Early outcome results of a phase I/II study for an IL-2/T-cell receptor fusion protein in combination with gemcitabine and cisplatin (GC) in patients with locally advanced or metastatic urothelial cancer.

e15010 Background: ALT-801 is a human IL-2/single-chain T-cell receptor fusion protein previously tested in a phase 1 in patients with advanced malignancy (Fishman 2011 CCR 17:7765) (CA097550). In various murine models, ALT-801 demonstrated potent activity against syngeneic and xenograft UC, suggesting sensitivity of this disease to IL-2 based immunotherapy (Wong, unpubl. data.). Although UC are sensitive to platinum-based chemotherapy, combinations such as gemcitabine (G) + cisplatin (C) are associated with CR rates of around 15%, and limited durability of responses with limited effects of retreatment. Methods: We report here initial efficacy results of co-administration of G (1000 mg/m2/dose, d 1 & 8), C (70 mg/m2/dose, d 1) and ALT-801 (escalating doses, d 3, 5, 8, 10) on a 21 day schedule, for 3 cycles, in patients with UC that was locally advanced, or metastatic, for whom GC chemotherapy would be considered. ALT-801 planned doses are 0.04 to 0.12 mg/kg/dose in 5 dose cohorts with a 3+3 escalation design. Subjects with at least stable disease after 3 courses may receive 4 additional weekly doses of ALT-801 alone. Results: To date, three Stage IV UC patients (1F, 2M; 59-63 yrs; 2 patients had predominantly nodal metastases and one patient liver metastases) completed treatment with 0.04 mg/kg ALT-801+GC. Two had previously undergone radical cystectomy and had then later failed following GC treatment. Grade 3/4 toxicities observed include neutropenia (2), thrombocytopenia (2), leukopenia (1), lymphopenia (1) and anemia (1), consistent with GC and ALT-801 known pharmacodynamic effects. All 3 had radiological CRs by week 13. One patient underwent radical cystectomy had a pCR. The next (0.06 mg/kg/dose) cohort has started treatment. A phase II expansion cohort is planned at the MTD. Conclusions: The early response pattern is encouraging in that ALT-801 may be a novel, active immunotherapy for UC. NCT01326871

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Phase I/II clinical trial of ALT-801, a T-cell receptor/IL-2 fusion protein, plus gemcitabine and cisplatin in urothelial cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.6_suppl.271 ◽

2013 ◽

Vol 31 (6_suppl) ◽

pp. 271-271 ◽

Cited By ~ 4

Author(s):

Mayer N. Fishman ◽

Julio Hadjenberg ◽

Timothy Kuzel ◽

Amit Mahipal ◽

Charles Joel Rosser ◽

...

Keyword(s):

Clinical Trial ◽

T Cell ◽

Fusion Protein ◽

T Cell Receptor ◽

Urothelial Cancer ◽

Objective Response ◽

Cell Receptor ◽

Active Immunotherapy ◽

Advanced Malignancy ◽

Metastatic Urothelial Cancer

271 Background: ALT-801, a T-cell receptor/IL-2 fusion protein, activates NK and CD4+ lymphocytes to secrete IFN-gamma which re-polarizes tumor associated macrophages from M1 to M2 in various murine tumor models. CD8+ memory cells also acquire an innate immune phenotype and become expanded upon ALT-801 activation. Via this novel mechanism, ALT-801 mounted effective immune responses and maintained immunological memory against urothelial cancer in these models. Pretreatment chemotherapy eliminated myeloid-derived suppressive cells, potentiating the anti-tumor effects of ALT-801 (Wong, unpublished data). Previous clinical studies with ALT-801 (advanced malignancy; Fishman 2011 CCR 17:7765) and ALT-801 + cisplatin (melanoma; NCT01029873) showed anti-tumor activity in these settings. Methods: We evaluated co-administration of gemcitabine [G] (1000 mg/m2/dose, day 1 and 8) and cisplatin [C] (70 mg/m2/dose, day 1) with ALT-801 (escalating doses; days 3, 5, 8, 10) for three 21-day cycles, in patients with metastatic urothelial cancer. Those with at least stable disease after 3 courses could receive 4 additional weekly doses of ALT-801 alone. ALT-801 doses were planned at 0.04 to 0.12 mg/kg/dose in 5 cohorts with a 3+3 escalation design. Results: The dose-escalation is completed, with a recommended dose of ALT-801 of 0.06 mg/kg/dose. The best objective response rate (ORR, RECIST v1.1) among 5 chemo-naïve subjects was 100% (2 CR, 3 PR) and among 5 previously treated patients 60% (1 CR, 2 PR), for an overall total of 80% (3 CR, 5 PR, 1 SD, 1 PD). One of 2 patients who underwent radical cystectomy was confirmed pathologically free of tumor. Responding lesions included bulky and extensive liver and pulmonary metastases, and adenopathy. ALT-801 at the 0.06 mg/kg/dose level in this combination was adequately-tolerated. Grade 3/4 toxicities including neutropenia, thrombocytopenia and lymphopenia, appear consistent with known G, C, and ALT-801 effects. Conclusions: ALT-801 is a novel and potentially active immunotherapy for urothelial cancer. More patients are in treatment on the open phase 2 expansion portion of the study (NCT01326871), and updated interim results are anticipated (CA097550). Clinical trial information: NCT01326871.

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Anti-graft-versus-host disease effect of DT390-anti-CD3sFv, a single- chain Fv fusion immunotoxin specifically targeting the CD3 epsilon moiety of the T-cell receptor

Blood ◽

10.1182/blood.v88.6.2342.bloodjournal8862342 ◽

1996 ◽

Vol 88 (6) ◽

pp. 2342-2353 ◽

Cited By ~ 24

Author(s):

DA Vallera ◽

A Panoskaltsis-Mortari ◽

C Jost ◽

S Ramakrishnan ◽

CR Eide ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Fusion Protein ◽

Enzymatic Activity ◽

T Cell Receptor ◽

Graft Versus Host Disease ◽

Cell Receptor ◽

Single Chain ◽

Graft Versus Host ◽

Single Chain Fv

In a recent study, we showed that an immunotoxin (IT) made with a conventional monoclonal antibody targeting the CD3 epsilon moiety of the T-cell receptor (TCR) had a potent, but partial, graft-versus-host disease (GVHD) effect (Vallera et al, Blood 86:4367, 1995). Therefore, in this current study, we determined whether a fusion immunotoxin made with anti-CD3 single-chain Fv (sFv), the smallest unit of antibody recognizing antigen, would have anti-GVHD activity. A fusion protein was synthesized from a construct made by splicing sFv cDNA from the hybridoma 145–2C11 to a truncated form of the diphtheria toxin (DT390) gene. DT390 encodes a molecule that retains full enzymatic activity, but excludes the native DT binding domain. The DT390-anti-CD3sFv hybrid gene was cloned into a vector under the control of an inducible promoter. The protein was expressed in Escherichia coli and then purified from inclusion bodies. The DT390 moiety of the protein had full enzymatic activity compared with native DT and DT390-anti-CD3sFv, with an IC50 of 1 to 2 nmol/L against phytohemagglutinin-stimulated and alloantigen-stimulated T cells. Specificity was shown (1) by blocking the IT with parental anti-CD3 antibody, but not with a control antibody; (2) by failure of DT390-anti-CD3sFv to inhibit lipopolysaccharide-stimulated murine B cells; (3) by failure of an Ig control fusion protein, DT390-Fc, to inhibit T-cell responses; and (4) with in vivo immunohistochemisty studies. GVHD was studied in a model in which C57BL/6 (H-2b)-purified lymph node T cells were administered to major histocompatibility complex (MHC) antigen disparate unirradiated C.B.-17 scid (H-2d) mice to assess GVHD effects in the absence of irradiation toxicity. Flow cytometry studies showed that donor T cells were expanded 57-fold and histopathologic analysis showed the hallmarks of a lethal model of GVHD. Control mice receiving phosphate-buffered saline showed 17% survival on day 80 after bone marrow transplantation, and mice receiving 2 micrograms DT390-Fc fusion toxin control administered in 2 daily doses for 6 days (days 0 through 5) had a 43% survival rate. In contrast, 86% of mice receiving the same dose of DT390-anti-CD3sFv were survivors on day 80, a significant improvement, although survivors still showed histopathologic signs of GVHD. These findings suggest that new anti-GVHD agents can be genetically engineered and warrant further investigation of fusion proteins for GVHD treatment.

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A soluble single-chain T-cell receptor IL-2 fusion protein retains MHC-restricted peptide specificity and IL-2 bioactivity

Cancer Immunology Immunotherapy ◽

10.1007/s00262-003-0450-3 ◽

2004 ◽

Vol 53 (4) ◽

pp. 345-357 ◽

Cited By ~ 26

Author(s):

Kimberlyn F. Card ◽

Shari A. Price-Schiavi ◽

Bai Liu ◽

Elizabeth Thomson ◽

Esperanza Nieves ◽

...

Keyword(s):

T Cell ◽

Fusion Protein ◽

T Cell Receptor ◽

Cell Receptor ◽

Single Chain ◽

Peptide Specificity

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Binding affinity and inhibitory properties of a single-chain anti-T cell receptor antibody.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)74449-4 ◽

1993 ◽

Vol 268 (34) ◽

pp. 25722-25727

Author(s):

B A Schodin ◽

D M Kranz

Keyword(s):

T Cell ◽

Binding Affinity ◽

T Cell Receptor ◽

Cell Receptor ◽

Single Chain ◽

Receptor Antibody

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Qualitative and quantitative contributions of the T cell receptor zeta chain to mature T cell apoptosis.

Journal of Experimental Medicine ◽

10.1084/jem.183.5.2109 ◽

1996 ◽

Vol 183 (5) ◽

pp. 2109-2117 ◽

Cited By ~ 62

Author(s):

B Combadière ◽

M Freedman ◽

L Chen ◽

E W Shores ◽

P Love ◽

...

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Cell Apoptosis ◽

Cell Receptor ◽

Single Chain ◽

Tcr Signaling ◽

T Cell Apoptosis ◽

Zeta Chain ◽

Src Homology ◽

Src Homology 2 Domain

Engagement of the T cell receptor (TCR) of mature T lymphocytes can lead either to activation/proliferation responses or programmed cell death. To understand the molecular regulation of these two fundamentally different outcomes of TCR signaling, we investigated the participation of various components of the TCR-CD3 complex. We found that the TCR-zeta chain, while not absolutely required, was especially effective at promoting mature T cell apoptosis compared with the CD3 epsilon, gamma, or delta chains. We also carried out mutagenesis to address the role of the immunoreceptor tyrosine-based activation motifs (ITAMs) that are the principal signaling components found three times in the TCR-zeta chain and once in each of the CD3 epsilon, gamma, or delta chains. We found that the ability of the TCR-zeta chain to promote apoptosis results both from a quantitative effect of the presence of multiple ITAMs as well as qualitatively different contributions made by individual ITAMs. Apoptosis induced by single chain chimeras revealed that the first zeta ITAM stimulated greater apoptosis than the third zeta ITAM, and the second zeta ITAM was unable to trigger apoptosis. Because microheterogeneity in the amino acid sequence of the various ITAM motifs found in the TCR-zeta and CD3 chains predicts interactions with distinct src-homology-2-domain signaling proteins, our results suggest the possibility that individual ITAM motifs might play unique roles in TCR responses by engaging specific signaling pathways.

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A functional recombinant single-chain T cell receptor fragment capable of selectively targeting antigen-presenting cells

Cancer Immunology Immunotherapy ◽

10.1007/s00262-002-0312-4 ◽

2002 ◽

Vol 51 (10) ◽

pp. 565-573 ◽

Cited By ~ 7

Author(s):

Malka Epel ◽

Joshua Ellenhorn ◽

Don Diamond ◽

Yoram Reiter

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Antigen Presenting Cells ◽

Cell Receptor ◽

Single Chain ◽

Antigen Presenting ◽

Receptor Fragment

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173 Expression of a membrane-tethered IL-15/IL-15 receptor fusion protein enhances the persistence of MSLN-targeted TRuC-T cells

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.173 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A185-A185

Author(s):

Michelle Fleury ◽

Derrick McCarthy ◽

Holly Horton ◽

Courtney Anderson ◽

Amy Watt ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Fusion Protein ◽

T Cell Receptor ◽

Cytokine Production ◽

Cell Receptor ◽

Great Promise ◽

And Function

BackgroundAdoptive cell therapies have shown great promise in hematological malignancies but have yielded little progress in the context of solid tumors. We have developed T cell receptor fusion construct (TRuC®) T cells, which are equipped with an engineered T cell receptor that utilizes the full complement of TCR signaling subunits and recognizes tumor-associated antigens independent of HLA. In clinical trials, mesothelin (MSLN)-targeting TRuC-T cells (TC-210 or gavo-cel) have shown unprecedented results in patients suffering from advanced mesothelioma and ovarian cancer. To potentially increase the depth of response, we evaluated strategies that can promote intra-tumoral T cell persistence and function. Among the common ??-chain cytokines, IL-15 uniquely supports the differentiation and maintenance of memory T cell subsets by limiting terminal differentiation and conferring resistance to IL-2 mediated activation-induced cell death (AICD). In the studies described here, we evaluated the potential of IL-15 as an enhancement to TRuC-T cell phenotype, persistence and function against MSLN+ targets.MethodsPrimary human T cells were activated and transduced with a lentiviral vector encoding an anti-MSLN binder fused to CD3ε alone or co-expressed with a membrane-tethered IL-15rα/IL-15 fusion protein (IL-15fu). Transduced T cells were expanded for 9 days and characterized for expression of the TRuC, IL-15rα and memory phenotype before subjecting them to in vitro functional assays to evaluate cytotoxicity, cytokine production, and persistence. In vivo efficacy was evaluated in MHC class I/II deficient NSG mice bearing human mesothelioma xenografts.ResultsIn vitro, co-expression of the IL-15fu led to similar cytotoxicity and cytokine production as TC-210, but notably enhanced T-cell expansion and persistence upon repeated stimulation with MSLN+ cell lines. Furthermore, the IL-15fu-enhanced TRuC-T cells sustained a significantly higher TCF-1+ population and retained a stem-like phenotype following activation. Moreover, the IL-15fu-enhanced TRuCs demonstrated robust in vivo expansion and intra-tumoral accumulation as measured by ex vivo analysis of TRuC+ cells in the tumor and blood, with a preferential expansion of CD8+ T cells. Finally, IL-15fu-enhanced TRuC-T cells could be observed in the blood long after the tumors were cleared.ConclusionsThese pre-clinical studies suggest that the IL-15fu can synergize with TC-210 to increase the potency and durability of response in patients with MSLN+ tumors.Ethics ApprovalAll animal studies were approved by the respective Institutional Animal Care and Use Committees.

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Safe and Effective Adoptive T-Cell Receptor Transfer with a High Affinity Single Chain p53(264–272)-Specific TCR

Blood ◽

10.1182/blood.v120.21.4226.4226 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4226-4226

Author(s):

Hakim Echchannaoui ◽

Jutta Petschenka ◽

Edite Antunes ◽

Matthias Theobald

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

T Cell Receptor ◽

Cell Receptor ◽

Cell Transfer ◽

Single Chain ◽

Adoptive T Cell Transfer ◽

High Affinity ◽

T Cell Transfer

Abstract Abstract 4226 Several studies have demonstrated the clinical efficacy of adoptive T cell therapy for targeting cancer. Using HLA-A2.1 transgenic mice, we have demonstrated the feasibility of T-cell receptor (TCR) gene transfer into T cells to circumvent self-tolerance to the widely expressed human p53(264–272) tumor-associated antigen and developed approaches to generate high-affinity CD8-independent TCR. A safety concern of TCR gene transfer is the pairing of endogenous and introduced TCR chains resulting in the potential generation of self-reactive T cells (off-target autoimmunity). Several strategies to favor matched TCR chains pairing and thus enhancing TCR cell surface expression, including optimization of TCR encoding nucleotide sequences, introduction of an additional inter-chain disulfide bond between the TCR α and β chain constant domains, coexpression of both TCR α and β encoding-genes using self-cleaving 2A virus peptide-based retroviral vectors have been applied. However, adoptive transfer of mouse T cells transduced with modified p53-specific TCRs into p53-deficient humanized (A2Kb) mice was inducing lethal autoimmunity due to the formation of self-reactive TCRs infiltrating vital organs, such as spleen, liver and bone marrow. Therefore, an optimized single chain (sc) p53-specific TCR was engineered to avoid the formation of mismatched TCR heterodimers. The safety and therapeutic efficiency of this approach were evaluated in humanized mouse models of adoptive T cell transfer and successfully demonstrated that optimized p53-specific scTCR-redirected T cells (i) do not induce OFF-target autoimmunity and (ii) mediate antitumor reactivity. Importantly, because the expression of p53 antigen on normal tissues raises the concern of potential on-target toxicity, we performed adoptive T cell transfer experiments in humanized mice expressing the Human p53 protein (Hupki mice) and did not observe any sign of TCR gene transfer-mediated GvHD in this model. In conclusion, these mouse studies suggest that the optimized p53(264–272)-specific scTCR could represent a safe and efficient approach for TCR-based gene therapy. Disclosures: No relevant conflicts of interest to declare.

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