T-cell engineered with a fully humanized B-cell maturation antigen-specific T-cell antigen coupler receptor effectively target multiple myeloma.

B-cell maturation antigen (BCMA) is a clinically validated target for multiple myeloma. T-cell engineered with chimeric antigen receptors (CARs) directed against BCMA have demonstrated robust therapeutic activity in clinical trials, but toxicities remain a significant concern for a subset of patients, supporting continued investigation of other engineered T-cell platforms that may offer equal efficacy with an improved toxicity profile. The authors recently described a BCMA-specific, T-cell-centric synthetic antigen receptor, the T-cell antigen coupler (TAC) receptor, that can be used to engineer T-cell with robust anti-myeloma activity. Here the authors describe the creation of a fully humanized BCMA-specific TAC receptor. Single-chain variable fragments (scFvs) were developed from BCMA-specific F(ab)s that were identified in a fully human phage display library. Twenty-four configurations of the F(ab)s were evaluated in a medium-throughput screening using primary T-cell, and a single F(ab), TRAC 3625, emerged as the most robust following in vitro and in vivo evaluation. An optimized BCMA-specific TAC receptor was developed through iterations of the BCMA-TAC design that evaluated a next-generation TAC scaffold sequence, different domains connecting the TAC to the 3625 scFv and different orientations of the TRAC 3625 heavy and light variable regions.

A randomized clinical trial of autologous T-cell therapy in multiple sclerosis: subset analysis and implications for trial design.

BACKGROUND: Tovaxin is an autologous T-cell immunotherapy under investigation for the treatment of MS. The product consists of in vitro expanded myelin-reactive T-cells manufactured against up to six immunodominant peptides derived from three myelin antigens. METHODS: A Phase 2b placebo controlled study (TERMS) was conducted in 150 subjects to gather safety and efficacy data in relapsing-remitting MS and clinically isolated syndrome subjects. RESULTS: Tovaxin had a favorable safety profile. Although no statistically significant clinical or radiological benefit of Tovaxin immunotherapy was identified in the modified intent-to-treat population, a prospective analysis of subjects with more active disease favored Tovaxin in terms of annualized relapse rate (ARR) and disability progression. An analysis also found a possible legacy effect of prior disease-modifying treatment (DMT) which may have contributed to a lowered ARR in the placebo group. DMT-naïve subjects treated with Tovaxin had a lower ARR compared to the placebo group, particularly in those with active baseline disease (ARR ≥ 1, ARR>1). However, clinical benefit was not was accompanied by a treatment-dependent improvement in MRI measures. CONCLUSIONS: Previous DMT exposure may reduce effect size and study power. Limiting subject selection to DMT-treatment-naïve individuals may be a reasonable approach to phase 2 or proof-of-concept studies of limited duration.

A phase 1/2 study of autologous neuroblastoma tumor cells genetically modified to secrete IL-2 in patients with high-risk neuroblastoma.

Autologous neuroblastoma (NB) tumor cells modified to secrete interleukin (IL)-2 (auto-IL-2) can be safely given to patients with advanced neuroblastoma and generate antitumor immune responses. As the benefits of tumor immunization may be greater in patients with minimal residual disease and thus rely on surrogate markers such as immune responses to measure effect, we studied the frequency of immune changes associated with vaccination. Thirteen patients (8 in first remission and 5 after treatment for recurrent NB) received 5 to 8 subcutaneous injections of auto-IL-2 at 0.3 x 10 cells/kg. The vaccine was well tolerated. Injection site biopsies revealed increased cellularity caused by infiltration of CD4 and CD8 lymphocytes, eosinophils, and dendritic cells. Enzyme-linked immunosorbent spot assays for interferon-gamma and IL-5 demonstrated that vaccination produced a rise in circulating CD4 and CD8 T cells responsive to stimulation by autologous tumor cells. Median event-free survival was 22 months for patients in first remission and 3 months for all others. Four patients treated in first remission remain alive and 3 without disease recurrence.

Phase I trial of vaccination with autologous neuroblastoma tumor cells genetically modified to secrete IL-2 and lymphotactin.

In murine models, transgenic chemokine-cytokine tumor vaccines overcome many of the limitations of single-agent immunotherapy by producing the sequence of T-cell attraction followed by proliferation of tumor antigen-activated clones. The safety and immunologic effects of this approach in humans were tested in 7 patients with relapsed or refractory neuroblastoma. They each received up to 8 subcutaneous injections of a vaccine combining lymphotactin--and interleukin-2 (IL-2)--secreting autologous neuroblastoma cells in a dose-escalating scheme. Adverse events were limited to grade 1 or 2 localized reactions in all patients, pain in 3 patients, and fever in 3 patients. Injection site biopsies revealed increased cellularity caused by infiltration of CD4 and CD8 lymphocytes, eosinophils, and dendritic cells with a decrease in dendritic cells from the first to the second vaccination. Systemically, vaccine was associated with increased tumor recognition as measured by enzyme-linked immunosorbent spot assays. Two patients had interferon-gamma predominant responses and 3 had IL-5 predominant responses. Only 1 patient received all 8 injections, 1 patient stopped the study early, and all other patients progressed before completion of the study. Hence, autologous tumor cell vaccines combining transgenic lymphotactin with IL-2 seem to have little toxicity in humans and can induce an antitumor immune response. In this setting, the immune response was insufficient to overcome active recurrent neuroblastoma.

Complement-fixing CD45 monoclonal antibodies to facilitate stem cell transplantation in mouse and man.

Broadening the applicability of stem cell therapies requires safer preparative regimens for patients. The CD45 antigen is present on all cells of the hematopoietic lineage, and using a murine model, we determined whether a lytic CD45 monoclonal antibody could produce persistent aplasia and whether it could facilitate syngeneic or allogeneic stem cell engraftment. After its systemic administration, we found that all leukocyte subsets in peripheral blood were markedly diminished, but only the effect on the lymphoid compartment was sustained and marrow progenitor cells were spared from destruction. Given the transient effects of the monoclonal antibody on myelopoiesis and the more persistent effects on lymphopoiesis, we asked whether this agent could contribute to donor hemopoietic engraftment after subablative transplantation. Treatment with anti-CD45 alone did not enhance syngeneic engraftment, consistent with its inability to destroy progenitor cells and permit competitive repopulation with syngeneic donor stem cells. By contrast, the combination of anti-CD45 and an otherwise inactive dose of total-body irradiation allowed engraftment of H2 fully allogeneic donor stem cells. We attribute this result to the recipient immunosuppression produced by depletion of CD45-positive lymphocytes. We next assessed a pair of unconjugated rat anti-human CD45 monoclonal antibodies (MAbs), YTH54.12 and YTH25.4, in a clinical trial in patients who were to receive stem cell transplantation for acute leukemia. The maximum tolerated dose of these MAbs, 400 microg/kg/day, produced a pattern of response identical to that seen in the mice, with marked reductions in circulating lymphoid and myeloid cells and sparing of early marrow progenitors. In two of three patients with active leukemia, the MAbs also produced a decrease in the percentage of leukemic blast cells in bone marrow. These pre-clinical and clinical results warrant further evaluation of anti-CD45 MAbs in subablative preparative regimens for stem cell transplantation.

Hematopoietic and immunomodulatory effects of lytic CD45 monoclonal antibodies in patients with hematologic malignancy.

The CD45 antigen is present on all cells of the hematopoietic lineage. In some rodent models, lytic CD45 monoclonal antibodies (MAbs) induce complete marrow aplasia. In others, only transient myelolymphodepletion are observed, which are nonetheless sufficient to permit engraftment with fully allogeneic stem cells after otherwise ineffective doses of radiation. The in vivo effects of unconjugated cytolytic CD45 MAbs on myeloid and lymphoid cells in humans are unknown, so it is unclear if they could contribute in a similar way to conventional ablative or to nonmyeloablative preparative regimens used for stem cell transplantation (SCT). We therefore assessed the safety, myeloreductive activities, and lymphoreductive activities of the unconjugated rat anti-human CD45 MAbs, YTH25.4 and YTH54.12, in subjects who were to undergo SCT for advanced hematologic malignancy. The MAb pair bind to contiguous but nonoverlapping epitopes on CD45 and work synergistically to fix complement and recruit cellular lytic mechanisms. The MAbs were given in increasing doses up to 1600 microg/kg during 4 days, after which the patients began their conventional transplantation preparative regimen. The maximum tolerated dose of these MAbs, 400 microg/kg/d, produced marked reduction in circulating lymphoid and myeloid cells while largely sparing marrow progenitors. In 2 of 3 patients who had active leukemia at the time of study, the MAbs reduced the percentage of leukemic blast cells in bone marrow. Seven of 14 patients are disease free 610 to 1555 days post-SCT. The in vivo myeloreductive and lymphoreductive properties of lytic CD45 MAb in humans, therefore, closely parallel the activity seen in a murine model and, therefore, may be of similar value.

Local and systemic effects of an allogeneic tumor cell vaccine combining transgenic human lymphotactin with interleukin-2 in patients with advanced or refractory neuroblastoma.

In murine models, transgenic chemokine-cytokine tumor vaccines overcome many of the limitations of single-agent immunotherapy by producing the sequence of T-cell attraction followed by proliferation. The safety and immunologic effects of this approach in humans were tested in 21 patients with relapsed or refractory neuroblastoma. They received up to 8 subcutaneous injections of a vaccine combining lymphotactin (Lptn)- and interleukin-2 (IL-2)-secreting allogeneic neuroblastoma cells in a dose-escalating scheme. Severe adverse reactions were limited to reversible panniculitis in 5 patients and bone pain in 1 patient. Injection-site biopsies revealed increased cellularity caused by infiltration of CD4+ and CD8+ lymphocytes, eosinophils, and Langerhans cells. Systemically, the vaccine produced a 2-fold (P =.035) expansion of CD4+ T cells, a 3.5-fold (P =.039) expansion of natural killer (NK) cells, a 2.1-fold (P =.014) expansion of eosinophils, and a 1.6-fold (P =.049) increase in serum IL-5. When restimulated in vitro by the immunizing cell line, T cells collected after vaccination showed a 2.3-fold increase (P =.02) of T-helper (TH2)-type CD3+IL-4+ cells. Supernatant collected from restimulated cells showed increased amounts of IL-4 (11.4-fold; P =.021) and IL-5 (8.7-fold; P =.002). Six patients had significant increases in NK cytolytic activity. Fifteen patients made immunoglobulin G (IgG) antibodies that bound to the immunizing cell line. Measurable tumor responses included complete remission in 2 patients and partial response in 1 patient. Hence, allogeneic tumor cell vaccines combining transgenic Lptn with IL-2 appear to have little toxicity in humans and can induce an antitumor immune response.

Transgenic expression of CD40 ligand produces an in vivo antitumor immune response against both CD40(+) and CD40(-) plasmacytoma cells.

Because tumor-specific antigens have been identified in multiple myeloma (MM), immunotherapy might provide an additional treatment modality for the disease. Expression of CD40 ligand (CD40L) proximate to the MM cells might serve this purpose, either by increasing their capacity to present self-antigens by activation through their CD40 receptor or by the recruitment of professional antigen-presenting cells (APCs) able to take up and present tumor-associated antigens. To distinguish between these possibilities and predict whether human CD40(-) myeloma might respond to this approach, we examined 3 murine plasmacytoma cell lines, 2 (MPC-11 and S107) expressing the CD40 molecule and 1 (X-24) lacking such expression. Syngeneic BALB/CBYJ mice were inoculated subcutaneously with tumor cells mixed with CL7.1 fibroblasts, retrovirally transduced to express either the mCD40L or the neo gene. For all 3 plasmacytoma cell lines, coinjection with CL7.1/mCD40L significantly reduced local tumor growth compared with controls. This effect was mediated by a systemic antitumor immune response, since mice immunized with tumor and CL7.1/mCD40L were resistant to subsequent challenge with tumor, and tumor growth inhibition was abolished when CD8(+) or CD4(+) lymphocytes were depleted. Because expression of CD40L gave equivalent protection from CD40(+) and CD40(-) tumors and transgenic-CD40L failed to up-regulate costimulatory molecules in either tumor, the protective effects of CD40L probably resulted from recruitment/activation of professional APCs rather than from CD40 activation of plasmacytoma cells. As further support of this concept, we found that mice were also well protected if CL7.1 and CD40L were injected together with apoptotic plasmacytoma cells from these tumors. Hence, transgenic CD40L expression may produce an antimyeloma immune response against either CD40(+) or CD40(-) tumors and may be of therapeutic value for both types of myeloma in humans.