Recent perspective on CAR and Fcγ-CR T cell immunotherapy for cancers: Preclinical evidence versus clinical outcomes.

The chimeric antigen receptor T cell (CAR-T cell) immunotherapy currently represents a hot research trend and it is expected to revolutionize the field of cancer therapy. Promising outcomes have been achieved using CAR-T cell therapy for haematological malignancies. Despite encouraging results, several challenges still pose eminent hurdles before being fully recognized. Directing CAR-T cells to target a single tumour associated antigen (TAA) as the case in haematological malignancies might be much simpler than targeting the extensive inhibitory microenvironments associated with solid tumours. This review focuses on the basic principles involved in development of CAR-T cells, emphasizing the differences between humoral IgG, T-cell receptors, CAR and Fcγ-CR constructs. It also highlights the complex inhibitory network that is usually associated with solid tumours, and tackles recent advances in the clinical studies that have provided great hope for the future use of CAR-T cell immunotherapy. While current Fcγ-CR T cell immunotherapy is in pre-clinical stage, is expected to provide a sound therapeutic approach to add to existing classical chemo- and radio-therapeutic modalities.

Quantitative detection of drug dose and spatial distribution in the lung revealed by Cryoslicing Imaging.

Administration of drugs via inhalation is an attractive route for pulmonary and systemic drug delivery. The therapeutic outcome of inhalation therapy depends not only on the dose of the lung-delivered drug, but also on its bioactivity and regional distribution. Fluorescence imaging has the potential to monitor these aspects already during preclinical development of inhaled drugs, but quantitative methods of analysis are lacking. In this proof-of-concept study, we demonstrate that Cryoslicing Imaging allows for 3D quantitative fluorescence imaging on ex vivo murine lungs. Known amounts of fluorescent substance (nanoparticles or fluorophore-drug conjugate) were instilled in the lungs of mice. The excised lungs were measured by Cryoslicing Imaging. Herein, white light and fluorescence images are obtained from the face of a gradually sliced frozen organ block. A quantitative representation of the fluorescence intensity throughout the lung was inferred from the images by accounting for instrument noise, tissue autofluorescence and out-of-plane fluorescence. Importantly, the out-of-plane fluorescence correction is based on the experimentally determined effective light attenuation coefficient of frozen murine lung tissue (10.0 ± 0.6 cm(-1) at 716 nm). The linear correlation between pulmonary total fluorescence intensity and pulmonary fluorophore dose indicates the validity of this method and allows direct fluorophore dose assessment. The pulmonary dose of a fluorescence-labeled drug (FcγR-Alexa750) could be assessed with an estimated accuracy of 9% and the limit of detection in ng regime. Hence, Cryoslicing Imaging can be used for quantitative assessment of dose and 3D distribution of fluorescence-labeled drugs or drug carriers in the lungs of mice.

Prediction of protein degradation during vibrating mesh nebulization via a high throughput screening method.

Maintaining the integrity of biopharmaceuticals is a major requirement for successful pulmonary delivery by nebulization. Sparing laborious nebulization tests, this study aimed to demonstrate the feasibility of a high throughput, material saving surrogate method to predict protein stability after nebulization. Detrimental conditions during nebulization with a PARI eFlow® vibrating mesh nebulizer were mimicked by vigorous agitation at elevated temperatures. Comparing the effect of several different excipients on the stability of the protein SM101 after nebulization and after the surrogate method revealed an excellent correlation regarding SM101 aggregation (R(2)=0.97). Design of experiment was used to develop an inhalable formulation of SM101 based entirely on the new surrogate method. Two lead formulation candidates were selected based on their predicted stability profile. The conservation of full SM101 stability and activity after nebulization was confirmed for an AKITA(2) vibrating mesh nebulizer. This study demonstrated that biopharmaceutical formulation development for nebulization is feasible by means of imitating nebulizer related detrimental factors, allowing an accelerated and more economic formulation development.

That's cool!--Nebulization of thermolabile proteins with a cooled vibrating mesh nebulizer.

Despite contrary reports, heating inside the medication reservoir was observed for several vibrating mesh nebulizers, which may be detrimental when nebulizing biopharmaceuticals. In this study we evaluated different strategies to reduce reservoir heating during nebulization with a PARI eFlow® regarding cooling efficiency, impact on nebulizer performance and on protein stability after nebulization. Passive cooling was achieved by solution pre-cooling, overcharging of the reservoir with 1 mL additional solution or intermittent nebulization. Active cooling was realized with a micro Peltier element attached to the nebulizer reservoir. Passive cooling was most effective when the reservoir was overcharged with pre-cooled solution reducing the average reservoir temperature (TRES AVG) by 8.4°C. Active cooling enabled nebulization at a constant reservoir temperature (TRES) as low as 15°C. TRES manipulation had a linear impact on nebulizer performance. While the output rate decreased with decreasing TRES, the inhalable fraction increased resulting in an inhalable aerosol rate constant over a large TRES range. The effect on protein stability depended on the susceptibility to thermal stress and was predicted by Tm values. For lactic dehydrogenase and SM101, both exhibiting a Tm below 60°C, cooling was protective in increasing the residual activity and reducing protein aggregation. A more thermostable IgG1 did not benefit from cooled nebulization. Nebulizer cooling is a prerequisite to retain the activity and stability of thermolabile proteins during vibrating mesh nebulization. It is best achieved by micro Peltier based active cooling or by simple passive cooling strategies.

Recombinant soluble human FcgammaR1A (CD64A) reduces inflammation in murine collagen-induced arthritis.

Binding of immune complexes to cellular FcgammaRs can promote cell activation and inflammation. In previous studies, a recombinant human (rh) soluble FcgammaR, rh-FcgammaRIA (CD64A), was shown to block inflammation in passive transfer models of immune complex-mediated disease. To assess whether rh-FcgammaRIA could block inflammation in a T cell- and B cell-dependent model of immune complex-mediated disease, the efficacy of rh-FcgammaRIA in collagen-induced arthritis was evaluated. Mice with established arthritis were treated with a single s.c. injection of rh-FcgammaRIA (0.2-2.0 mg/dose) given every other day for 11 days. Relative to mice injected with vehicle alone, mice treated with rh-FcgammaRIA exhibited lower serum concentrations of IL-6, anti-type II collagen Abs, and total IgG2a. These changes were correlated with lower levels of paw swelling and joint damage in the rh-FcgammaRIA-treated mice and occurred in the presence of a significant murine Ab response to rh-FcgammaRIA. Comparison of the serum rh-FcgammaRIA concentration vs time profiles for rh-FcgammaRIA administered at two dose levels by i.v. and s.c. injection revealed that the bioavailabilty of s.c. administered rh-FcgammaRIA was 27-37%. Taken together, these data show that rh-FcgammaRIA is an effective inhibitor of inflammation in a model of established arthritis in mice.

Systemic administration of an Fcgamma-Fc(epsilon)-fusion protein in house dust mite sensitive nonhuman primates.

Crosslinking Fc(epsilon)RI and FcgammaRIIB receptors inhibits mast cell and basophil activation, decreasing mediator release. In this study, a fusion protein incorporating human Fcgamma and Fc(epsilon) domains, hGE2, was shown to inhibit degranulation of human mast cells and basophils, and to exhibit efficacy in a nonhuman primate model of allergic asthma. hGE2 increased the provocative concentration of dust mite aeroallergen that induced an early phase asthmatic response. The treatment effect lasted up to 4 weeks and was associated with reduction in the number of circulating basophils and decreased expression of Fc(epsilon)RI on repopulating basophils. Repeat hGE2 dosing induced production of serum antibodies against human Fcgamma and Fc(epsilon) domains and acute anaphylaxis-like reactions. Immune serum induced histamine release from human IgE or hGE2-treated cord blood-derived mast cells and basophils in vitro. These results indicate that repeat administration with hGE2 induced an antibody response to the human molecule that resulted in activation rather than inhibition of allergic responses.

A novel Fc gamma receptor ligand augments humoral responses by targeting antigen to Fc gamma receptors.

Generating efficient antibody (Ab) responses against weak antigens remains challenging. Ab responses require antigen (Ag) uptake by antigen-presenting cells (APC), followed by presentation of processed Ag to T cells. Limited uptake of antigenic peptides by APC constrains Ab responses. Here we improve vaccine efficacy by targeting Ag to Fcgamma receptors (FcgammaR) using R4, a recombinant FcgammaR ligand. R4 has four repeats per chain of the hinge region and CH2 domain (HCH2) of human IgG1. HCH2 encompasses the FcgammaR binding site. The repeats are linked to the human IgG1 framework. To test R4 in augmenting Ag uptake, we expressed human serum albumin domain 1 (HSA1) at the N terminus of R4 to produce HSA1R4. HSA1R4 (50 microg) administered to mice in Ribi adjuvant induces up to 1100-fold higher HSA1-specific IgG titers than HSA1 (p<0.001). HSA1R4 (250 ng) induces up to 130 times more anti-HSA1 Ab than HSA1Fc, a protein with HSA1 linked to the IgG1 framework (p<0.001). HSA-reactive T cells proliferate more briskly to HSA1R4 than to HSA1Fc (p<0.008). Immunization with HSA1R4 yields greater T cell reactivity to HSA1 ex vivo than immunization with HSA1Fc (p<0.004). Linking antigenic peptides to linear HCH2 polymers may facilitate vaccine development.

The RANKL inhibitor OPG-Fc increases cortical and trabecular bone mass in young gonad-intact cynomolgus monkeys.

UNLABELLED: Weekly treatment of gonad-intact cynomolgus monkeys (for up to 6 months) with the RANKL inhibitor OPG-Fc reduced bone turnover markers and increased volumetric cortical and trabecular BMD and BMC at radial and tibial metaphyses. OPG-Fc was well tolerated in this study without evidence of change in measured toxicologic parameters vs. control. INTRODUCTION: RANKL is the primary mediator of osteoclast formation, function, and survival. The catabolic effects of RANKL are inhibited by OPG, a soluble decoy receptor for RANKL. We investigated the safety and pharmacology of OPG-Fc in gonad-intact cynomolgus monkeys. METHODS: Males and females were treated weekly with vehicle (n = 5/sex) or OPG-Fc (15 mg/kg) by s.c. (n = 5/sex) or i.v. (n = 3/sex) injection for 6 months. RESULTS: Routine toxicologic investigations, hematologic parameters, body and organ weights, and ophthalmologic and electrocardiographic findings were not affected by OPG-Fc treatment. Because s.c. and i.v. dosing of OPG-Fc caused similar effects, these groups were combined for analyses. The following endpoints were significantly different in males and/or females treated with OPG-Fc relative to sex-matched vehicle controls after 6 months (p < 0.05). Biochemical markers of bone turnover (urine N-telopeptide and serum osteocalcin) were significantly decreased with OPG-Fc treatment. Cortical and trabecular volumetric BMD and BMC, cortical thickness, and cross-sectional moment of inertia were significantly increased by OPG-Fc treatment at the proximal tibia and distal radius metaphyses. Increases in cortical thickness were associated with significantly greater periosteal circumference. CONCLUSIONS: OPG-Fc increased cortical and trabecular BMD and BMC in young gonad-intact cynomolgus monkeys.

A two-component modular approach for enhancing T-cell activation utilizing a unique anti-FcgammaRI-streptavidin construct and microspheres coated with biotinylated-antigen.

The professional antigen presenting cell (APC) plays an essential role in the initiation and propagation of the acquired immune response. Thus, much work has been done in designing strategies that target vaccine antigen (Ag) to APC. Utilizing recombinant DNA technology, we have created a unique two-component system that delivers biotinylated Ag to the Fc gamma receptor type I (FcgammaRI) on APC. Our studies demonstrate that we can successfully engineer FcgammaRI-specific targeting element proteins that simultaneously bind both biotin and recognize FcgammaRI. Additionally, we are able to engineer biotinylated Ag, which form functional elements when adsorbed onto latex microspheres. Furthermore, the targeting and functional element components bind to each other and successfully form two-component immunogens. T-cell activation in response to targeted Ag-laden microspheres is 10- to 100-fold greater than the response to the non-targeted Ag-laden microspheres. This enhancement is 100- to 1000-fold greater than the responses generated to soluble Ag. Thus, our results suggest that specific targeting of Ag-laden microspheres to FcgammaRI may significantly enhance the adjuvant properties of microparticulate delivery systems. Further development of this system may help to elucidate the mechanisms involved in generating enhanced responses to APC-targeted vaccines and significantly advance vaccine technology.

Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences.

Many biological functions, including control of the homeostasis and maternofetal transfer of serum gamma-globulins, are mediated by the MHC class I-related neonatal FcR (FcRn). A correlation exists in mice between the binding affinity of IgG1/Fc fragments to FcRn at pH 6.0 and their serum t(1/2). To expand this observation, phage display of mutagenized Fc fragments derived from a human IgG1 was used to increase their affinity to both murine and human FcRn. Ten variants were identified that have a higher affinity toward murine and human FcRn at pH 6.0, with DeltaDeltaG (DeltaG(wild type) - DeltaG(mutant)) from 1.0 to 2.0 kcal/mol and from 0.6 to 2.4 kcal/mol, respectively. Those variants exhibit a parallel increase in binding at pH 7.4 to murine, but not human, FcRn. Although not degraded in blood in vitro, accumulated in tissues, nor excreted in urine, their serum concentration in mice is decreased. We propose that higher affinity to FcRn at pH 7.4 adversely affects release into the serum and offsets the benefit of the enhanced binding at pH 6.0.

Soluble CD16 in the treatment of murine lupus nephritis.

To determine if soluble CD16 (sCD16) could alter the expression of lupus-like disease, groups of 10 female NZB/NZW mice (age 16-20 weeks) were given sCD16 three times a week for 5 weeks (control; 100 microg; 200 microg/dose) after onset of proteinuria. Results of this study indicate that the administration of sCD16 after onset of disease lowered anti-DNA levels, delayed the development of proteinuria, and significantly prolonged survival while the mice were on treatment. These results indicate that sCD16 alters the expression of autoantibodies and the progression of renal disease in NZB/NZW mice, suggesting that therapies directed at Fc receptors may be useful in the treatment of SLE.

Generation of HER-2/neu-specific cytotoxic neutrophils in vivo: efficient arming of neutrophils by combined administration of granulocyte colony-stimulating factor and Fcgamma receptor I bispecific antibodies.

Abs are able to induce inflammatory antitumor responses by recruiting IgG Fc receptor (FcgammaR)-bearing cytotoxic effector cells. We recently described the capacity of the high affinity FcgammaRI (CD64) to trigger cytotoxic activity of neutrophils (PMN) during granulocyte CSF (G-CSF) treatment. To take advantage of FcgammaRI as a cytotoxic trigger molecule on PMN, two Ab constructs were prepared. We show that a chimeric human IgG1 Ab (Ch520C9) and an anti-FcgammaRI bispecific Ab (BsAb; 22x520C9), both directed to the proto-oncogene product HER-2/neu, interact with FcgammaRI. In addition, both Ab constructs mediate enhanced lysis of HER-2/neu-expressing tumor cells by G-CSF-primed PMN. However, engagement of FcgammaRI by Ch520C9 was inhibited by human serum IgG, thereby abrogating the enhanced Ch520C9-mediated cytotoxicity. BsAb 22x520C9, which binds FcgammaRI outside the ligand binding domain, effectively recruits the cytotoxic potential of FcgammaRI on G-CSF-primed PMN regardless of the presence of human serum. These results indicate that under physiologic conditions, serum IgG impairs activation of FcgammaRI-mediated cytotoxicity by conventional antitumor Abs. The IgG blockade can be circumvented with anti-FcgammaRI BsAbs. Using human FcgammaRI transgenic mice we demonstrate that BsAb 22x520C9 is able to engage FcgammaRI in vivo. BsAb 22x520C9 injected i.v. was readily detected on circulating PMN of G-CSF-treated transgenic animals. In addition, we showed that PMN remain "armed" with BsAb 22x520C9 during migration to inflammatory sites, and that after isolation such PMN specifically lyse HER-2/neu-expressing tumor cells. These results point to the possibility of targeting anti-FcgammaRI BsAbs to G-CSF-primed PMN in vivo, endowing them with specific anti-tumor activity.