Page 112 - Haematologica-April 2018

Z. Li et al.
sive communication between the tumor and immune sys- tem in tumor microenvironments have also been effective in HL patients.11-13
To date, the main utility of identifying membrane- bound CD83 has been to define activated dendritic cells (DC), but CD83 is also expressed on the surface of some activated B cells, T cells, macrophages and neutrophils.14- 18 In addition to a membrane-bound form, there is a membrane cleaved soluble (s) form of CD83. We report- ed that lymphoma tumor cells (HL and non-Hodgkin lymphoma [NHL]) expressed CD83 and released sCD83 into serum.19,20 Recombinant sCD83 protein has immune inhibitory function in mice and humans.21,22 Recently, CD83 was identified as one of the four classifiers to dis- tinguish HL with anaplastic lymphoma kinase (ALK)- anaplastic large cell lymphoma.23 Despite its potential as a relatively specific target, CD83 has not been investigat- ed as a therapeutic target on either HL or NHL. We gen- erated a human anti-human CD83 antibody, 3C12C, which prevents graft-versus-host disease (GvHD) but pre- serves anti-tumor T-cell function in mice after transplan- tation with human peripheral blood mononuclear cell (PBMC).24,25 The availability of this potential therapeutic anti-CD83 antibody prompted us to investigate CD83 biology in HL. We show herein that an antibody that detects CD83 in paraffin sections stains Hodgkin and Reed-Sternberg (HRS) cells in most HL lymph node biop- sy samples, that HL tumor cells secrete sCD83, and the serum sCD83 level in HL patients correlates with the clinical response. The 3C12C antibody, and its toxin con- jugate, killed HL lines and 3C12C depleted CD83 target cells in non-human primate studies without any evi- dence of toxicity.
Methods
HL tissue section and serum samples
Lymph node biopsies and serum of HL patients were collected after approval by the Sydney Local Health District (SLHD) Human Research Ethics Committee, consistent with the Declaration of Helsinki. Archival paraffin embedded lymph node biopsies were obtained from 35 HL patients at initial diagnosis (Table 1), while serum samples were collected from six HL patients at diagnosis and during chemotherapy.
Table 1. Characteristics of 35 Hodgkin lymphoma patients.
Age at enrolment (mean, range)
Sex
Male; n (%) Female; n (%)
Histologic subtype
cHL-Nodular sclerosis (NS)
cHL-Mixed cellularity (MC) cHL-Lymphocyte rich (LR) cHL-unspecified (CHL-U)
Nodular lymphocyte predominant (NLP)
Stage at onset I
II III IV
cHL: classic Hodgkin lymphoma.
35 (17-71)
18 (51.4%) 17 (48.6%)
21 (60.0%) 7 (20.0%) 1 (2.9%) 2 (5.7%) 4 (11.4%)
3 (8.6%) 19 (54.3%) 5 (14.3%) 8 (22.9%)
656
Immunohistochemistry
Immunohistochemical staining was performed on 3mm sections of formalin fixed paraffin embedded lymph node biopsies of HL patients or non-human primates. The primary antibodies used were mouse anti-human CD20 (L26, Dako), CD83 monoclonal antibodies (mAb; F5, Santa Cruz Biotechnology), CD30 (Ber-H2, Dako), and staining was performed on a Leica Bond III Autostainer (Leica Biosystems) using a Bond Polymer Refine Detection kit for visualization with 3, 3’-diaminobenzidine (DAB). Images were taken with an Olympus BX51 microscopy with an Olympus PP71 camera using Olympus labSens software.
sCD83 analysis
For the analysis of sCD83 levels, KM-H2, L428 and HDLM2 cells were cultured at concentrations of 106 cells/ml in complete roswell park memorial institute (RPMI) medium containing 10% fetal calf serum, 2mM glutaMAXTM, 100U/ml penicillin, 100μg/ml streptomycin (Thermo Fisher Scientific) at 37°C, in 5% CO2. Cell culture supernatant were collected 24 hours after fresh medium
change. Human sCD83 was analyzed by a sCD83 ELISA kit (Sino Biological Inc.).
3C12C conjugation with monomethyl auristatin
E (3C12C-MMAE) and cytotoxicity on CD83+ cell lines
3C12C is a human immunoglobulin G1 (IgG1) anti-human CD83 mAb selected from a phage display library26 and further engineered to improve affinity.25,27 To produce 3C12C-MMAE, a lysosomal cathepsin B-cleavable, self-emolative dipeptide (ValCit) maleimide linker was prepared from MMAE for conjugation to partially reduced 3C12C using a similar method to brentuximab vedotin.28 The cytotoxic activity was assessed by 7-amino-actino- mycin D (7AAD, Thermo Fisher Scientific) staining using flow cytometry.
3C12C trials in non-human primates
The SLHD Animal Research Ethics Committee approved the study of five non-human primates (Papio Hamadryas baboon), which received intravenous human-IgG (Intragam, CSL) (10 mg/kg) or 3C12C mAb (1, 5, 10, 10 mg/kg) at days 0, 7, 14 and 21. PBMC were analyzed for immune cell populations including Dendritic cells (DC), T cells and B cells on a Fortessa X20 flow cytometer (BD Biosciences). Liver and kidney function were assessed by measuring alkaline phosphatase (ALP), aspartate transaminase (AST) and creatinine in serum samples using the Cobas 8000 (Roche). Lymph nodes were taken from 3C12C (10mg/kg) or human IgG (10mg/kg) treated animals at day 28 for immunohistological staining.
Statistical Analysis
Mean values with standard error of mean (SEM) bars are shown in graphs. Statistical analyses were performed using Prism 6.0 (GraphPad Software). A Mann-Whitney or one-way analysis of variance (ANOVA) test with Greenhouse-Geisser correction for multiple comparisons were used. Differences with P<0.05 were considered significant.
Antibody Dependent Cell Cytotoxicity (ADCC) Assays
Target HL cells labeled with 25mM Calcein-AM (Life Technologies) were co-cultured at a ratio of 1:25 with human PBMC of a healthy donor used as effector cells. Supernatants were collected after three hours to measure released calcein using an enzyme-linked immunosorbent assay (ELISA) Reader (Perkin Elmer). The percentage of specific cytolysis was calculated as described.25
haematologica | 2018; 103(4)