Optimization of therapeutic T cell expansion in G-Rex device and applicability to large-scale production for clinical use.

Our center performs experimental clinical studies with advanced therapy medicinal products (ATMPs) based on polyclonal T cells, all of which are currently expanded in standard T-flasks. Given the need to increase the efficiency and safety of large-scale T cell expansion for clinical use, we have optimized the method to expand in G-Rex devices both cytokine-induced killer cells (CIKs) from peripheral or cord blood and blinatumomab-expanded T cells (BETs). We show that the G-Rex reproducibly allowed the expansion of >30 × 106 CD3+ cells/cm2 of gas-permeable membrane in a mean of 10 to 11 days in a single unit, without manipulation, except for addition of cytokines and sampling of supernatant for lactate measurement every 3 to 4 days. In contrast, 21 to 24 days, twice-weekly cell resuspension and dilution into 48 to 72 T-flasks were required to complete expansions using the standard method. We show that the CIKs produced in G-Rex (CIK-G) were phenotypically very similar, for a large panel of markers, to those expanded in T-flasks, although CIK-G products had lower expression of CD56 and higher expression of CD27 and CD28. Functionally, CIK-Gs were strongly cytotoxic in vitro against the NK cell target K562 and the REH pre-B ALL cell line in the presence of blinatumomab. CIK-Gs also showed therapeutic activity in vivo in the Ph+ pre-B ALL-2 model in mice. The expansion of both CIKs and BETs in G-Rex was validated in good manufacturing practices (GMP) conditions, and we plan to use G-Rex for T cell expansion in future clinical studies.

BL-01, an Fc-bearing, tetravalent CD20 × CD5 bispecific antibody, redirects multiple immune cells to kill tumors in vitro and in vivo.

BACKGROUND AIMS: The authors describe here a novel therapeutic strategy combining a bispecific antibody (bsAb) with cytokine-induced killer (CIK) cells. METHODS: The authors have designed, produced and purified a novel tetravalent IgG1-like CD20 × CD5 bsAb called BL-01. The bsAb is composed of a fused heavy chain and two free light chains that pair correctly to the heavy chain sequences thanks to complementary mutations in the monoclonal antibody 2 CH1/CL sequences. RESULTS: The authors show that BL-01 can bind specifically to CD20 and CD5 with an affinity of 4-6 nM, demonstrating correct pairing of two light chains to the fused heavy chain. The CD20 × CD5 BL-01 bsAb has a functional human IgG1 Fc and can induce up to 65% complement-dependent cytotoxicity of a CD20+ lymphoma cell line in the presence of human complement, similar to anti-CD20 rituximab. The bsAb also induces significant natural killer cell activation and antibody-dependent cytotoxicity of up to 25% as well as up to 65% phagocytosis by human macrophages in the presence of CD20+ tumor cells. The BL-01 bsAb binds to CD20 and CD5 simultaneously and can redirect CIK cells in vitro to kill CD20+ targets, increasing the cytotoxicity of CIK cells by about 3-fold. The authors finally show that the CD20 × CD5 BL-01 bsAb synergizes with CIK cells in vivo in controlling tumor growth and prolonging survival of nonobese diabetic/severe combined immunodeficiency mice inoculated with a patient-derived, aggressive diffuse large B-cell lymphoma xenograft. CONCLUSIONS: The authors suggest that the efficacy of bsAb in vivo is due to the combined activation of innate immunity by Fc and redirection of CIK cells to kill the tumor target.

Cord blood-derived cytokine-induced killer cells combined with blinatumomab as a therapeutic strategy for CD19+ tumors.

BACKGROUND: Cytokine-induced killer cells (CIKs) are an advanced therapeutic medicinal product (ATMP) that has shown therapeutic activity in clinical trials but needs optimization. We developed a novel strategy using CIKs from banked cryopreserved cord blood units (CBUs) combined with bispecific antibody (BsAb) blinatumomab to treat CD19+ malignancies. METHODS: CB-CIKs were expanded in vitro and fully characterized in comparison with peripheral blood (PB)-derived CIKs. RESULTS: CB-CIKs, like PB-CIKs, were mostly CD3+ T cells with mean 45% CD3+CD56+ and expressing mostly TCR(T cell receptor)αß with a TH1 phenotype. CB-CIK cultures had, however, a larger proportion of CD4+ cells, mostly CD56-, as well as a greater proportion of naïve CCR7+CD45RA+ and a lower percentage of effector memory cells, compared with PB-CIKs. CB-CIKs were very similar to PB-CIKs in their expression of a large panel of co-stimulatory and inhibitory/exhaustion markers, except for higher CD28 expression among CD8+ cells. Like PB-CIKs, CB-CIKs were highly cytotoxic in vitro against natural killer (NK) cell targets and efficiently lysed CD19+ tumor cells in the presence of blinatumomab, with 30-60% lysis of target cells at very low effector:target ratios. Finally, both CB-CIKs and PB-CIKs, combined with blinatumomab, showed significant therapeutic activity in an aggressive PDX Ph+ CD19+ acute lymphoblastic leukemia model in NOD-SCID mice, without sign of toxicity or graft-versus-host disease. The improved expansion protocol was finally validated in good manufacturing practice conditions, showing reproducible expansion of CIKs from cryopreserved cord blood units with a median of 28.8 × 106 CIK/kg. DISCUSSION: We conclude that CB-CIKs, combined with bispecific T-cell-engaging antibodies, offer a novel, effective treatment strategy for leukemia.

Novel pyrrole carboxamide inhibitors of JAK2 as potential treatment of myeloproliferative disorders.

Compound 1, a hit from the screening of our chemical collection displaying activity against JAK2, was deconstructed for SAR analysis into three regions, which were explored. A series of compounds was synthesized leading to the identification of the potent and orally bioavailable JAK2 inhibitor 16 (NMS-P830), which showed an encouraging tumour growth inhibition in SET-2 xenograft tumour model, with evidence for JAK2 pathway suppression demonstrated by in vivo pharmacodynamic effects.

A novel method using blinatumomab for efficient, clinical-grade expansion of polyclonal T cells for adoptive immunotherapy.

Current treatment of chronic lymphocytic leukemia (CLL) patients often results in life-threatening immunosuppression. Furthermore, CLL is still an incurable disease due to the persistence of residual leukemic cells. These patients may therefore benefit from immunotherapy approaches aimed at immunoreconstitution and/or the elimination of residual disease following chemotherapy. For these purposes, we designed a simple GMP-compliant protocol for ex vivo expansion of normal T cells from CLL patients' peripheral blood for adoptive therapy, using bispecific Ab blinatumomab (CD3 × CD19), acting both as T cell stimulator and CLL depletion agent, and human rIL-2. Starting from only 10 ml CLL peripheral blood, a mean 515 × 10(6) CD3(+) T cells were expanded in 3 wk. The resulting blinatumomab-expanded T cells (BET) were polyclonal CD4(+) and CD8(+) and mostly effector and central memory cells. The Th1 subset was slightly prevalent over Th2, whereas Th17 and T regulatory cells were <1%. CMV-specific clones were detected in equivalent proportion before and after expansion. Interestingly, BET cells had normalized expression of the synapse inhibitors CD272 and CD279 compared with starting T cells and were cytotoxic against CD19(+) targets in presence of blinatumomab in vitro. In support of their functional capacity, we observed that BET, in combination with blinatumomab, had significant therapeutic activity in a systemic human diffuse large B lymphoma model in NOD-SCID mice. We propose BET as a therapeutic tool for immunoreconstitution of heavily immunosuppressed CLL patients and, in combination with bispecific Ab, as antitumor immunotherapy.

Pyrrole-3-carboxamides as potent and selective JAK2 inhibitors.

We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally bioavailable JAK2 inhibitor 28 (NMS-P953). Compound 28 displayed significant tumour growth inhibition in SET-2 xenograft tumour model, with a mechanism of action confirmed in vivo by typical modulation of known biomarkers, and with a favourable pharmacokinetic and safety profile.

The Polo-Like Kinase 1 (PLK1) inhibitor NMS-P937 is effective in a new model of disseminated primary CD56+ acute monoblastic leukaemia.

CD56 is expressed in 15-20% of acute myeloid leukaemias (AML) and is associated with extramedullary diffusion, multidrug resistance and poor prognosis. We describe the establishment and characterisation of a novel disseminated model of AML (AML-NS8), generated by injection into mice of leukaemic blasts freshly isolated from a patient with an aggressive CD56(+) monoblastic AML (M5a). The model reproduced typical manifestations of this leukaemia, including presence of extramedullary masses and central nervous system involvement, and the original phenotype, karyotype and genotype of leukaemic cells were retained in vivo. Recently Polo-Like Kinase 1 (PLK1) has emerged as a new candidate drug target in AML. We therefore tested our PLK1 inhibitor NMS-P937 in this model either in the engraftment or in the established disease settings. Both schedules showed good efficacy compared to standard therapies, with a significant increase in median survival time (MST) expecially in the established disease setting (MST = 28, 36, 62 days for vehicle, cytarabine and NMS-P937, respectively). Importantly, we could also demonstrate that NMS-P937 induced specific biomarker modulation in extramedullary tissues. This new in vivo model of CD56(+) AML that recapitulates the human tumour lends support for the therapeutic use of PLK1 inhibitors in AML.

4,5-Dihydro-1H-pyrazolo[4,3-h]quinazolines as potent and selective Polo-like kinase 1 (PLK1) inhibitors.

A series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives was optimized as Polo-like kinase 1 inhibitors. Extensive SAR afforded a highly potent and selective PLK1 compound. The compound showed good antiproliferative activity when tested in a panel of tumor cell lines with PLK1 related mechanism of action and with good in vivo antitumor efficacy in two xenograft models after i.v. administration.