Zelig Eshhar

A Phase I Study on Adoptive Immunotherapy Using Gene-Modified T Cells for Ovarian Cancer

Purpose: A phase I study was conducted to assess the safety of adoptive immunotherapy using gene-modified autologous T cells for the treatment of metastatic ovarian cancer. Experimental Design: T cells with reactivity against the ovarian cancer–associated antigen α-folate receptor (FR) were generated by genetic modification of autologous T cells with a chimeric gene incorporating an anti-FR single-chain antibody linked to the signaling domain of the Fc receptor γ chain. Patients were assigned to one of two cohorts in the study. Eight patients in cohort 1 received a dose escalation of T cells in combination with high-dose interleukin-2, and six patients in cohort 2 received dual-specific T cells (reactive with both FR and allogeneic cells) followed by immunization with allogeneic peripheral blood mononuclear cells. Results: Five patients in cohort 1 experienced some grade 3 to 4 treatment-related toxicity that was probably due to interleukin-2 administration, which could be managed using standard measures. Patients in cohort 2 experienced relatively mild side effects with grade 1 to 2 symptoms. No reduction in tumor burden was seen in any patient. Tracking 111In-labeled adoptively transferred T cells in cohort 1 revealed a lack of specific localization of T cells to tumor except in one patient where some signal was detected in a peritoneal deposit. PCR analysis showed that gene-modified T cells were present in the circulation in large numbers for the first 2 days after transfer, but these quickly declined to be barely detectable 1 month later in most patients. An inhibitory factor developed in the serum of three of six patients tested over the period of treatment, which significantly reduced the ability of gene-modified T cells to respond against FR+ tumor cells. Conclusions: Large numbers of gene-modified tumor-reactive T cells can be safely given to patients, but these cells do not persist in large numbers long term. Future studies need to employ strategies to extend T cell persistence. This report is the first to document the use of genetically redirected T cells for the treatment of ovarian cancer.

A Herceptin-Based Chimeric Antigen Receptor with Modified Signaling Domains Leads to Enhanced Survival of Transduced T Lymphocytes and Antitumor Activity

To generate chimeric Ag receptors (CARs) for the adoptive immunotherapy of cancer patients with ErbB2-expressing tumors, a single-chain Ab derived from the humanized mAb 4D5 Herceptin (trastuzumab) was initially linked to T cell signaling domains derived from CD28 and the CD3ζ to generate a CAR against ErbB2. Human PBLs expressing the 4D5 CAR demonstrated Ag-specific activities against ErbB2+ tumors. However, a gradual loss of transgene expression was noted for PBLs transduced with this 4D5 CAR. When the CD3ζ signaling domain of the CAR was truncated or mutated, loss of CAR expression was not observed, suggesting that the CD3ζ signaling caused the transgene decrease, which was supported by the finding that T cells expressing 4D5 CARs with CD3ζ ITAM mutations were less prone to apoptosis. By adding 4-1BB cytoplasmic domains to the CD28-CD3ζ signaling moieties, we found increased transgene persistence in 4D5 CAR-transduced PBLs. Furthermore, constructs with 4-1BB sequences demonstrated increased cytokine secretion and lytic activity in 4D5 CAR-transduced T cells. More importantly, PBLs expressing this new version of the 4D5 CAR could not only efficiently lyse the autologous fresh tumor digests, but they could strongly suppress tumor growth in a xenogenic mouse model.

Detection of a cytokeratin determinant common to diverse epithelial cells by a broadly cross-reacting monoclonal antibody.

A monoclonal antibody derived from a mouse immunized with bovine epidermal prekeratin has been characterized by its binding to cytoskeletal polypeptides separated by one‐ or two‐dimensional gel electrophoresis and by immunofluorescence microscopy. This antibody (KG 8.13) binds to a determinant present in a large number of human cytokeratin polypeptides, notably some polypeptides (Nos. 1, 5, 6, 7, and 8) of the ‘basic cytokeratin subfamily’ defined by peptide mapping, as well as a few acidic cytokeratins such as the epidermis‐specific cytokeratins Nos. 10 and 11 and the more widespread cytokeratin No. 18. This antibody reacts specifically with a wide variety of epithelial tissues and cultured epithelial cells, in agreement with previous findings that at least one polypeptide of the basic cytokeratin subfamily is present in all normal and neoplastic epithelial cells so far examined. The antibody also reacts with corresponding cytokeratin polypeptides in a broad range of species including man, cow, chick, and amphibia but shows only limited reactivity with only a few rodent cytokeratins. The value of this broad‐range monoclonal antibody, which apparently recognizes a stable cytokeratin determinant ubiquitous in human epithelia, for the immunohistochemical identification of epithelia and carcinomas is discussed.

Photodynamic therapy with Pd-Bacteriopheophorbide (TOOKAD): successful in vivo treatment of human prostatic small cell carcinoma xenografts.

Small cell carcinoma of the prostate (SCCP), although relatively rare, is the most aggressive variant of prostate cancer, currently with no successful treatment. It was therefore tempting to evaluate the response of this violent malignancy and its bone lesions to Pd‐Bacteriopheophorbide (TOOKAD)‐based photodynamic therapy (PDT), already proven by us to efficiently eradicate other aggressive non‐epithelial solid tumors. TOOKAD is a novel bacteriochlorophyll‐derived, second‐generation photosensitizer recently, developed by us for the treatment of bulky tumors. This photosensitizer is endowed with strong light absorbance (ϵ0 ∼ 105 mol−1 cm−1) in the near infrared region (λ=763nm), allowing deep tissue penetration. The TOOKAD‐PDT protocol targets the tumor vasculature leading to inflammation, hypoxia, necrosis and tumor eradication. The sensitizer clears rapidly from the circulation within a few hours and does not accumulate in tissues, which is compatible with the treatment of localized tumor and isolated metastases. Briefly, male CD1‐nude mice were grafted with the human SCCP (WISH‐PC2) in 3 relevant anatomic locations: subcutaneous (representing tumor mass), intraosseous (representing bone metastases) and orthotopically within the murine prostate microenvironment. The PDT protocol consisted of i.v. administration of TOOKAD (4 mg/kg), followed by immediate illumination (650–800 nm) from a xenon light source or a diode laser emitting at 770 nm. Controls included untreated animals or animals treated with light or TOOKAD alone. Tumor volume, human plasma chromogranin A levels, animal well being and survival were used as end points. In addition, histopathology and immunohistochemistry were used to define the tumor response. Subcutaneous tumors exhibited complete healing within 28–40 days, reaching an overall long‐term cure rate of 69%, followed for 90 days after PDT. Intratibial WISH‐PC2 lesions responded with complete tumor elimination in 50% of the treated mice at 70–90 days after PDT as documented histologically. The response of the orthotopic model was also analyzed histologically with similar results. The study with this model suggests that TOOKAD‐based PDT can reach large tumors and is a feasible, efficient and well‐tolerated approach for minimally invasive treatment of local and disseminated SCCP.

Lymphoid Cell Surface Interaction Structures Detected Using Cytolysis‐Inhibiting Monoclonal Antibodies

We screened monoclonal antibodies obtained by xenogeneic immunization for their capacity to inhibit T cell-mediated cytolysis. These antibodies fell into two classes according to the cell structures they recognized, of 30-35 K and 94-180 K apparent molecular weight, respectively. The main features of these structures and of their interaction with the corresponding antibodies were reviewed. The inhibition of cytolysis by these antibodies was shown to occur mainly at the effector cell level, at the recognition stage of cytolysis, and to depend on the nature of target cells, effector cells, and link between these cells. T cell functions other than cytolysis were also inhibited by some of these antibodies. We considered various possible mechanisms to account for the inhibition of cytolysis by these mAb. We favor an hypothesis based on inhibition by these mAb of lymphoid cell surface interaction structures. This hypothesis was discussed within the general framework of cell interaction structures in immunological and non-immunological experimental systems.

Tumor-specific T-bodies: towards clinical application

Abstract Unlike antibodies, T cells are well suited to penetrate and destroy solid tumors. The T-body approach combines antibody recognition and T cells effector function. It is based on T cells expressing chimeric receptors composed of antibody-derived Fv or scFv as their extracellular recognition elements joined to lymphocyte triggering molecules. This receptors can redirect the specificity of T cells in an MHC independent manner. Upon encountering their target cells, T-bodies are able to undergo specific stimulation for interleukin/cytokine production, and kill hapten-modified or tumor cells in model systems both in vitro and in vivo. T cells expressing chimeric receptors made of antitumor antibodies are able to discriminate between a tumor and normal cell with negligible bystander cytotoxicity. Further studies should be carried out to evaluate and optimize the persistence, homing patterns and reactivation potential of T-bodies in the body.

Reorganization of arrays of prekeratin filaments during mitosis. Immunofluorescence microscopy with multiclonal and monoclonal prekeratin antibodies.

Abstract Indirect immunofluorescent labelling of different epithelial cell lines for intermediate filaments of the prekeratin type revealed prominent changes in the organization of prekeratin during mitosis. In three out of four cell lines tested (Henle-407, A-431 and HeLa cells) the filamentous prekeratin networks disappeared at the initiation of mitosis and the immunofluorescent labelling was concentrated in small cytoplasmic bodies. This observation was obtained with both polyspecific rabbit anti-bovine prekeratin antibodies and with monospecific antibodies produced by mouse hybridomas. In a fourth cell line, PtK 2 , prekeratin filaments were retained throughout mitosis, mainly in the mitotic poles, whereas the central areas of the cells were apparently devoid of filaments. The addition of colchicine to the different cultured cells induced alterations in the organization of prekeratin filaments which were usually manifested by the formation of thicker filament bundles. It did not induce the formation of the prekeratin-cytoplasmic bodies in interphase cells. However, upon prolonged incubation in the presence of colchicine, there was an increase in the number of mitotically arrested cells and a parallel increase in the number of cells containing prekeratin cytoplasmic bodies. It is thus proposed that the state of organization of prekeratin in these cells is cell-cycle-dependent and may be modulated to permit radical shape changes as those occurring during mitosis.

Suppression of Human Prostate Tumor Growth in Mice by a Cytolytic d-, l-Amino Acid Peptide Membrane Lysis, Increased Necrosis, and Inhibition of Prostate-Specific Antigen Secretion

Gene-encoded host defense peptides are used as part of the innate immunity, and many of them act by directly lysing the cell membrane of the pathogen. A few of these peptides showed anticancer activity in vitro but could not be used in vivo because of their inactivation by serum. We designed a 15-amino acid peptide, composed of d- and l-amino acids (diastereomer), which targets both androgen-independent and androgen-dependent human prostate carcinoma cell lines (CL1, 22RV1, and LNCaP). Most importantly, we observed a complete arrest of growth in CL1 and 22RV1 xenografts treated intratumorally with the diastereomer. This was also accompanied by a lowering of prostate-specific antigen serum levels secreted by the 22RV1 xenograft. Furthermore, the diastereomer synergized with conventional chemotherapeutics. In contrast, the parental all l-amino acids peptide was highly active only in vitro and could not discriminate between tumor and nontumor cells. Fluorescent confocal microscopy, histopathologic examination, and cell permeability studies (depolarization of transmembrane potential and release of an encapsulated dye) suggest a necrotic mechanism of killing, after a threshold concentration of peptide has been reached. Its destructive killing effect and the simple sequence of the diastereomer make it an attractive chemotherapeutic candidate possessing a new mode of action, with potential to be developed additionally for the treatment of prostate carcinoma.

Inhibition of Tumor Growth and Elimination of Multiple Metastases in Human Prostate and Breast Xenografts by Systemic Inoculation of a Host Defense–Like Lytic Peptide

We report on a short host defense-like peptide that targets and arrests the growth of aggressive and hormone-resistant primary human prostate and breast tumors and prevents their experimental and spontaneous metastases, respectively, when systemically inoculated to immunodeficient mice. These effects are correlated with increased necrosis of the tumor cells and a significant decrease in the overall tumor microvessel density, as well as newly formed capillary tubes and prostate-specific antigen secretion (in prostate tumors). Growth inhibition of orthotopic tumors derived from stably transfected highly fluorescent human breast cancer cells and prevention of their naturally occurring metastases were visualized in real time by using noninvasive whole-body optical imaging. The exclusive selectivity of the peptide towards cancer derives from its specific binding to surface phosphatidylserine and the killing of the cancer cells via cytoplasmic membrane depolarization. These data indicate that membrane disruption can provide a therapeutic means of inhibiting tumor growth and preventing metastases of various cancers.

Monoclonal antibodies against defined determinants of acetylcholine receptor

The nicotinic acetylcholine receptor (AChR) is a major autoantigen in the neuromuscular disease myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) [l-3]. Cellular and humoral immune responses to AChR have been demonstrated in both the human and the experimental diseases [I ,2,4-71. The involvement of anti-acetylcholine receptor antibodies in the pathogenesis of MG and EAMG has been proposed on clinical and experimental ground. Passive transfer of immunoglobulins from myasthenic patients [8] and from animals with EAMG [9] resulted in my~~enia gravislike symptoms. AChR is a complexed multideterminant immunogen which raises a heterogeneous immune response. In order to analyze the structure of the AChR molecule and for identifying the molecular entity which governs its myasthenic activity, large quantities of antibodies with defined specificity are required. Such antibodies also provide a useful tool for study~g the role of antibodies in myasthenia and their mechanism of action, To this end we have used the method of lymphocyte hybridization developed [lo]. Here we describe the preparation of monoclonal anti-AChR antibodies with restricted specificity towards defined determinants, secreted by hybridomas obtained by hybridi~tion of anti-AC!hR antibodies producing cells with a myeloma line.