Functional Analysis of LDLR (Low-Density Lipoprotein Receptor) Variants in Patient Lymphocytes to Assess the Effect of Evinacumab in Homozygous Familial Hypercholesterolemia Patients With a Spectrum of LDLR Activity

Abstract

Objective: Homozygous familial hypercholesterolemia is a rare disease usually caused by LDLR (low-density lipoprotein receptor) mutations. Homozygous familial hypercholesterolemia is characterized by markedly elevated LDL-C (low-density lipoprotein cholesterol) levels and an extremely high risk of premature atherosclerotic cardiovascular disease. A phase 2, proof-of-concept study (NCT02265952) demonstrated that evinacumab, a fully human monoclonal antibody to ANGPTL3 (angiopoietin-like 3 protein), reduced LDL-C levels in 9 patients with genotypically confirmed homozygous familial hypercholesterolemia and was well tolerated. The aim of this study was to analyze the effects of evinacumab on LDLR activity in lymphocytes purified from patients in the proof-of-concept study. Approach and Results: LDLR activity was assessed in patient lymphocytes before and after treatment with evinacumab and versus lymphocytes carrying wild-type LDLR, and also in an LDLR-defective Chinese hamster ovary cell line (CHO-ldlA7) transfected with plasmids encoding the LDLR variants. Overall mean peak reduction in LDL-C with evinacumab was -58±18%, occurring between Week 4 and Week 12. Mutations identified in the 9 patients were shown to be pathogenic, with loss of LDLR activity versus wild type. Two of the LDLR variants, p.(Cys681*) and p.(Ala627Profs*38), were class 2 type mutations that are retained in the endoplasmic reticulum. Six variants were class 3 type mutations with impaired LDL-C binding activity: p.(Trp87Gly), occurring in 2 patients, p.(Gln254Pro), p.(Ser177Leu), p.(Gly335Val), and p.(Ser306Leu). Evinacumab had no effect on LDLR activity.

Conclusions: These results suggest that evinacumab is effective for lowering LDL-C in patients with homozygous familial hypercholesterolemia, and the inhibition of ANGPTL3 in humans lowers LDL-C in a mechanism independent of the LDLR.

Keywords: hypercholesterolemia; lipoproteins; mutations; proof of concept study; rare disease.

Figure 1.

LDLR (low-density lipoprotein receptor) activity in lymphocytes from 9 homozygous familial hypercholesterolemia (HoFH) patients before and after evinacumab treatment. LDLR expression (A); LDL binding (B); and LDL uptake (C). LDLR activity and expression was quantified by flow cytometry as described in the Materials and Methods section. The values represent the mean of 3 independent experiments; error bars represent ±SD. wt=wild-type (non-HoFH) control; c.[191-?_694+?del] and p.(Trp87*)=LDLR defective heterozygous patient controls (see Methods). (1;2) represents the mean±SD of patients 1 and 2 which carry the same LDLR variants; the lymphocytes from these 2 patients were analyzed independently. The sex of the patients 1 to 9 from which the lymphocytes were sourced is indicated in the Table. HoFH indicates homozygous familial hypercholesterolemia; LDL, low-density lipoprotein; and LDLR, low-density lipoprotein receptor. *P<0.001 comparing wt with each variant before and after treatment. No statistical significance was found when after treatment vs before treatment LDLR activity data (expression, binding, and uptake) of each variant was compared.

Figure 2.

Expression of wild-type LDLR and LDLR variants in Chinese hamster ovary-ldlA7 transfected cells. LDLR (low-density lipoprotein receptor) expression (upper) and GAPDH expression (A and C; lower); LDLR to GAPDH densitometric analysis (B and D). Cells were transfected with the corresponding plasmids carrying the LDLR mutations of interest and immunoblot analysis was performed, as described in Materials and Methods. The relative band intensity of mature LDLR protein expression was calculated as the ratio of 160 kDa LDLR band intensity to that of GAPDH. A representative experiment from 3 independently performed assays is shown in A. Levels of significance were determined by a 2-tailed Student t test, and a CI of >95% (P<0.05) was used to establish statistical significance. No statistically significant differences were found among LDLR expression for variants analyzed in A and B, but differences were significant for variants analyzed in C and D. wt indicates wild-type.

Figure 3.

LDLR (low-density lipoprotein receptor) activity in Chinese hamster ovary-ldlA7-transfected cells. LDLR expression (A); LDL (low-density lipoprotein) binding (B); and LDL uptake (C). LDLR activity and expression was quantified by flow cytometry, as described in the Materials and Methods section. The values represent the mean of 3 independent experiments; error bars represent ±SD. wt indicates wild-type. *P<0.001 comparing wt with each variant.

Figure 4.

Class 2 type mutation assignment of variants found in homozygous familial hypercholesterolemia patients. LDLR (low-density lipoprotein receptor) expression and colocalization with calregulin in the endoplasmic reticulum (A); LDL (low-density lipoprotein)-LDLR binding activity (B); and LDL internalization activity (C). Confocal laser scanning microscopy was used to analyze LDLR activity, as described in the Materials and Methods section. wt indicates wild-type.

Figure 5.

Class 3 type mutation assignment of variants found in homozygous familial hypercholesterolemia patients. LDLR (low-density lipoprotein receptor) expression at cellular membrane (A); LDL (low-density lipoprotein)-LDLR binding activity (B); and LDL internalization activity (C). Confocal laser scanning microscopy was used to analyze LDLR activity, as described in the Materials and Methods section. wt indicates wild-type.