Functional Activity of Cytokine-Induced Killer Cells Enhanced by CAR-CD19 Modification or by Soluble Bispecific Antibody Blinatumomab.

Strategies to increase the anti-tumor efficacy of cytokine-induced killer cells (CIKs) include genetic modification with chimeric antigen receptors (CARs) or the addition of soluble T-cell engaging bispecific antibodies (BsAbs). Here, CIKs were modified using a transposon system integrating two distinct anti-CD19 CARs (CAR-MNZ and CAR-BG2) or combined with soluble CD3xCD19 BsAb blinatumomab (CIK + Blina). CAR-MNZ bearing the CD28-OX40-CD3ζ signaling modules, and CAR-BG2, designed on the Tisagenlecleucel CAR sequence (Kymriah®), carrying the 4-1BB and CD3ζ signaling elements, were employed. After transfection and CIK expansion, cells expressed CAR-CD19 to a similar extent (35.9% CAR-MNZ and 17.7% CAR-BG2). In vitro evaluations demonstrated robust proliferation and cytotoxicity (~50% cytotoxicity) of CARCIK-MNZ, CARCIK-BG2, and CIK + Blina against CD19+ target cells, suggesting similar efficacy. All effectors formed an increased number of synapses, activated NFAT and NFkB, and secreted IL-2 and IFN-É£ upon encountering targets. CIK + Blina displayed strongest NFAT and IFN-É£ induction, whereas CARCIK-BG2 demonstrated superior synapse formation. All the effectors have shown therapeutic activity in vivo against the CD19+ Daudi tumor model, with CARCIK cells showing a more durable response compared to CIK + Blina, likely due to the short half-life of Blina in this model.

Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1.

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC50 in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb.

Optimization and validation of in vivo flow cytometry chimeric antigen receptor T cell detection method using CD19his indirect staining.

CD19-targeted chimeric antigen receptor T (CAR-T) cell therapy has shown unprecedented results in patients with B cell relapsed/refractory acute lymphoblastic leukemia (R/R-ALL) and B cell non-Hodgkin lymphomas where no other curative options are available. In vivo monitoring of CAR-T cell kinetics is fundamental to understand the correlation between CAR-T cells expansion and persistence with treatment response and toxicity development. The aim of this study was to define a robust, sensitive, and universal method for CAR-T cell detection using flow cytometry. We set up and compared with each other three assays for CD19 CAR-T cell detection, all based on commercially available reagents. All methods used a recombinant human CD19 protein fragment recognized by the single-chain variable fragment of the CAR construct. The two indirect staining assays (CD19his + APC-conjugated antihistidine antibody and CD19bio + APC-conjugated antibiotin antibody) showed better sensitivity and specificity compared with the direct staining with CD19-FITC, and CD19his had a better cost-effective profile. We validated CAR detection with CD19his with parallel quantitative real-time polymerase chain reaction data and we could demonstrate a strong positive correlation. We also showed that CD19his staining can be easily included in a multicolor flow cytometry panel to achieve additional information about the cell phenotype of CAR-T cell positive subpopulations. Finally, this method can be used for different anti-CD19 CAR-T cell products and for different sample sources. These data demonstrate that detection of CAR-T cells by CD19his flow cytometry staining is a reliable, robust, and broadly applicable tool for in vivo monitoring of CAR-T cells.

Genetic defects of gamma-secretase genes in a multiple myeloma patient with high and dysregulated BCMA surface density: A case report.

Multiple myeloma (MM) cells from 1 out of 20 patient expressed high basal levels of membrane B-cell maturation antigen (BCMA, TNFRSF17, CD269), which was not upregulated by gamma-secretase inhibitor, suggesting a defective BCMA shedding by gamma-secretase. Genetic analyses of the patient's bone marrow DNA showed no mutations within the BCMA coding region, but rather partial deletion of PSEN1 and amplification of PSEN2, which encode alternative catalytic units of gamma-secretase. Altogether the data suggest that pt#12 MM cells express high and dysregulated BCMA with no shedding, due to genetic alterations of one or more gamma-secretase subunits.

Safety and Preliminary Efficacy of Mesenchymal Stromal Cell (ORBCEL-M) Therapy in Diabetic Kidney Disease: A Randomized Clinical Trial (NEPHSTROM).

SIGNIFICANCE STATEMENT: Mesenchymal stromal cells (MSCs) may offer a novel therapy for diabetic kidney disease (DKD), although clinical translation of this approach has been limited. The authors present findings from the first, lowest dose cohort of 16 adults with type 2 diabetes and progressive DKD participating in a randomized, placebo-controlled, dose-escalation phase 1b/2a trial of next-generation bone marrow-derived, anti-CD362 antibody-selected allogeneic MSCs (ORBCEL-M). A single intravenous (iv) infusion of 80×10 6 cells was safe and well-tolerated, with one quickly resolved infusion reaction in the placebo group and no subsequent treatment-related serious adverse events (SAEs). Compared with placebo, the median annual rate of decline in eGFR was significantly lower with ORBCEL-M, although mGFR did not differ. The results support further investigation of ORBCEL-M in this patient population in an appropriately sized phase 2b study. BACKGROUND: Systemic therapy with mesenchymal stromal cells may target maladaptive processes involved in diabetic kidney disease progression. However, clinical translation of this approach has been limited. METHODS: The Novel Stromal Cell Therapy for Diabetic Kidney Disease (NEPHSTROM) study, a randomized, placebo-controlled phase 1b/2a trial, assesses safety, tolerability, and preliminary efficacy of next-generation bone marrow-derived, anti-CD362-selected, allogeneic mesenchymal stromal cells (ORBCEL-M) in adults with type 2 diabetes and progressive diabetic kidney disease. This first, lowest dose cohort of 16 participants at three European sites was randomized (3:1) to receive intravenous infusion of ORBCEL-M (80×10 6 cells, n =12) or placebo ( n =4) and was followed for 18 months. RESULTS: At baseline, all participants were negative for anti-HLA antibodies and the measured GFR (mGFR) and estimated GFR were comparable between groups. The intervention was safe and well-tolerated. One placebo-treated participant had a quickly resolved infusion reaction (bronchospasm), with no subsequent treatment-related serious adverse events. Two ORBCEL-M recipients died during follow-up of causes deemed unrelated to the trial intervention; one recipient developed low-level anti-HLA antibodies. The median annual rate of kidney function decline after ORBCEL-M therapy compared with placebo did not differ by mGFR, but was significantly lower by eGFR estimated by the Chronic Kidney Disease Epidemiology Collaboration and Modification of Diet in Renal Disease equations. Immunologic profiling provided evidence of preservation of circulating regulatory T cells, lower natural killer T cells, and stabilization of inflammatory monocyte subsets in those receiving the cell therapy compared with placebo. CONCLUSIONS: Findings indicate safety and tolerability of intravenous ORBCEL-M cell therapy in the trial's lowest dose cohort. The rate of decline in eGFR (but not mGFR) over 18 months was significantly lower among those receiving cell therapy compared with placebo. Further studies will be needed to determine the therapy's effect on CKD progression. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrial.gov NCT02585622 .

Role of Fc Core Fucosylation in the Effector Function of IgG1 Antibodies.

The presence of fucose on IgG1 Asn-297 N-linked glycan is the modification of the human IgG1 Fc structure with the most significant impact on FcÉ£RIII affinity. It also significantly enhances the efficacy of antibody dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in vitro, induced by IgG1 therapeutic monoclonal antibodies (mAbs). The effect of afucosylation on ADCC or antibody dependent phagocytosis (ADCP) mediated by macrophages or polymorphonuclear neutrophils (PMN) is less clear. Evidence for enhanced efficacy of afucosylated therapeutic mAbs in vivo has also been reported. This has led to the development of several therapeutic antibodies with low Fc core fucose to treat cancer and inflammatory diseases, seven of which have already been approved for clinical use. More recently, the regulation of IgG Fc core fucosylation has been shown to take place naturally during the B-cell immune response: A decrease in α-1,6 fucose has been observed in polyclonal, antigen-specific IgG1 antibodies which are generated during alloimmunization of pregnant women by fetal erythrocyte or platelet antigens and following infection by some enveloped viruses and parasites. Low IgG1 Fc core fucose on antigen-specific polyclonal IgG1 has been linked to disease severity in several cases, such as SARS-CoV 2 and Dengue virus infection and during alloimmunization, highlighting the in vivo significance of this phenomenon. This review aims to summarize the current knowledge about human IgG1 Fc core fucosylation and its regulation and function in vivo, in the context of both therapeutic antibodies and the natural immune response. The parallels in these two areas are informative about the mechanisms and in vivo effects of Fc core fucosylation, and may allow to further exploit the desired properties of this modification in different clinical contexts.

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.

Third-party bone marrow-derived mesenchymal stromal cell infusion before liver transplantation: A randomized controlled trial.

Mesenchymal stromal cells (MSC) have emerged as a promising therapy to minimize the immunosuppressive regimen or induce tolerance in solid organ transplantation. In this randomized open-label phase Ib/IIa clinical trial, 20 liver transplant patients were randomly allocated (1:1) to receive a single pretransplant intravenous infusion of third-party bone marrow-derived MSC or standard of care alone. The primary endpoint was the safety profile of MSC administration during the 1-year follow-up. In all, 19 patients completed the study, and none of those who received MSC experienced infusion-related complications. The incidence of serious and non-serious adverse events was similar in the two groups. Circulating Treg/memory Treg and tolerant NK subset of CD56bright NK cells increased slightly over baseline, albeit not to a statistically significant extent, in MSC-treated patients but not in the control group. Graft function and survival, as well as histologic parameters and intragraft expression of tolerance-associated transcripts in 1-year protocol biopsies were similar in the two groups. In conclusion, pretransplant MSC infusion in liver transplant recipients was safe and induced mild positive changes in immunoregulatory T and NK cells in the peripheral blood. This study opens the way for a trial on possible tolerogenic efficacy of MSC in liver transplantation. ClinicalTrials.gov identifier: NCT02260375.

Tolerance to Bone Marrow Transplantation: Do Mesenchymal Stromal Cells Still Have a Future for Acute or Chronic GvHD?

Mesenchymal Stromal Cells (MSCs) are fibroblast-like cells of mesodermal origin present in many tissues and which have the potential to differentiate to osteoblasts, adipocytes and chondroblasts. They also have a clear immunosuppressive and tissue regeneration potential. Indeed, the initial classification of MSCs as pluripotent stem cells, has turned into their identification as stromal progenitors. Due to the relatively simple procedures available to expand in vitro large numbers of GMP grade MSCs from a variety of different tissues, many clinical trials have tested their therapeutic potential in vivo. One pathological condition where MSCs have been quite extensively tested is steroid resistant (SR) graft versus host disease (GvHD), a devastating condition that may occur in acute or chronic form following allogeneic hematopoietic stem cell transplantation. The clinical and experimental results obtained have outlined a possible efficacy of MSCs, but unfortunately statistical significance in clinical studies has only rarely been reached and effects have been relatively limited in most cases. Nonetheless, the extremely complex pathogenetic mechanisms at the basis of GvHD, the fact that studies have been conducted often in patients who had been previously treated with multiple lines of therapy, the variable MSC doses and schedules administered in different trials, the lack of validated potency assays and clear biomarkers, the difference in MSC sources and production methods may have been major factors for this lack of clear efficacy in vivo. The heterogeneity of MSCs and their different stromal differentiation potential and biological activity may be better understood through more refined single cell sequencing and proteomic studies, where either an "anti-inflammatory" or a more "immunosuppressive" profile can be identified. We summarize the pathogenic mechanisms of acute and chronic GvHD and the role for MSCs. We suggest that systematic controlled clinical trials still need to be conducted in the most promising clinical settings, using better characterized cells and measuring efficacy with specific biomarkers, before strong conclusions can be drawn about the therapeutic potential of these cells in this context. The same analysis should be applied to other inflammatory, immune or degenerative diseases where MSCs may have a therapeutic potential.

The Role of Complement in the Mechanism of Action of Therapeutic Anti-Cancer mAbs.

Unconjugated anti-cancer IgG1 monoclonal antibodies (mAbs) activate antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells and antibody-dependent cellular phagocytosis (ADCP) by macrophages, and these activities are thought to be important mechanisms of action for many of these mAbs in vivo. Several mAbs also activate the classical complement pathway and promote complement-dependent cytotoxicity (CDC), although with very different levels of efficacy, depending on the mAb, the target antigen, and the tumor type. Recent studies have unraveled the various structural factors that define why some IgG1 mAbs are strong mediators of CDC, whereas others are not. The role of complement activation and membrane inhibitors expressed by tumor cells, most notably CD55 and CD59, has also been quite extensively studied, but how much these affect the resistance of tumors in vivo to IgG1 therapeutic mAbs still remains incompletely understood. Recent studies have demonstrated that complement activation has multiple effects beyond target cell lysis, affecting both innate and adaptive immunity mediated by soluble complement fragments, such as C3a and C5a, and by stimulating complement receptors expressed by immune cells, including NK cells, neutrophils, macrophages, T cells, and dendritic cells. Complement activation can enhance ADCC and ADCP and may contribute to the vaccine effect of mAbs. These different aspects of complement are also briefly reviewed in the specific context of FDA-approved therapeutic anti-cancer IgG1 mAbs.

Combined Anti-Cancer Strategies Based on Anti-Checkpoint Inhibitor Antibodies.

Therapeutic monoclonal antibodies for the treatment of cancer came of age in 1997, with the approval of anti-CD20 Rituximab. Since then, a wide variety of antibodies have been developed with many different formats and mechanisms of action. Among these, antibodies blocking immune checkpoint inhibitors (ICI) have revolutionized the field, based on the novelty of their concept and their demonstrated efficacy in several types of cancer otherwise lacking effective immunotherapy approaches. ICI are expressed by tumor, stromal or immune cells infiltrating the tumor microenvironment, and negatively regulate anti-tumor immunity. Antibodies against the first discovered ICI, CTLA-4, PD-1 and PD-L1, have shown significant activity in phase III studies against melanoma and other solid cancers, alone or in combination with chemotherapy or radiotherapy. However, not all cancers and not all patients respond to these drugs. Therefore, novel antibodies targeting additional ICI are currently being developed. In addition, CTLA-4, PD-1 and PD-L1 blocking antibodies are being combined with each other or with other antibodies targeting novel ICI, immunostimulatory molecules, tumor antigens, angiogenic factors, complement receptors, or with T cell engaging bispecific antibodies (BsAb), with the aim of obtaining synergistic effects with minimal toxicity. In this review, we summarize the biological aspects behind such combinations and review some of the most important clinical data on ICI-specific antibodies.

Monoclonal Antibody Monitoring: Clinically Relevant Aspects, A Systematic Critical Review.

Monoclonal antibody (mAb) therapy does not usually lead to a clinical response in all patients and resistance may increase over time after repeated mAb administration. This lack or loss of response to the treatment may originate from different and little-known epigenetic, biomolecular, or pathophysiological mechanisms, although an inadequate serum concentration is perhaps the most likely cause, even if not widely recognized and investigated yet. Patient factors that influence the pharmacokinetics (PK) of a mAb should be taken into account. Multiple analyses of patient-derived PK data have identified various factors influencing the clearance of mAbs. These factors include the presence of antidrug antibodies, low serum albumin, high serum levels of C-reactive protein, high body weight, and gender differences among others. The same clearance processes involved in systemic clearance after intravenous administration are also involved in local first-pass catabolism after subcutaneous administration of mAbs. Therapeutic drug monitoring has been proposed as a way to understand and respond to the variability in clinical response and remission. For both classes of mAbs with anti-inflammatory and antitumor effects, dose-guided optimization based on the measurement of serum concentrations in individual patients could be the next step for a personalized and targeted mAb therapy.

Human neutrophils express low levels of FcγRIIIA, which plays a role in PMN activation.

We have identified a rare healthy FcγRIIIB (CD16B)-null donor completely lacking FCGR3B RNA and protein expression and dissected the role of the different neutrophil Fcγ receptors in the response to therapeutic anti-CD20 monoclonal antibodies. We observed that polymorphonuclear neutrophils (PMNs) from FcγRIIIB wild-type (WT) individuals or the null donor were more effectively activated by chronic lymphocytic leukemia (CLL) B-cell targets opsonized with glycoengineered anti-CD20 antibodies compared with fully core-fucosylated anti-CD20 antibodies, suggesting the presence and role of FcγRIIIA (CD16A) on PMNs. Indeed, we demonstrated by reverse-transcription polymerase chain reaction, flow cytometry, and western blot analysis that PMNs from FcγRIIIB WT donors and the null individual express low levels of FcγRIIIA on their surfaces. FcγRIIIA is a functional and activating molecule on these cells, because anti-CD16 F(ab')2 antibodies alone were able to activate highly purified PMNs from the FcγRIIIB-null donor. Use of blocking anti-CD16 and anti-CD32 antibodies showed that FcγRIIIA is also a major mediator of phagocytosis of CD20-opsonized beads by FcγRIIIB WT and null PMNs. In contrast, trogocytosis of antibody-opsonized CLL B cells by PMNs was mediated primarily by FcγRIIIB in WT PMNs and by FcγRIIA in null PMNs. We conclude that FcγRIIIA is an important player in PMN functions, whereas FcγRIIIB is dispensable for activation and phagocytosis. We discuss the clinical implications of these findings.

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.

Utility of routine evaluation of sterility of cellular therapy products with or without extensive manipulation: Best practices and clinical significance.

BACKGROUND: We analyzed the results of routine sterility testing performed in our center over the last 10 years, in the context both hematopoietic stem cell transplantation (HSCT) and Advanced Therapeutic Medicinal Products (ATMPs). METHODS: For sterility tests 14-day cultures were performed in culture media detecting aerobic and anaerobic microorganisms. RESULTS: In this study, 22/1643 (1.3%) of apheretic products for autologous or allogeneic HSCT were contaminated, whereas 14/73 bone marrow (BM) harvests (17.8%) were positive. In 22 cases, the contaminated HSCs were infused to patients, but there was no evidence of any adverse impact of contamination on the hematologic engraftment or on infections. Indeed none of the five positive hemocultures detected in patients following infusion could be linked to the contaminated stem cell product. Our Cell Factory also generated 286 ATMPs in good manufacturing practice (GMP) conditions since 2007 and all final products were sterile. In three cases of mesenchymal stromal cell expansions, the starting BM harvests were contaminated, but the cell products at the end of expansion were sterile, presumably thanks to the presence of an antibiotic in the culture medium. DISCUSSION: The decreased rate of contamination of cell harvests observed with time suggests that routine sterility testing and communication of the results to the collecting centers may improve clinical practices. Furthermore, we recommend the use of antibiotics in the medium for ATMP expansion, to decrease the likelihood of expanding microorganisms within clean rooms. Finally we discuss the costs of sterility testing of ATMPs by GMP-approved external laboratories.

Direct targeting of cancer cells with antibodies: What can we learn from the successes and failure of unconjugated antibodies for lymphoid neoplasias?

Following approval in 1997 of the anti-CD20 antibody rituximab for the treatment of B-NHL and CLL, many other unconjugated IgG1 MAbs have been tested in pre-clinical and clinical trials for the treatment of lymphoid neoplasms. Relatively few have been approved however and these are directed against a limited number of target antigens (CD20, CD52, CCR4, CD38, CD319). We review here the known biological properties of these antibodies and discuss which factors may have led to their success or may, on the contrary, limit their clinical application. Common factors of the approved MAbs are that the target antigen is expressed at relatively high levels on the neoplastic targets and their mechanism of action is mostly immune-mediated. Indeed most of these MAbs induce ADCC and phagocytosis by macrophages, and many also activate complement, leading to target cell lysis. In contrast direct cell death induction is not a common feature but may enhance efficacy in some cases. Interestingly, a key factor for the success of several MAbs appears to be their capacity to skew immunity towards an anti-tumour mode, by inhibiting/depleting suppressor cells and/or activating immune cells within the microenvironment, independently of FcγRs. We also expose here some of the strategies employed by industry to expand the clinical use of these molecules beyond their original indication. Interestingly, due to the central role of lymphocytes in the control of the immune response, several of the antibodies are now successfully used to treat many different autoimmune diseases and have also been formally approved for some of these new indications. There is little doubt that this trend will continue and that the precise mechanisms of therapeutic MAbs will be further dissected and better understood in the context of both tumour immunology and autoimmunity.

Phase II Study of Sequential Infusion of Donor Lymphocyte Infusion and Cytokine-Induced Killer Cells for Patients Relapsed after Allogeneic Hematopoietic Stem Cell Transplantation.

Seventy-four patients who relapsed after allogeneic stem cell transplantation were enrolled in a phase IIA study and treated with the sequential infusion of donor lymphocyte infusion (DLI) followed by cytokine-induced killer (CIK) cells. Seventy-three patients were available for the intention to treat analysis. At least 1 infusion of CIK cells was given to 59 patients, whereas 43 patients received the complete cell therapy planned (58%). Overall, 12 patients (16%) developed acute graft-versus-host disease (aGVHD) of grades I to II in 7 cases and grades III to IV in 5). In 8 of 12 cases, aGVHD developed during DLI treatment, leading to interruption of the cellular program in 3 patients, whereas in the remaining 5 cases aGVHD was controlled by steroids treatment, thus allowing the subsequent planned administration of CIK cells. Chronic GVHD (cGVHD) was observed in 11 patients (15%). A complete response was observed in 19 (26%), partial response in 3 (4%), stable disease in 8 (11%), early death in 2 (3%), and disease progression in 41 (56%). At 1 and 3 years, rates of progression-free survival were 31% and 29%, whereas rates of overall survival were 51% and 40%, respectively. By multivariate analysis, the type of relapse, the presence of cGVHD, and a short (<6 months) time from allogeneic hematopoietic stem cell transplantation to relapse were the significant predictors of survival. In conclusion, a low incidence of GVHD is observed after the sequential administration of DLI and CIK cells, and disease control can be achieved mostly after a cytogenetic or molecular relapse.