for the development of antibodies specific to our protein of interest. Through the fusion of mouse splenocytes and myeloma cells we were able to create hybridoma cells that had the properties a B-cell allowing for the production and secretion antibodies of and had the immortality conferred by the myeloma cell partner. The antibodies produced from this hybridoma fusion were screened for reactivity against MUCβ recombinant protein and against cell lysates from MUC4-expressing cells to identify antibody clones with high binding affinity. These clones were further purified and taken through specificity testing. We began testing for specificity by Western blotting cell lysates from MUC4-expressing and non-expressing cells lines. Determination of MUC4 molecular weights occurred through the use of both horizontal agarose gels for the detection of full length MUC4 and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) to detect the smaller MUC4β fragment. The specificity testing further extended to testing on the native conformation of the MUC4 protein in flow cytometry and confocal microscopy. The specific detection of MUC4 in PC patient samples was also performed using immunohistochemistry (IHC). The binding affinity of the mAbs was determined in surface plasmon resonance (SPR), which demonstrate binding with nanomolar affinity. Overall, the mAbs were validated for MUC4β specificity and could be used for further cell-based functional testing. To determine the therapeutic potential of MUC4β antibodies, we propose to study their applications for inhibiting MUC4-mediated cell-based functions. Cell-based growth and motility assays were studied as part of the functional application of the MUC4β mAbs. Each assay was performed across several MUC4-expressing and non-expressing cell lines. Growth inhibition was only found to occur to a significant degree for the MUC4-expressing cell line CD18. The inhibition of growth was not found to occur through the induction of apoptosis. The inhibition of motility was only observed to occur to a significant degree for the cell lines engineered to express MUC4. Additional testing included use of the anti-MUC4β mAbs in combination with the chemotherapy gemcitabine against the cell line CD18. This combination demonstrated and additive effect at least equal to each of the therapeutics alone. Together these results demonstrate that the anti-MUC4β mAbs may have the capacity to be used as therapeutic agents and warrant further study in vivo

The Current Landscape of Antibody-based Therapies in Solid Malignancies

Over the past three decades, monoclonal antibodies (mAbs) have revolutionized the landscape of cancer therapy. Still, this benefit remains restricted to a small proportion of patients due to moderate response rates and resistance emergence. The field has started to embrace better mAb-based formats with advancements in molecular and protein engineering technologies. The development of a therapeutic mAb with long-lasting clinical impact demands a prodigious understanding of target antigen, effective mechanism of action, gene engineering technologies, complex interplay between tumor and host immune system, and biomarkers for prediction of clinical response. This review discusses the various approaches used by mAbs for tumor targeting and mechanisms of therapeutic resistance that is not only caused by the heterogeneity of tumor antigen, but also the resistance imposed by tumor microenvironment (TME), including inefficient delivery to the tumor, alteration of effector functions in the TME, and Fc-gamma receptor expression diversity and polymorphism. Further, this article provides a perspective on potential strategies to overcome these barriers and how diagnostic and prognostic biomarkers are being used in predicting response to mAb-based therapies. Overall, understanding these interdependent parameters can improve the current mAb-based formulations and develop novel mAb-based therapeutics for achieving durable clinical outcomes in a large subset of patients

L'écriture de la vieillesse dans la littérature allemande contemporaine

Chronique sous la direction de Mihaela AilincaiInternational audienc

Development and characterization of carboxy-terminus specific monoclonal antibodies for understanding MUC16 cleavage in human ovarian cancer

<div><p>MUC16 is overexpressed in ovarian cancer and plays important roles in invasion and metastasis. Previously described monoclonal antibodies against cell surface expressed MUC16 recognize the N-terminal tandemly repeated epitopes present in cancer antigen 125 (CA125). MUC16 is cleaved at a specific location, thus, releasing CA125 into the extracellular space. Recent reports have indicated that the retained carboxy-terminal (CT) fragment of MUC16 might play an important role in tumorigenicity in diverse types of cancers. However, limited data is available on the fate and existence of CT fragment on the surface of the cancer cell. Herein, we characterize two monoclonal antibodies (mAbs) showing specificity to the retained juxtamembrane region of MUC16. For the first time, we demonstrate that MUC16 is cleaved in ovarian cancer cells (NIH:OVCAR-3 [OVCAR-3]) and that the cleaved MUC16 subunits remain associated with each other. Immunohistochemical analyses on different grades of ovarian tumor tissues indicated differential reactivity of CA125 and MUC16 CT mAbs. The CA125 (M11) mAb detected 32/40 (80%), while the CT mAb (5E6) detected 33/40 (82.5%) of total ovarian cancer cases. For serous and serous papillary cases, the CA125 (M11) mAb stained 27/31 cases (87%), while CT mAb (5E6) stained 29/31 cases (93.5%). The CT mAb(s) accurately predict expression of MUC16 since their epitopes are not tandemly repeated and their reactivity may not be dependent on O-linked glycosylation. These antibodies can serve as valuable reagents for understanding MUC16 cleavage and may also serve as potential therapeutic agents for treatment of ovarian cancer.</p></div

Variations in the first steps of photosynthesis for the diatom Cyclotella meneghiniana grown under different light conditions

In this work we have applied picosecond and steady-state fluorescence measurements to study excitation energy transfer and trapping in intact Cyclotella meneghiniana diatom cells grown at different light intensities. Different excitation and detection wavelengths were used to discriminate between Photosystem I and II (PSI and PSII) kinetics and to study excitation energy transfer from the outer antenna to the core of PSI and PSII. It is found that the light-harvesting fucoxanthin chlorophyll proteins (FCPs) transfer their excitation energy predominantly to PSII. It is also observed that the PSII antenna is slightly richer in red-absorbing fucoxanthin than the FCPs associated with PSI. The average excitation trapping time in PSI is around 75 ps whereas this time is around 450 ps for PSII in cells grown in 20 µmol of photons per m2 per s. The latter time decreases to 425 ps for 50 µmol of photons and 360 ps for 140 µmol of photons. It is concluded that cells grown under higher photon flux densities have a smaller antenna size than the ones grown in low light. At the same time, the increase of growth light intensity leads to a decrease of the relative amount of PSI. This effect is accompanied by a substantial increase in the amount of chlorophyll a that is not active in excitation energy transfer and most probably attached to inactivated/disassembled PSII units

Narrowing down the epitope recognized by MUC16 CT mAbs.

<p>(<b>A</b>) The last 12 and 29 amino acids from TM domain of MUC16 were deleted in the F114HA construct, tagged with FLAG at N-terminus and HA at C-terminus, to generate 2 deletion constructs (Δ12 and Δ29). The Δ12 and Δ29 constructs were transfected into HEK293T cells and immunoblotted with the indicated antibodies. (<b>B</b>) Schematic representation of MUC16 C-terminal region indicating various domains, cleavage site and approximate location of the putative epitope recognized by mAbs 5E6and 3H1. <u>Key</u>: Purple—represents the last SEA domain. Red portion—indicates the start of the transmembrane domain. Boxed region—indicates the putative epitope. Underlined region—represents the predicted putative cleavage site.</p

Generation and characterization of monoclonal antibodies (mAbs) to MUC16 C-terminal (CT) domain.

<p>(<b>A</b>) Structure of MUC16 CT domain indicating the two membrane-proximal cleavage sites. The fragment used for generation of hybridomas is indicated by a line with double arrow heads. This fragment incorporates the last putative cleavage site. Other important domains are indicated: TM- Transmembrane domain; Cyt. Tail- Cytoplasmic tail. (<b>B</b>) Binding of selected anti-MUC16 CT monoclonal antibodies with purified MUC16 CT protein using an indirect ELISA. MUC16 CT protein was coated in ELISA wells and hybridoma supernatants of the indicated antibodies were added. Antibody binding was detected using a secondary antibody labeled with horseradish peroxidase and TMB substrate. (<b>C</b>) Flow cytometry analysis showing relative binding of anti-MUC16 CT mAbs (5E6 and 3H1) to MIA PaCa-2 cells transfected either with control vector or MUC16 CT FL 321 construct (last 321 amino acids of MUC16). Binding was also analyzed on OVCAR-3 (MUC16_HIGH) and OVCAR-5 (MUC16_LOW) cells. Anti- tandem repeat mAb M11 served as a positive control. Cells were stained with the indicated antibodies and the signal was detected using Alexa-Fluor 488 anti-mouse IgG secondary antibody. A mouse IgG1 antibody served as the irrelevant isotype control and is indicated by the gray shaded curve. (<b>D</b>) Flow cytometry analysis of OVCAR-3 cells using mAbs pre-incubated with either MUC16 peptide or irrelevant control peptide. (<b>E and F</b>) OVCAR-3 and OVCAR-5 cells were seeded on coverslips, fixed with 4% Paraformaldehyde in PBS and were either permeabilized with 0.1% Triton X-100 in PBS (E) or not permeabilized (F) and incubated with 10 μg/ml of indicated mAbs. Signal was detected using Alexa Fluor 488 conjugated secondary antibody. Coverslips were placed on glass slides containing a drop of anti-fade Vectashield mounting medium and observed under a ZEISS confocal laser scanning microscope (magnification, 630X).</p

MUC16 CT mAb 5E6 exhibits heterogeneous staining on human ovarian cancer tissues.

<p>Sections of ovarian cancer tissues indicating strong membranous and cytoplasmic staining in tumor cells (A), focal apical membranous staining on tumor cells (B), and strong intra-luminal and membranous staining (C) of MUC16 CT. In all cases, the surrounding stroma was negative for MUC16 CT expression. Original magnification 200х.</p

Partial epitope mapping using MUC16 CT constructs transfected into HEK293T cells.

<p><b>(A</b>) Schematic representation of different lengths of MUC16 CT fragments with C-terminal HA-tag cloned into the p3X-FLAG-CMV9 vector (Empty vector) with a preprotrypsin leader peptide (LP). The predicted cleavage sites in the last (site #1, PLARRVDR) and penultimate (site #2, DSVLV) SEA domains and the transmembrane (TM) domain are indicated. (<b>B</b>) Partial epitope mapping using various constructs of MUC16 CT (given in (<b>A</b>)) transfected into HEK293T cells was performed. Lysates from transfected cells were immunoblotted with the indicated antibodies. (<b>C</b>) The Flag tagged F114HA MUC16 CT construct was domain swapped with the various domains of MUC4 as indicated. (<b>D</b>) The constructs described in (<b>C</b>) were transfected into HEK293T cells. Lysates from these cells were immunoblotted with the respective antibodies.</p