Steven M. Albelda

Integrins and other cell adhesion molecules.

Cell‐cell and cell‐substratum interactions are mediated through several different families of receptors. In addition to targeting cell adhesion to specific extracellular matrix proteins and ligands on adjacent cells, these receptors influence many diverse processes including cellular growth, differentiation, junction formation, and polarity. Several families of adhesion receptors have been identified. These include: 1) the integrins, heterodimeric molecules that function both as cell‐substratum and cell‐cell adhesion receptors; 2) the adhesion molecules of the immunoglobulin superfamily, which are involved in cell‐cell adhesion and especially important during embryogenesis, wound healing, and the inflammatory response; 3) the Cadherins, developmentally regulated, calcium‐dependent homophilic cell‐cell adhesion proteins; 4) the LEC‐CAMs, cell adhesion molecules with lectin‐like domains that mediate white blood cell/endothelial cell adhesion; and 5) homing receptors that target lymphocytes to specific lymphoid tissue. In this review we summarize recent data describing the structure and function of some of these cell adhesion molecules (with special emphasis on the integrin family) and discuss the possible role of these molecules in development, inflammation, wound healing, coagulation, and tumor metastasis.— Albelda, S. M., Buck, C. A. Integrins and other cell adhesion molecules. FASEB J. 4: 2868‐2880; 1990.

Polarization of Tumor-Associated Neutrophil Phenotype by TGF-β: “N1” versus “N2” TAN

TGF-beta blockade significantly slows tumor growth through many mechanisms, including activation of CD8(+) T cells and macrophages. Here, we show that TGF-beta blockade also increases neutrophil-attracting chemokines, resulting in an influx of CD11b(+)/Ly6G(+) tumor-associated neutrophils (TANs) that are hypersegmented, more cytotoxic to tumor cells, and express higher levels of proinflammatory cytokines. Accordingly, following TGF-beta blockade, depletion of these neutrophils significantly blunts antitumor effects of treatment and reduces CD8(+) T cell activation. In contrast, in control tumors, neutrophil depletion decreases tumor growth and results in more activated CD8(+) T cells intratumorally. Together, these data suggest that TGF-beta within the tumor microenvironment induces a population of TAN with a protumor phenotype. TGF-beta blockade results in the recruitment and activation of TANs with an antitumor phenotype.

Adhesion molecules and inflammatory injury.

Neutrophil‐endothelial cell interactions are mediated by interacting sets of cell adhesion molecules (CAMs) and chemoattractant/activator molecules to form an “adhesion cascade.” The initial phase of inflammation, a transient slowing of neutrophils in postcapillary venules, is mediated by selectins. Subsequently, firm adhesion of neutrophils to the vessel wall occurs via interaction of the CD11/GD18 (β2) integrins to endothelial ligands such as intercellular adhesion molecule‐1 (ICAM‐1). This binding requires activation of CD11/GD18 by exposure of the neutrophil to a variety of activating/chemoattractant molecules, such as platelet‐activating factor or interleukin‐8. Finally, transmigration into tissues occurs, a process that requires both a chemotactic stimulus and engagement of platelet‐endothelial cell adhesion molecule‐1 (PECAM‐1). Several approaches have been used to probe the role of CAMs in vivo. These include the use of blocking antibodies, chimeric selectin‐immunoglobulin proteins, sialyl Lewisx oligosaccharides and peptides, along with the study of humans and animals with genetically determined adhesion deficiencies. These studies demonstrate that CAM blockade can effectively inhibit inflammation; however, there appear to be clear differences in the adhesion requirements for particular types of inflammation. By understanding the CAM/chemoattractant profiles involved in specific disease states, it may be possible to precisely and effectively target therapy to a wide variety of inflammatory diseases.—Albelda, S. M., Smith, C. W., Ward, P. A. Adhesion molecules and inflammatory injury. FASEB J. 8: 504‐512; 1994.

Gemcitabine Selectively Eliminates Splenic Gr-1+/CD11b+ Myeloid Suppressor Cells in Tumor-Bearing Animals and Enhances Antitumor Immune Activity

Purpose: Myeloid suppressor (Gr-1+/CD11b+) cells accumulate in the spleens of tumor-bearing mice where they contribute to immunosuppression by inhibiting the function of CD8+ T cells and by promoting tumor angiogenesis. Elimination of these myeloid suppressor cells may thus significantly improve antitumor responses and enhance effects of cancer immunotherapy, although to date few practical options exist. Experimental Design: The effect of the chemotherapy drug gemcitabine on the number of (Gr-1+/CD11b+) cells in the spleens of animals bearing large tumors derived from five cancer lines grown in both C57Bl/6 and BALB/c mice was analyzed. Suppressive activity of splenocytes from gemcitabine-treated and control animals was measured in natural killer (NK) cell lysis and Winn assays. The impact of myeloid suppressor cell activity was determined in an immunogene therapy model using an adenovirus expressing IFN-β. Results: This study shows that the chemotherapeutic drug gemcitabine, given at a dose similar to the equivalent dose used in patients, was able to dramatically and specifically reduce the number of myeloid suppressor cells found in the spleens of animals bearing large tumors with no significant reductions in CD4+ T cells, CD8+ T cells, NK cells, macrophages, or B cells. The loss of myeloid suppressor cells was accompanied by an increase in the antitumor activity of CD8+ T cells and activated NK cells. Combining gemcitabine with cytokine immunogene therapy using IFN-β markedly enhanced antitumor efficacy. Conclusions: These results suggest that gemcitabine may be a practical strategy for the reduction of myeloid suppressor cells and should be evaluated in conjunction with a variety of immunotherapy approaches.

Cross-Talk between Myeloid-Derived Suppressor Cells and Macrophages Subverts Tumor Immunity toward a Type 2 Response

Although the immune system has the potential to protect against malignancies, many individuals with cancer are immunosuppressed. Myeloid-derived suppressor cells (MDSC) are elevated in many patients and animals with tumors, and contribute to immune suppression by blocking CD4+ and CD8+ T cell activation. Using the spontaneously metastatic 4T1 mouse mammary carcinoma, we now demonstrate that cross-talk between MDSC and macrophages further subverts tumor immunity by increasing MDSC production of IL-10, and by decreasing macrophage production of IL-12. Cross-talk between MDSC and macrophages requires cell-cell contact, and the IL-12 decrease is dependent on MDSC production of IL-10. Treatment with the chemotherapeutic drug gemcitabine, which reduces MDSC, promotes rejection of established metastatic disease in IL-4Rα−/− mice that produce M1 macrophages by allowing T cell activation, by maintaining macrophage production of IL-12, and by preventing increased production of IL-10. Therefore, MDSC impair tumor immunity by suppressing T cell activation and by interacting with macrophages to increase IL-10 and decrease IL-12 production, thereby promoting a tumor-promoting type 2 response, a process that can be partially reversed by gemcitabine.

Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains

Mesothelin is a cell-surface molecule over-expressed on a large fraction of carcinomas, and thus is an attractive target of immunotherapy. A molecularly targeted therapy for these cancers was created by engineering T cells to express a chimeric receptor with high affinity for human mesothelin. Lentiviral vectors were used to express a single-chain variable fragment that binds mesothelin and that is fused to signaling domains derived from T-cell receptor zeta, CD28, and CD137 (4–1BB). When stimulated by mesothelin, lentivirally transduced T cells were induced to proliferate, express the antiapoptotic gene Bcl-XL, and secrete multiple cytokines, all features characteristic of central memory T cells. When transferred intratumorally or intravenously into NOD/scid/IL2rγ−/− mice engrafted with large pre-established tumors, the engineered T cells reduced the tumor burden, and in some cases resulted in complete eradication of the tumors at low effector-to-target ratios. Incorporation of the CD137 signaling domain specifically reprogrammed cells for multifunctional cytokine secretion and enhanced persistence of T cells. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of poorly immunogenic tumors. Genetically redirected T cells have promise of targeting T lymphocytes to tumor antigens, confer resistance to the tumor microenvironment, and providing immunosurveillance.

Mesothelin-Specific Chimeric Antigen Receptor mRNA-Engineered T Cells Induce Antitumor Activity in Solid Malignancies

Beatty, Haas, and colleagues report antitumor activity in two patients treated with autologous T cells transfected with mRNA encoding a chimeric antigen receptor that recognizes mesothelin and contains the CD3-ζ and 4-1BB costimulatory domains (CARTmeso). The short-lived CARTmeso cells induced novel antiself antibodies and a broadly directed epitope spreading. Off-target toxicity due to the expression of target antigens in normal tissue represents a major obstacle to the use of chimeric antigen receptor (CAR)-engineered T cells for treatment of solid malignancies. To circumvent this issue, we established a clinical platform for engineering T cells with transient CAR expression by using in vitro transcribed mRNA encoding a CAR that includes both the CD3-ζ and 4-1BB costimulatory domains. We present two case reports from ongoing trials indicating that adoptive transfer of mRNA CAR T cells that target mesothelin (CARTmeso cells) is feasible and safe without overt evidence of off-tumor on-target toxicity against normal tissues. CARTmeso cells persisted transiently within the peripheral blood after intravenous administration and migrated to primary and metastatic tumor sites. Clinical and laboratory evidence of antitumor activity was shown in both patients, and the CARTmeso cells elicited an antitumor immune response revealed by the development of novel antiself antibodies. These data show the potential of using mRNA-engineered T cells to evaluate, in a controlled manner, potential off-tumor on-target toxicities and show that short-lived CAR T cells can induce epitope spreading and mediate antitumor activity in patients with advanced cancer. Thus, these findings support the development of mRNA CAR-based strategies for carcinoma and other solid tumors. Cancer Immunol Res; 2(2); 112–20. ©2013 AACR.

Molecular and functional aspects of PECAM-1/CD31

Among vascular cell adhesion molecules, platelet-endothelial cell adhesion molecule (PECAM-1/CD31) has the distinctive feature of being expressed on several of the major cell types associated with the vascular compartment. This makes it uniquely positioned to mediate multiple and important cell-cell interactions involving platelets, leukocytes and endothelial cells. Thus, PECAM-1 may represent a potential target for new therapeutic agents directed at a variety of pathological states.

Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor

Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor-reactive T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production have limited clinical application of T cells expressing chimeric antigen receptors (CAR). T lymphocytes can be gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe platform for adoptive immunotherapy, we first optimized the vector backbone for RNA in vitro transcription to achieve high-level transgene expression. CAR expression and function of RNA-electroporated T cells could be detected up to a week after electroporation. Multiple injections of RNA CAR-electroporated T cells mediated regression of large vascularized flank mesothelioma tumors in NOD/scid/γc(-/-) mice. Dramatic tumor reduction also occurred when the preexisting intraperitoneal human-derived tumors, which had been growing in vivo for >50 days, were treated by multiple injections of autologous human T cells electroporated with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes showing that autologous T cells from cancer patients can be engineered to provide an effective therapy for a disseminated tumor in a robust preclinical model. Multiple injections of RNA-engineered T cells are a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors.

A novel endocytic pathway induced by clustering endothelial ICAM-1 or PECAM-1

Antibody conjugates directed against intercellular adhesion molecule (ICAM-1) or platelet-endothelial cell adhesion molecule (PECAM-1) have formed the basis for drug delivery vehicles that are specifically recognized and internalized by endothelial cells. There is increasing evidence that ICAM-1 and PECAM-1 may also play a role in cell scavenger functions and pathogen entry. To define the mechanisms that regulate ICAM-1 and PECAM-1 internalization, we examined the uptake of anti-PECAM-1 and anti-ICAM-1 conjugates by endothelial cells. We found that the conjugates must be multimeric, because monomeric anti-ICAM-1 and anti-PECAM-1 are not internalized. Newly internalized anti-ICAM-1 and anti-PECAM-1 conjugates did not colocalize with either clathrin or caveolin, and immunoconjugate internalization was not reduced by inhibitors of clathrin-mediated or caveolar endocytosis, suggesting that this is a novel endocytic pathway. Amiloride and protein kinase C (PKC) inhibitors, agents known to inhibit macropinocytosis, reduced the internalization of clustered ICAM-1 and PECAM-1. However, expression of dominant-negative dynamin-2 constructs inhibited uptake of clustered ICAM-1. Binding of anti-ICAM-1 conjugates stimulated the formation of actin stress fibers by human umbilical vein endothelial cells (HUVEC). Latrunculin, radicicol and Y27632 also inhibited internalization of clustered ICAM-1, suggesting that actin rearrangements requiring Src kinase and Rho kinase (ROCK) were required for internalization. Interestingly, these kinases are part of the signal transduction pathways that are activated when circulating leukocytes engage endothelial cell adhesion molecules, suggesting the possibility that CAM-mediated endocytosis is regulated using comparable signaling pathways.