Distinct Cell Surface Molecules Research Articles

Abstract 2984: Design and characterization of bispecific engineered toxin bodies for targeted cancer therapy

Abstract Engineered Toxin Bodies (ETBs) are a distinct class of targeted immunotoxins in development as anti-cancer therapeutics by Molecular Templates. ETBs drive a potent and targeted response mediated by antibody-like binding, induced internalization, and enzymatic ribosomal inhibition via the delivery of a Shiga-like toxin subunit A (SLTA) that has been proprietarily modified to avoid innate and adaptive immune recognition. MTEM has a pipeline of scFv targeted ETBs, including its clinical lead MT-3724. MT-3724 targets CD20 for treatment of B-cell lymphoma and has shown promising signs of activity in heavily pretreated patients. The novel MOA inherent to ETB therapeutics allows for activity in relapsed/refractory settings, as previously acquired resistance mechanisms should not hinder efficacy. To further expand the therapeutic benefit of the platform, MTEM is characterizing ETBs that are targeted through multiple binding domains. Bispecific ETBs that target two epitopes on the same receptor, or two distinct cell surface molecules both expressed on cancer cells, may allow for enhanced activity profiles. These possibilities include: (i) activity in the presence of a competitive binding protein (ii) sustained activity when one target molecule is shed or downregulated, (iii) synergistic binding events to increase overall potency, and (iv) increased specificity towards cancer over normal tissue. MTEM has performed in vitro experiments to explore the pairing of binding domains into a single fusion protein, resulting in two targeting arms and a single SLTA subunit. Included in these efforts are the design and characterization of ETBs harboring a single domain antibody derived from camelids, or VHH domains, paired in tandem with other VHH domains or with scFv binding domains to produce both bispecific and biparatopic ETBs. Bispecific ETBs have been generated to engage a variety of target combinations, relevant to both solid and hematologic cancer treatment. In vitro characterization studies conducted include cellular cytotoxicity assays and on-cell binding assays. Bispecific ETBs highlighted in this presentation demonstrate functionality of each targeting arm within a single ETB molecule. In addition, we have shown that one arm can bind target and elicit cytotoxicity even when the other target is blocked. Furthermore, we are learning that some bispecific combinations are cooperative and achieve activities in vitro not observed with the parent single-target ETBs from which they were derived. MTEM is exploring therapeutically relevant target combinations towards the development of a bispecific clinical lead. By pairing the biology and potency of ETB-mediated killing with the expanded targeting possibilities afforded by bispecific molecules, we aim to address unmet medical needs in cancer therapy. Citation Format: Aimee Iberg, Garrett L. Cornelison, Caleigh Howard, Paul Amador, Steven Rivera, Michael Jamaleddine, Garrett L. Robinson, Erin K. Willert. Design and characterization of bispecific engineered toxin bodies for targeted cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2984.

Dual-Affinity Re-Targeting proteins direct T cell-mediated cytolysis of latently HIV-infected cells.

Enhancement of HIV-specific immunity is likely required to eliminate latent HIV infection. Here, we have developed an immunotherapeutic modality aimed to improve T cell-mediated clearance of HIV-1-infected cells. Specifically, we employed Dual-Affinity Re-Targeting (DART) proteins, which are bispecific, antibody-based molecules that can bind 2 distinct cell-surface molecules simultaneously. We designed DARTs with a monovalent HIV-1 envelope-binding (Env-binding) arm that was derived from broadly binding, antibody-dependent cellular cytotoxicity-mediating antibodies known to bind to HIV-infected target cells coupled to a monovalent CD3 binding arm designed to engage cytolytic effector T cells (referred to as HIVxCD3 DARTs). Thus, these DARTs redirected polyclonal T cells to specifically engage with and kill Env-expressing cells, including CD4+ T cells infected with different HIV-1 subtypes, thereby obviating the requirement for HIV-specific immunity. Using lymphocytes from patients on suppressive antiretroviral therapy (ART), we demonstrated that DARTs mediate CD8+ T cell clearance of CD4+ T cells that are superinfected with the HIV-1 strain JR-CSF or infected with autologous reservoir viruses isolated from HIV-infected-patient resting CD4+ T cells. Moreover, DARTs mediated CD8+ T cell clearance of HIV from resting CD4+ T cell cultures following induction of latent virus expression. Combined with HIV latency reversing agents, HIVxCD3 DARTs have the potential to be effective immunotherapeutic agents to clear latent HIV-1 reservoirs in HIV-infected individuals.

The Nogo Receptor NgR1 Mediates Infection by Mammalian Reovirus

Neurotropic viruses, including mammalian reovirus, must disseminate from an initial site of replication to the central nervous system (CNS), often binding multiple receptors to facilitate systemic spread. Reovirus engages junctional adhesion molecule A (JAM-A) to disseminate hematogenously. However, JAM-A is dispensable for reovirus replication in the CNS. We demonstrate that reovirus binds Nogo receptor NgR1, a leucine-rich repeat protein expressed in the CNS, to infect neurons. Expression of NgR1 confers reovirus binding and infection of nonsusceptible cells. Incubating reovirus virions with soluble NgR1 neutralizes infectivity. Blocking NgR1 on transfected cells or primary cortical neurons abrogates reovirus infection. Concordantly, reovirus infection is ablated in primary cortical neurons derived from NgR1 null mice. Reovirus virions bind to soluble JAM-A and NgR1, while infectious disassembly intermediates (ISVPs) bind only to JAM-A. These results suggest that reovirus uses different capsid components to bind distinct cell-surface molecules, engaging independent receptors to facilitate spread and tropism.

Advanced immunophenotyping: more is better!

Recent advances in multicolor flow cytometry have made possible the simultaneous analysis of up to 12 distinct cell surface molecules. This technical advance increases sensitivity and specificity for more accurate diagnosis and classification of various malignancies. In turn, this allows for a better assessment of disease prognosis and the establishment of a treatment regimen. In addition, multicolor flow cytometry provides a sensitive technique for investigating the occurrence of minimal residual disease. Studies of leukemias and lymphomas are particularly suitable for investigation by flow cytometry. Studies on solid tumors such as breast cancer, or on tumor infiltrating immune cells, are also possible if appropriate processing techniques are used to generate single-cell suspensions without altering cell surface receptor expression. In addition to analytical immunophenotyping, isolation of even very small (malignant) cell populations by multicolor flow cytometry provides a highly pure source for DNA and RNA analyses. Such combined sorting and molecular approaches provide a comprehensive basis for studying the molecular mechanisms underlying malignant transformation. Importantly, they also enable distinct diagnoses to be made when cell surface marker immunophenotyping alone is inconclusive. To exemplify the importance of combining various immunophenotyping tools, such as multicolor flow cytometry and gene expression analyses, with the classical tools of histopathology and differential blood count, we provide data on the analysis of a mouse model of human chronic B-cell leukemia (B-CLL): the New Zealand Black mouse. We relied for this study on gene expression databases generated by the National Cancer Institute for various human B-cell malignancies. B-CLL is one of the most common forms of leukemia in the Western world. No therapies currently exist against this usually slow-progressing but always fatal malignancy, making the development of a good animal model an important task. The data show that although the New Zealand Black mouse shows a spontaneous expansion of a CD5+ B-cell population (a hallmark of human B-CLL), it does not fit the classification of B-CLL by either phenotypic or genetic analysis criteria. In conclusion, increasing the number of simultaneous measurements for immunophenotyping and cell sorting by flow cytometry and combining it with comprehensive gene expression studies supports and expands the power of classical analysis tools.

GRP78, a coreceptor for coxsackievirus A9, interacts with major histocompatibility complex class I molecules which mediate virus internalization.

It is becoming apparent that over the years cell infection by virus seems to have evolved into a multistep process in which many viruses employ distinct cell surface molecules for their attachment and cell entry. In this study the attachment and entry pathway of coxsackievirus A9 (CAV-9), a member of the Picornaviridae family, was investigated. It has been known that, although integrin alpha(v)beta3 is utilized as a receptor, its presence alone is insufficient for CAV-9 infection and that CAV-9 also requires a 70-kDa major histocompatibility complex class I (MHC-I)-associated protein (MAP-70) as a coreceptor molecule. We document by protein isolation and peptide sequencing that the 70-kDa protein is GRP78, a member of the heat shock protein 70 family of stress proteins. Furthermore we show by using fluorescence resonance energy transfer (FRET) that GRP78 is also expressed on the cell surface and associates with MHC-I molecules. In addition CAV-9 infection of permissive cells requires GRP78 and also MHC-I molecules, which are essential for virus internalization. The identification of GRP78 as a coreceptor for CAV-9 and the revelation of GRP78 and MHC-I associations have provided new insights into the life cycle of CAV-9, which utilizes integrin alpha(v)beta3 and GRP78 as receptor molecules whereas MHC-I molecules serve as the internalization pathway of this virus to mammalian cells.

Dendritic cells genetically engineered to express Fas ligand induce donor-specific hyporesponsiveness and prolong allograft survival.

Polarization of an immune response toward tolerance or immunity is dictated by the interactions between T cells and dendritic cells (DC), which in turn are modulated by the expression of distinct cell surface molecules, and the cytokine milieu in which these interactions are taking place. Genetic modification of DC with genes coding for specific immunoregulatory cell surface molecules and cytokines offers the potential of inhibiting immune responses by selectively targeting Ag-specific T cells. In this study, the immunomodulatory effects of transfecting murine bone marrow-derived DC with Fas ligand (FasL) were investigated. In this study, we show that FasL transfection of DC markedly augmented their capacity to induce apoptosis of Fas+ cells. FasL-transfected DC inhibited allogeneic MLR in vitro, and induced hyporesponsiveness to alloantigen in vivo. The induction of hyporesponsiveness was Ag specific and was dependent on the interaction between FasL on DC and Fas on T cells. Finally, we show that transfusion of FasL-DC significantly prolonged the survival of fully MHC-mismatched vascularized cardiac allografts. Our findings suggest that DC transduced with FasL may facilitate the development of Ag-specific unresponsiveness for the prevention of organ rejection. Moreover, they highlight the potential of genetically engineering DC to express other genes that affect immune responses.

A Cell Surface ADP-ribosyltransferase Modulates T Cell Receptor Association and Signaling

ART-1, a cell surface ADP-ribosyltransferase, is imbedded in the membrane by a glycosylphosphatidylinositol anchor. Function of this enzyme in mouse T lymphocytes is to transfer ADP-ribose groups from NAD to arginine residues, exposed on the extracellular domain of cell surface molecules. As a consequence, T cell responses are modulated. To explore the precise action of the enzyme, the T cell lymphoma EL-4 was transfected with the ART-1 gene, and its effects were examined. It is shown that ART-1 ADP-ribosylates distinct cell surface molecules, causing inhibition of T cell receptor signaling, concomitant to suppression of p56(lck) kinase activation. These effects are explained by failure of T cell receptors and co-receptors to associate into a contiguous and functional receptor cluster.

Cutting Edge: Negative Regulation of Human T Cell Activation by the Receptor-Type Protein Tyrosine Phosphatase CD148

AbstractT cell activation represents a balance between positive and negative signals delivered via distinct cell surface molecules. Many cytoplasmic protein tyrosine phosphatases are involved in regulating cellular responses by antagonizing the action of protein tyrosine kinases. CD148 is a receptor-type protein tyrosine phosphatase expressed by all human mononuclear cells. We have investigated the effect of CD148 on TCR-mediated activation of human T cells. Overexpression of wild-type, but not a phosphatase-deficient, CD148 in Jurkat T cells inhibited TCR-mediated activation, evidenced by reduced expression of the early activation Ag CD69, inhibition of tyrosine phosphorylation of many intracellular proteins including the critical protein tyrosine kinase ZAP-70, and impairment of mitogen-activated protein kinase activation. Taken together, these results suggest that CD148 is an important phosphatase involved in negatively regulating the proximal signaling events during activation of Ag-specific T cells.

Cell adhesion: more than just glue (review).

The ability of cells to interact with each other and their surroundings in a co-ordinated manner depends on multiple adhesive interactions between neighbouring cells and their extracellular environment. These adhesive interactions are mediated by a family of cell surface proteins, termed cell adhesion molecules. Fortunately these adhesion molecules fall into distinct families with adhesive interactions varying in strength from strong binding involved in the maintenance of tissue architecture to more transient, less avid, dynamic interactions observed in leukocyte biology. Adhesion molecules are extremely versatile cell surface receptors which not only stick cells together but provide biochemical and physical signals that regulate a range of diverse functions, such as cell proliferation, gene expression, differentiation, apoptosis and migration. In addition, like many other cell surface molecules, they have been usurped as portals of entry for pathogens, including prions. How the mechanical and chemical messages generated from adhesion molecules are integrated with other signalling pathways (such as receptor tyrosine kinases and phosphatases) and the role that aberrant cell adhesion plays in developmental defects and disease pathology are currently very active areas of research. This review focuses on the biochemical features that define whether a cell surface molecule can act as an adhesion molecule, and discusses five specific examples of how cell adhesion molecules function as more than just 'sticky' receptors. The discussion is confined to the signalling events mediated by members of the integrin, cadherin and immunoglobulin gene superfamilies. It is suggested that, by controlling the membrane organization of signalling receptors, by imposing spatial organization, and by regulating the local concentration of cytosolic adapter proteins, intercellular and cell-matrix adhesion is more than just glue holding cells together. Rather dynamic 'conversations' and the formation of multi-protein complexes between adhesion molecules, growth factor receptors and matrix macromolecules can now provide a molecular explanation for the long-observed but poorly understood requirement for a number of seemingly distinct cell surface molecules to be engaged for efficient cell function to occur.

Regulation of macrophage gene expression following invasion by Mycobacterium tuberculosis

Introduction: Mycobacteria are intracellular pathogens which survive and grow in host macrophages. M. tuberculosis bacilli enter the macrophage via binding to several distinct cell surface molecules. Following phagocytosis, sustained intracellular bacterial growth depends on the ability to avoid destruction by macrophage-mediated host defences such as lysosomal enzymes, reactive oxygen and the reactive nitrogen intermediates. We used differential display reverse transcription polymerase chain reaction (DD RT-PCR) to identify host genes which are regulated during infection and hence which might be involved in the host-parasite cross talk. Results: Live M. tuberculosis (strain H 37Rv) was used to infect Balb/c peritoneal murine macrophages. mRNA from infected and uninfected macrophages was isolated at different time intervals after phagocytosis and subjected to DD RT-PCR. Oligo dT 12NV and random 10mer primers were used for PCR amplification of cDNA. Macrophage genes which appeared to be differently regulated during infection were subjected to further reamplification by PCR in order to clone and sequence them. The differential expression of the selected bands was further analysed by an RNA protection assay and a Northern blot. Results: Several differentially regulated bands were identified. One band, of 158 bp, was down regulated after infection. Sequencing of this band revealed a high level of homology (95% identity) to mouse cytochrome c oxidase subunit VIIc. The downregulation was specific for live virulent Mtb, while live BCG, heat killed Mtb and latex beads-mediated phagocytosis did not affect the transcriptional level of this enzyme. Conclusions: The cytochrome oxidase enzyme complex of the inner mytochondrial membrane catalyzes the reaction between ferrocytochrome c and oxygen. The reaction is the terminal event in the electron transport scheme. Downregulation of cytochrome c oxidase subunit VIIc could interfere with: (1) the host apoptotic programme; or (2) the host respiratory burst.

Mast cells and mast cell growth factor: possible role in auricular thrombosis

Although mast cells have been implicated in many vascular and inflammatory reactions, the mechanisms underlying such mast cell-dependent processes have not been fully clarified. Recently we have established an association between mast cells and local thrombus formation. The most useful model for studying molecular mechanisms of mast cell-thrombosis interactions appeared to be auricular thrombosis. In the present review we summarize our findings in patients with auricular thrombosis with regard to functionally important molecules that can either be expressed and released by cardiac mast cells or interact with mast cells via distinct cell surface molecules. The potential prothrombolytic function of mast cells in atrial thrombosis is discussed. Biomedical Reviews 1995; 4: 29-34.

Changes in metastasis formation and distinct cell surface molecules in adhesion variants of a metastatic murine tumor.

Changes in metastasis formation and distinct cell surface molecules in adhesion variants of a metastatic murine tumor.

A Thy-1.1-specific monoclonal alloantibody activates both mouse and rat T cells

In an attempt to further evaluate the role of Thy-1 in the antigen-independent triggering of mouse T cells, we have examined the activating properties of two Thy-1.1-specific mouse monoclonal antibodies (mAb). These reagents were established from an (A.TH × A.TL)F1 hybrid mouse ( Thy-1 b ) immunized with IL-2 producing (BALB/c ( Thy-1 b ) × BW5147 ( Thy-1 a ) T hybridoma cells. Although both mAb recognized the same Thy-1.1 determinant, one mAb of the γ3,κ class (H171-146) was found to induce several T hybridoma cells to produce IL-2, and AKR thymocytes or cloned helper T cells to proliferate, whereas another mAb of the γ1,κ class (H171-112) failed to do so even in the presence of phorbol myristic acetate (PMA). Increased IL-2 responses of T hybridoma cells were observed when the cell bound Thy-1.1-specific mAb were crosslinked by goat anti-mouse Ig (GaMIg) antibodies. Both a T-cell activating rat anti-Thy-1.2 mAb and the anti-Thy-1.1 mAb H171-146, although directed at distinct cell surface molecules, synergistically stimulated IL-2 production by T hybridoma cells. In addition, the mouse mAb H171-146 was found to stimulate LOU/M rat thymocytes to proliferate in the presence of exogenous IL-2. These data demonstrate that T cells can use Thy-1 as a signal-transducing molecule in both mouse and rat species, and support the notion that the activating properties of Thy-1.1-specific mAb are influenced by their heavy chain isotypes.

Monoclonal antibodies against human monocytes. II. Recognition of two distinct cell surface molecules.

Three monoclonal antibodies (63D3, 63D2, and 61D3) with reactivity against human monocytes have been studied. They reacted with most adherent mononuclear cells but not with pure populations of peripheral blood B and T lymphocytes. Two of these antibodies, 63D3 and 63D2, competed in fluorescence inhibition experiments, recognized a monocyte surface antigen of about 200,000 daltons, and were idiotypically related. The third antibody, 61D3, did not compete with the others in fluorescence inhibition experiments, recognized a different surface molecule, and was idiotypically distinct from 63D3 and 63D2. Whereas 63D3 and 63D2 reacted very weakly with some granulocytes, 61D3 did not, suggesting that it is specific for monocytes alone.