NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma.

Despite recent therapeutic advances, multiple myeloma (MM) remains largely incurable. Here we report results of a phase I/II trial to evaluate the safety and activity of autologous T cells engineered to express an affinity-enhanced T cell receptor (TCR) recognizing a naturally processed peptide shared by the cancer-testis antigens NY-ESO-1 and LAGE-1. Twenty patients with antigen-positive MM received an average 2.4 × 10(9) engineered T cells 2 d after autologous stem cell transplant. Infusions were well tolerated without clinically apparent cytokine-release syndrome, despite high IL-6 levels. Engineered T cells expanded, persisted, trafficked to marrow and exhibited a cytotoxic phenotype. Persistence of engineered T cells in blood was inversely associated with NY-ESO-1 levels in the marrow. Disease progression was associated with loss of T cell persistence or antigen escape, in accordance with the expected mechanism of action of the transferred T cells. Encouraging clinical responses were observed in 16 of 20 patients (80%) with advanced disease, with a median progression-free survival of 19.1 months. NY-ESO-1-LAGE-1 TCR-engineered T cells were safe, trafficked to marrow and showed extended persistence that correlated with clinical activity against antigen-positive myeloma.

Rapid immune recovery and graft-versus-host disease-like engraftment syndrome following adoptive transfer of Costimulated autologous T cells.

PURPOSE: Previously, we showed that adoptive transfer of in vivo vaccine-primed and ex vivo (anti-CD3/anti-CD28) costimulated autologous T cells (ex-T) at day +12 after transplant increased CD4 and CD8 T-cell counts at day +42 and augmented vaccine-specific immune responses in patients with myeloma. Here, we investigated the safety and kinetics of T-cell recovery after infusing ex-T at day +2 after transplant. EXPERIMENTAL DESIGN: In this phase I/II two-arm clinical trial, 50 patients with myeloma received autografts after high-dose melphalan followed by infusions of ex-T at day +2 after transplant. Patients also received pretransplant and posttransplant immunizations using a pneumococcal conjugate vaccine only (arm B; n = 24) or the pneumococcal conjugate vaccine plus an HLA-A2-restricted microltipeptide vaccine for HLA-A2(+) patients (arm A; n = 26). RESULTS: The mean number of T cells infused was 4.26 x 10(10) (range, 1.59-5.0). At day 14 after transplant, the median CD3, CD4, and CD8 counts were 4,198, 1,545, and 2,858 cells/microL, respectively. Interleukin (IL)-6 and IL-15 levels increased early after transplant and IL-15 levels correlated significantly to day 14 T-cell counts. Robust vaccine-specific B- and T-cell responses were generated. T-cell infusions were well tolerated with no effect on hematopoietic recovery. Eight patients (16%) developed a T-cell "engraftment syndrome" characterized by diarrhea and fever that was clinically and histopathologically indistinguishable from grade 1 to 3 acute graft-versus-host disease (GVHD) of the gastrointestinal tract (seven patients) and/or grade 1 to 2 cutaneous GVHD (four patients). CONCLUSIONS: Adoptive T-cell transfers achieve robust T-cell recovery early after transplant and induce moderate-to-severe autologous GVHD in a subset of patients.

Phase I trial of first-line bortezomib/thalidomide plus chemotherapy for induction and stem cell mobilization in patients with multiple myeloma.

BACKGROUND: In preclinical studies, bortezomib was shown to suppress tumor growth, sensitize malignant cells to apoptosis, and reverse chemotherapy resistance. PATIENTS AND METHODS: We evaluated the addition of escalating doses of bortezomib 0.7, 1, and 1.3 mg/m2 intravenously on days 1, 4, and 8 to DT-PACE (cisplatin 10 mg/m2, doxorubicin 10 mg/m2, cyclophosphamide 400 mg/m2, and etoposide 40 mg/m2 per day by intravenous continuous infusion on days 1-4) plus oral dexamethasone 40 mg on days 1-4 and thalidomide 200 mg on days 1-8 in newly diagnosed patients with multiple myeloma. Peripheral blood stem cells were collected after cycle 1. Twelve patients completed the study, and all received autologous stem cell transplantation (SCT). RESULTS: Hematologic toxicities were predictable, with 3 episodes of neutropenic fever. Grade >/= 2 nonhematologic toxicities included diarrhea (n = 1), deep vein thrombosis (n = 2), hypotension (n = 2), syncope (n = 1), and peripheral neuropathy (n = 3). The median number of CD34+ cells collected was 20.57 x 10 superset6 CD34+ cells/kg. After 2 cycles, 10 of 12 patients exhibited a partial response or better. Best response after autologous SCT, complete response/near complete response was exhibited in 9 patients, and partial response was exhibited in 3 patients. At a median of 20 months, 4 patients experienced relapse and 1 had died. Bortezomib/DT-PACE compared favorably with DT-PACE with regard to leukapheresis days, total CD34+ cell collection, and engraftment. CONCLUSION: This novel strategy of simultaneous proteasome inhibition in combination with thalidomide and chemotherapy was effective and safe, allowing for adequate stem cell collection and early autologous SCT; its impact on overall survival, especially in patients with high-risk myeloma, awaits further investigation.

Restoration of immunity in lymphopenic individuals with cancer by vaccination and adoptive T-cell transfer.

Immunodeficiency is a barrier to successful vaccination in individuals with cancer and chronic infection. We performed a randomized phase 1/2 study in lymphopenic individuals after high-dose chemotherapy and autologous hematopoietic stem cell transplantation for myeloma. Combination immunotherapy consisting of a single early post-transplant infusion of in vivo vaccine-primed and ex vivo costimulated autologous T cells followed by post-transplant booster immunizations improved the severe immunodeficiency associated with high-dose chemotherapy and led to the induction of clinically relevant immunity in adults within a month after transplantation. Immune assays showed accelerated restoration of CD4 T-cell numbers and function. Early T-cell infusions also resulted in significantly improved T-cell proliferation in response to antigens that were not contained in the vaccine, as assessed by responses to staphylococcal enterotoxin B and cytomegalovirus antigens (P < 0.05). In the setting of lymphopenia, combined vaccine therapy and adoptive T-cell transfer fosters the development of enhanced memory T-cell responses.

Biological activity of IgE specific for cross-reactive carbohydrate determinants.

BACKGROUND: The clinical relevance of IgE specific for cross-reactive carbohydrate determinants (CCDs) has been a matter of controversy. Until now, no convincing experiments have been performed to test the biologic significance of individual multivalent allergens that carry multiple carbohydrate epitopes. OBJECTIVE: We sought to contribute to the understanding of the role of CCD-specific IgE antibodies and to study whether CCD-specific IgE antibodies are able to activate basophils using different multivalent glycoproteins as antigens. METHODS: Purified natural tomato beta-fructofuranosidase (nLyc e 2) and rLyc e 2.02 expressed in Escherichia coli were compared by means of histamine release tests. In addition, native and deglycosylated horseradish peroxidase and a neoglycoprotein consisting of a defined glycopeptide (Manalpha1-6[Xylbeta1-2]Manbeta1-4GlcNAcbeta1-4[Fucalpha1-3]GlcNAc) coupled to BSA were used in histamine release assays using stripped basophils from normal donors resensitized with IgE from CCD-reactive patients with food allergy to tomato. RESULTS: Ten CCD-positive and 2 CCD-negative sera from patients with tomato allergy underwent histamine release testing by using the glycoproteins and nonglycosylated controls as antigens, respectively. All sera induced histamine release with tomato extract (up to 100%), confirming the allergic status of the donors. Four of the CCD-positive sera induced releases ranging from 12% to 82% with all of the glycoproteins but not with the nonglycosylated or monovalent controls. All other sera showed no response or only very weak response to the glycoproteins. CONCLUSION: Approximately one third of the CCD-positive sera from patients with tomato allergy have biologically relevant CCD-specific IgE antibodies. Therefore the general claim that CCD-specific IgE is clinically irrelevant has to be reconsidered critically. Hence IgE specific for CCDs should be taken into consideration in the diagnosis and therapy of certain allergies. In the subgroup of patients sensitized to CCDs, the use of natural allergens should be preferred over the use of recombinant allergens expressed in prokaryotic organisms.

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.