Basics of advanced therapy medicinal product development in academic pharma and the role of a GMP simulation unit.

Following successes of authorized chimeric antigen receptor T-cell products being commercially marketed in the United States and European Union, product development of T-cell-based cancer immunotherapy consisting of cell-based advanced therapy medicinal products (ATMPs) has gained further momentum. Due to their complex characteristics, pharmacological properties of living cell products are, in contrast to classical biological drugs such as small molecules, more difficult to define. Despite the availability of many new advanced technologies that facilitate ATMP manufacturing, translation from research-grade to clinical-grade manufacturing in accordance with Good Manufacturing Practices (cGMP) needs a thorough product development process in order to maintain the same product characteristics and activity of the therapeutic product after full-scale clinical GMP production as originally developed within a research setting. The same holds true for transferring a fully developed GMP-grade production process between different GMP facilities. Such product development from the research to GMP-grade manufacturing and technology transfer processes of established GMP-compliant procedures between facilities are challenging. In this review, we highlight some of the main obstacles related to the product development, manufacturing process, and product analysis, as well as how these hinder rapid access to ATMPs. We elaborate on the role of academia, also referred to as 'academic pharma', and the added value of GMP production and GMP simulation facilities to keep innovation moving by reducing the development time and to keep final production costs reasonable.

Cognate CD4 T-cell licensing of dendritic cells heralds anti-cytomegalovirus CD8 T-cell immunity after human allogeneic umbilical cord blood transplantation.

UNLABELLED: Reactivation of human cytomegalovirus (CMV) is hazardous to patients undergoing allogeneic cord blood transplantation (CBT), lowering survival rates by approximately 25%. While antiviral treatment ameliorates viremia, complete viral control requires CD8+ T-cell-driven immunity. Mouse studies suggest that cognate antigen-specific CD4+ T-cell licensing of dendritic cells (DCs) is required to generate effective CD8+ T-cell responses. For humans, this was not fully understood. We here show that CD4+ T cells are essential for licensing of human DCs to generate effector and memory CD8+ T-cell immunity against CMV in CBT patients. First, we show in CBT recipients that clonal expansion of CMV-pp65-specific CD4+ T cells precedes the rise in CMV-pp65-specific CD8+ T cells. Second, the elicitation of CMV-pp65-specific CD8+ T cells from rare naive precursors in cord blood requires DC licensing by cognate CMV-pp65-specific CD4+ T cells. Finally, also CD8+ T-cell memory responses require CD4+ T-cell-mediated licensing of DCs in our system, by secretion of gamma interferon (IFN-γ) by pp65-specific CD4+ T cells. Together, these data show that human DCs require licensing by cognate antigen-specific CD4+ T cells to elicit effective CD8+ T-cell-mediated immunity and fight off viral reactivation in CBT patients. IMPORTANCE: Survival rates after stem cell transplantation are lowered by 25% when patients undergo reactivation of cytomegalovirus (CMV) that they harbor. Immune protection against CMV is mostly executed by white blood cells called killer T cells. We here show that for generation of optimally protective killer T-cell responses that respond to CMV, the early elicitation of help from a second branch of CMV-directed T cells, called helper T cells, is required.

Predictors of dose escalation of adalimumab in a prospective cohort of Crohn's disease patients.

BACKGROUND: Adalimumab is effective for the induction and maintenance of remission in Crohn's disease (CD)-patients. AIM: To find predictors for adalimumab dose escalation at initiation of adalimumab. METHODS: Crohn's disease patients in a single tertiary referral centre who started adalimumab between July 2007 and March 2010 at an induction dose (week 0 160 mg subcutaneously (sc), week 2 80 mg sc) and maintenance dose of 40 mg sc thereafter every other week were followed prospectively. Patients on adalimumab for at least 3 months were included. The number of patients needing dose escalation was assessed. Patients that needed dose escalation were compared with patients that did not need dose escalation. RESULTS: Of 199 CD patients treated with adalimumab and followed prospectively, 122 patients (M/F 54/68, median age 35 years, range 18-66 years, median CDAI 164, range 6-468) were treated for 3 months. In total 38% of these patients (46/122) needed a dose escalation within a median time of 21 weeks after adalimumab introduction (range 4-105). Body mass index (BMI) (P < 0.03) and secondary non-response to infliximab (IFX) (P < 0.06) were identified as predictors for dose escalation. Concomitant use of immunomodulators at initiation of adalimumab and the presence of autoantibodies to IFX did not predict dose escalation. CONCLUSIONS: Over one-third adalimumab-treated patients are dose escalated within a median of 5 months. Higher BMI and secondary non-response to IFX treatment are predictive for a dose escalation during adalimumab treatment.

High intra-uterine exposure to infliximab following maternal anti-TNF treatment during pregnancy.

BACKGROUND: Typically, inflammatory bowel disease (IBD) patients are in their reproductive years, raising questions about safely using antitumour necrosis factor antibodies like infliximab (IFX) during pregnancy. IgG antibodies naturally cross the placenta, especially during the last trimester. To prevent foetal intra-uterine exposure, stopping IFX treatment at gestational week 30 is recommended. However, whether this limits intra-uterine and early postnatal IFX exposure is unestablished. AIM: To determine the intra-uterine exposure to IFX following maternal treatment with IFX. METHODS: Four pregnant IBD patients intentionally continued IFX during pregnancy. IFX levels were assessed in newborns' cord blood and the mothers' peripheral blood at delivery. The children's development during the first 3-6 months, infections, vaccine reactions and antibody responses to vaccinations against Haemophilus influenzae type b and Pneumococcus were assessed. RESULTS: The patients stopped IFX therapy at gestational week 21, 26, 26 and 30, respectively. In three infants, therapeutic IFX levels were present in cord blood at levels of 5.5-13.7 µg/mL and were two- to three-fold higher than in the peripheral blood of their mothers. During the 3- to 6-month follow-up, the children developed normally without signs of infections or allergic reactions, and had normal antibody titres after routine childhood vaccinations. CONCLUSION: The use of IFX until gestational week 30 leads to foetal intra-uterine exposure to IFX at levels that exceed those in the mothers' peripheral blood. Although no short-term complications were detected, the high IFX levels observed in newborns raise concerns about unknown effects of IFX on the developing immune system.

Ultrafine but not fine particulate matter causes airway inflammation and allergic airway sensitization to co-administered antigen in mice.

BACKGROUND: Airborne particulate matter (PM) is an important factor associated with the enhanced prevalence of respiratory allergy. The PM adjuvant activity on allergic sensitization is a possible mechanism of action involved, and the induction of airway inflammation is suggested to be of importance in PM-induced adjuvant activity. OBJECTIVE: Because differently sized PM have different toxic potentials, we studied the role of particle size in the induction of airway inflammation and allergic sensitization. This was done using fine (0.250 and 0.260 micro m) and ultrafine (0.029 and 0.014 micro m) titanium dioxide (TiO(2)) and carbon black particles (CBP) with known differences in airway toxicity. METHODS: Mice were intranasally exposed to ovalbumin (OVA) alone or in combination with one of the different particles. The induction of airway inflammation and the immune adjuvant activity were studied in the lungs and lung-draining peribronchial lymph nodes (PBLN) at day 8. OVA-specific antibodies were measured at day 21, and the development of allergic airway inflammation was studied after OVA challenges (day 28). RESULTS: When administered at the same total particle mass (200 micro g), exposure to ultrafine TiO(2) and CBP-induced airway inflammation, and had immune adjuvant activity. The latter was shown by increasing both the PBLN cell numbers and the production of OVA-specific T-helper type 2 (Th2) cytokines (IL-4, IL-5, IL-10 and IL-13). Whereas OVA-specific IgE and IgG1 levels in serum were only increased in animals exposed to the ultrafine TiO(2), allergic airway inflammation could be detected in both ultrafine TiO(2)-and CBP-treated groups after challenges with OVA. CONCLUSION: Our data show that only the ultrafine particles, with a small diameter and a large total surface area/mass, cause airway inflammation and have immune adjuvant activity in the current model supporting the hypothesis that particle toxicity is site-dependent and related to adjuvant activity.

Coadministration of antigen and particles optimally stimulates the immune response in an intranasal administration model in mice.

Some particulate matter is known to affect human health, yet the mechanism(s) by which it acts is largely unknown. One of the factors that may play a role in the immune- stimulating activity of particles is binding of allergen to particles. This may turn the particles into allergen carriers, resulting in antigen deposition within the altered inflammatory microenvironment created by the particles. We compared the efficacy of simultaneous versus separate administration of antigen and particles during sensitization in an intranasal exposure model in BALB/c mice. Sensitization consisted of three separate doses (10 microg) of TNP-OVA at Days 1, 2, and 3. Two hundred micrograms of carbon black particles (CBP) were administered either 1 day before sensitization (Day 0), 1 day after sensitization (Day 4), or during sensitization. The latter was performed either at Day 1 (200 microg) or at Days 1, 2, and 3 (67 microg/day). At Day 10 a challenge with 10 microg of TNP-OVA was performed, and at Day 15 the immune response was assessed. The total number of cells as well as antibody-forming cells (AFC) in lymph nodes draining the lung (peribronchial lymph nodes [PBLN]) were determined, and immunoglobulin levels in blood were assessed. Cell numbers of PBLN increased significantly in all particle-treated groups compared to controls. The number of TNP-specific IgG1-forming cells in the groups receiving particles during sensitization was significantly higher than control level. Only groups receiving particles during or before sensitization displayed significantly higher IgG1 levels than controls, in contrast to the group receiving particles after sensitization. Only in animals receiving three doses of 67 microg during sensitization did TNP-specific IgE increase significantly compared to controls. IgG2a did not show significant differences compared to controls, indicating that the response is predominantly Th2 mediated. These data indicate that coadministration of particles at all time points of antigen dosing is the most effective way to stimulate an immune response in our model compared to separate particle and antigen dosing. Also, administration shortly before antigen administration was effective in stimulating an immune response, suggesting that time-dependent processes are involved in immune-stimulating activity of particles, supporting the important role of the altered inflammatory microenvironment created by the particles.

Diesel exhaust, carbon black, and silica particles display distinct Th1/Th2 modulating activity.

Certain particulate air pollutants may play an important role in the increasing prevalence of respiratory allergy by stimulating T helper 2 cell (Th2)-mediated immune responses to common antigens. The study described here examined different particles, diesel exhaust particles (DEP), carbon black particles (CBP), and silica particles (SIP) for their immunomodulating capacity in both primary and secondary immune responses in female BALB/C mice. The primary response was studied after subcutaneous injection of 1 mg of particle together with 10 microgram of reporter antigen TNP-OVA (2,4,6-trinitrophenyl coupled to ovalbumin) into the hind paw. Interferon-gamma (IFN-gamma) and interleukin 4 (IL-4) production was assessed in the popliteal lymph node (PLN) at Day 2 and Day 5 after injection by flow cytometry and ELISA. The number of IL-4-containing CD4(+) T cells increased between Day 2 and Day 5 in DEP- and CBP-exposed mice, in contrast to SIP-treated animals. IL-4 production by cultured PLN cells was also significantly increased for DEP- and CBP-treated animals. The secondary response was studied in different organs after an intranasal challenge with TNP-OVA (50 microgram), which was given 4 weeks after the initial subcutaneous injection. Five days after challenge the number of antibody-forming cells (AFCs) was assessed in peribronchial lymph nodes (PBLN), spleen, bone marrow, and PLN, and antibody levels were determined in weekly obtained blood samples. It appeared that all particles acted as adjuvant, but the different particles stimulated distinct types of immune responses to TNP-OVA. DEP-treated animals show high IgG1 and IgE levels in serum and high IgG1 and IgE-forming AFC numbers in PBLN, bone marrow, and spleen. CBP-treated animals show even higher IgG1 and IgE levels and AFC numbers, and in addition display IgG2a production. SIP-injected animals display predominantly IgG2a responses. It is concluded that DEP are able to skew the immune response toward the T helper 2 (Th2) side, whereas SIP stimulate a Th1 response and CBP have a mixed activity, stimulating both Th1 and Th2 responses in this model.