Tumor cell-macrophage interactions increase angiogenesis through secretion of EMMPRIN

Tumor macrophages are generally considered to be alternatively/M2 activated to induce secretion of pro-angiogenic factors such as VEGF and MMPs. EMMPRIN (CD147, basigin) is overexpressed in many tumor types, and has been shown to induce fibroblasts and endothelial cell expression of MMPs and VEGF. We first show that tumor cell interactions with macrophages resulted in increased expression of EMMPRIN and induction of MMP-9 and VEGF. Human A498 renal carcinoma or MCF-7 breast carcinoma cell lines were co-cultured with the U937 monocytic-like cell line in the presence of TNFalpha (1 ng/ml). Membranal EMMPRIN expression was increased in the co-cultures (by 3-4 folds, p<0.01), as was the secretion of MMP-9 and VEGF (by 2-5 folds for both MMP-9 and VEGF, p<0.01), relative to the single cultures with TNFalpha. Investigating the regulatory mechanisms, we show that EMMPRIN was post-translationally regulated by miR-146a, as no change was observed in the tumoral expression of EMMPRIN mRNA during co-culture, expression of miR-146a was increased and its neutralization by its antagomir inhibited EMMPRIN expression. The secretion of EMMPRIN was also enhanced (by 2-3 folds, p<0.05, only in the A498 co-culture) via shedding off of the membranal protein by a serine protease that is yet to be identified, as demonstrated by the use of wide range protease inhibitors. Finally, soluble EMMPRIN enhanced monocytic secretion of MMP-9 and VEGF, as inhibition of its expression levels by neutralizing anti-EMMPRIN or siRNA in the tumor cells lead to subsequent decreased induction of these two pro-angiogenic proteins. These results reveal a mechanism whereby tumor cell-macrophage interactions promote angiogenesis via an EMMPRIN-mediated pathway

Data from: Immunogenicity of glycans on biotherapeutic drugs produced in plant expression systems - the taliglucerase alfa story

Plants are a promising alternative for the production of biotherapeutics. Manufacturing in-planta adds plant specific glycans. To understand immunogenic potential of these glycans, we developed a validated method to detect plant specific glycan antibodies in human serum. Using this assay, low prevalence of pre-existing anti-plant glycan antibodies was found in healthy humans (13.5%) and in glucocerebrosidase-deficient Gaucher disease (GD) patients (5%). A low incidence (9% in naïve patient and none in treatment experienced patients) of induced anti-plant glycan antibodies was observed in GD patients after up to 30 months replacement therapy treatment with taliglucerase alfa, a version of human glucocerebrosidase produced in plant cells. Detailed evaluation of clinical safety and efficacy endpoints indicated that anti-plant glycan antibodies did not affect the safety or efficacy of taliglucerase alfa in patients. This study shows the benefit of using large scale human trials to evaluate the immunogenicity risk of plant derived glycans, and indicates no apparent risk related to anti-plant glycan antibodies

Immunogenicity of glycans on biotherapeutic drugs produced in plant expression systems—The taliglucerase alfa story

<div><p>Plants are a promising alternative for the production of biotherapeutics. Manufacturing <i>in-planta</i> adds plant specific glycans. To understand immunogenic potential of these glycans, we developed a validated method to detect plant specific glycan antibodies in human serum. Using this assay, low prevalence of pre-existing anti-plant glycan antibodies was found in healthy humans (13.5%) and in glucocerebrosidase-deficient Gaucher disease (GD) patients (5%). A low incidence (9% in naïve patient and none in treatment experienced patients) of induced anti-plant glycan antibodies was observed in GD patients after up to 30 months replacement therapy treatment with taliglucerase alfa, a version of human glucocerebrosidase produced in plant cells. Detailed evaluation of clinical safety and efficacy endpoints indicated that anti-plant glycan antibodies did not affect the safety or efficacy of taliglucerase alfa in patients. This study shows the benefit of using large scale human trials to evaluate the immunogenicity risk of plant derived glycans, and indicates no apparent risk related to anti-plant glycan antibodies.</p></div

Glycan composition of TGA and HRP.

<p>Glycan composition of TGA and HRP.</p

Schematic representation of the assay.

<p>A stepwise format (1–5) was developed for the binding of serum antibodies to TGA (A) with HRP and (B) without prior HRP incubation, showing a response reduction in the presence of HRP.</p

A Consort flowchart of the clinical studies included in the study.

<p>Consort flowchart shows all three studies with original enrolment, number of excluded subjects and the final amount of subjects included in the immunogenicity tests.</p

Interaction of assay controls with TGA with and without HRP.

<p>Assay controls were tested on TGA coated plates, with and without HRP competitor and mean absorbance was measured by OD. PC showed high initial OD and inhibition of ~80% with the addition of HRP, indicating recognition of plant glycans. NC showed high OD, both with and without HRP, indicating recognition of protein backbone. Normal serum pool (NSP) represented serum background levels with low OD, both with and without HRP, indicating low reactivity to TGA. Results, presented as a box plot graph, include data from 3 independent runs, showing individual measurements together with mean.</p

Efficacy parameters and mean response by anti-plant glycan antibody—Status of available data at end of study.

<p>Efficacy parameters and mean response by anti-plant glycan antibody—Status of available data at end of study.</p

Assay cut-point determination.

<p>Percent (%) Immunodepletion by HRP of healthy individual serum samples. The data show three independent runs and their mean± standard deviation. Individuals 5 and 40 (highlighted) were identified as outliers, and were excluded from the calculation.</p

Total ADA and anti-plant glycan antibodies prevalence found in GD patient population treated with TGA.

<p>(A) Data evaluated from ERT-naïve patient samples and (B) ERT-experienced patient samples. *One patient of the ERT-experienced group, was counted for both baseline ADA and treatment induced ADA, since he was ADA positive at baseline and had a treatment-boosted following treatment with TGA (had ≥6-fold higher titer after TGA treatment).</p