Overcoming resistance to rituximab in relapsed non-Hodgkin lymphomas by antibody-polymer drug conjugates actively targeted by anti-CD38 daratumumab

Article abstract of DOI:10.1016/j.jconrel.2020.08.042

B-cell non-Hodgkin lymphomas (B-NHL) represent the most common type of hematologic malignancies in the Western hemisphere. The therapy of all B-NHL is based on the combination of different genotoxic cytostatics and anti-CD20 monoclonal antibody (mAb) rituximab. Unfortunately, many patients relapse after the mentioned front-line treatment approaches. The therapy of patients with relapsed/refractory (R/R) B-NHL represents an unmet medical need. We designed, developed and tested novel actively targeted hybrid mAb-polymer-drug conjugate (APDC) containing anti-CD20, anti-CD38 or anti-CD19 mAbs. Biocompatible copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA) with cytostatic agent doxorubicin attached via stimuli-sensitive hydrazone bond were employed for the mAb grafting. Anti-lymphoma efficacy of the APDC nanotherapeutics was evaluated in vivo on a panel of three patient-derived lymphoma xenografts derived from two patients with R/R B-NHL and one patient with so far untreated B-NHL. In both PDX models derived from patients with R/R B-NHL, the targeting with anti-CD38 antibody daratumumab demonstrated highly improved anti-lymphoma efficacy compared to the targeting with anti-CD20 rituximab, two experimental anti-CD19 antibodies and non-targeted controls. The results represent a proof-of-concept of a new algorithm of personalized anti-tumor therapy based on highly innovative APDC biomaterials.

Full text of DOI:10.1016/j.jconrel.2020.08.042

Journal of Controlled Release 328 (2020) 160–170
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Overcoming resistance to rituximab in relapsed non-Hodgkin lymphomas by
antibody-polymer drug conjugates actively targeted by anti-CD38
daratumumab
a,1
b,c,a,1
a
b,c
b
Ondřej Lidický , Pavel Klener
, Daniela Machová , Petra Vočková , Eva Pokorná ,
a
a,⁎
d
e
Karel Helman , Cory Mavis , Olga Janoušková , Tomáš Etrych
a
b
c
Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovský Sq. 2, 162 06 Prague 6, Czech Republic
Institute of Pathological Physiology, First Faculty of Medicine, Charles University, U Nemocnice 5, 128 53 Prague 2, Czech Republic
First Department of Internal Medicine- Hematology, University General Hospital and First Faculty of Medicine, Charles University, U Nemocnice 2, 128 08 Prague 2,
Czech Republic
d
e
Faculty of Informatics and Statistics, University of Economics, Prague, Czech Republic
Department of Medicine Lymphoma/Myeloma, Roswell Park, Comprehensive Cancer Center, Elm & Carlton Sts, Buffalo, NY 14263, United States of America
A R T I C L E I N F O
A B S T R A C T
Keywords:
B-cell non-Hodgkin lymphomas (B-NHL) represent the most common type of hematologic malignancies in the
Western hemisphere. The therapy of all B-NHL is based on the combination of different genotoxic cytostatics and
anti-CD20 monoclonal antibody (mAb) rituximab. Unfortunately, many patients relapse after the mentioned
front-line treatment approaches. The therapy of patients with relapsed/refractory (R/R) B-NHL represents an
unmet medical need. We designed, developed and tested novel actively targeted hybrid mAb-polymer-drug
conjugate (APDC) containing anti-CD20, anti-CD38 or anti-CD19 mAbs. Biocompatible copolymers based on N-
Non-Hodgkin lymphoma
Active targeting
Drug delivery
APDC
Polymer nanomaterials
Rituximab
(
2-hydroxypropyl)methacrylamide (HPMA) with cytostatic agent doxorubicin attached via stimuli-sensitive
Daratumumab
CD38
hydrazone bond were employed for the mAb grafting. Anti-lymphoma efficacy of the APDC nanotherapeutics
was evaluated in vivo on a panel of three patient-derived lymphoma xenografts derived from two patients with
R/R B-NHL and one patient with so far untreated B-NHL. In both PDX models derived from patients with R/R B-
NHL, the targeting with anti-CD38 antibody daratumumab demonstrated highly improved anti-lymphoma ef-
ficacy compared to the targeting with anti-CD20 rituximab, two experimental anti-CD19 antibodies and non-
targeted controls. The results represent a proof-of-concept of a new algorithm of personalized anti-tumor therapy
based on highly innovative APDC biomaterials.
1
. Introduction
Non-Hodgkin lymphomas (NHL), the most common hematologic
cell Cytotoxicity (CDC) [7]. While DCD is triggered by binding of the
mAb to CD20 antigen, ADCC and CDC depend on Fc fragment-mediated
activation of the complement cascade and immune cells, respectively.
Natural killer cells and macrophages belong to major effector cells of
ADCC. Despite the fact that the outcome of patients with B-NHL im-
proved considerably during the last 20 years, approximately half of all
patients experience relapse after achievement of remission [8]. Such
patients are subject to diverse chemotherapy salvage regimens that
implement different cytostatics, e.g. platinum derivatives and high-dose
cytarabine [9,10]. In most instances these different chemotherapy re-
gimens are combined with the same anti-CD20 antibody rituximab [9].
General usage of rituximab in the relapse setting is a consequence of
two facts: first, despite the fact that a proportion of patients might
malignancies, are heterogeneous lymphoid tumors of immune cells
[
1,2]. Current front-line therapy is based on diverse im-
munochemotherapy regimen, e.g. CHOP (C-cyclophosphamid, H-dox-
orubicin, O-oncovin, P-prednisone) in combination with anti-CD20
antibody rituximab (R-CHOP) [3–5]. Rituximab is a chimeric mono-
clonal antibody (mAb) targeting surface CD20 antigen presented on
normal mature B-cells and virtually all lymphoma cells of B-cell origin
[
6]. After cell surface opsonization by rituximab several mechanisms
lead to lymphoma cell death including Direct Cell Death (DCD), Anti-
body Dependent Cell Cytotoxicity (ADCC) and Complement-Dependent
⁎
Corresponding author.
E-mail address: etrych@imc.cas.cz (T. Etrych).
1
Both authors contributed equally.
https://doi.org/10.1016/j.jconrel.2020.08.042
Received 16 June 2020; Received in revised form 11 August 2020; Accepted 21 August 2020
Available online 27 August 2020
0
168-3659/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(
http://creativecommons.org/licenses/BY-NC-ND/4.0/).

O. Lidický, et al.
Journal of Controlled Release 328 (2020) 160–170
present with CD20-negative disease by standard immunohistochemistry
after failure of rituximab-based upfront therapy, not all patients are
subject to lymphoma re-biopsy at the time of relapse. And second, de-
spite a number of trials there is currently no effective immunotherapy
approved for the treatment of relapsed B-NHL other than anti-CD20
antibodies (namely rituximab, ofatumumab and obinutuzumab)
(4-cyanovaleric acid) (ABIK), 4,5- dihydrothiazole-2-thiol (TT), 4-(di-
methylamino)pyridine (DMPA), dimethyl sulfoxide (DMSO), N-(2-
aminoethyl)maleimide trifluoroacetate, dimethylformamide (DMF),
trifluoroacetic acid (TFA), triisopropyl silan (TIPS), 5,5′-disulfanylbis(2-
nitrobenzoic acid) (Ellman's reagent), doxorubicin hydrochloride
(Dox·HCl), dithiothreitol (DTT), 5,5′-dithiobis(2-nitrobenzoic acid)
(Ellman's reagent), cysteine, ethylenediaminetetraacetic acid (EDTA),
phthalaldehyde (OPA), N,N-diisopropylethylamine (DIPEA) were pur-
chased from Sigma-Aldirch. 2,4,6-trinitrobenzene-1-sulfonic acid
(TNBSA) was purchased from Serva. Chimeric anti-human CD20 anti-
body rituximab (mAb20) (MabThera®, Roche, Great Britain), human
anti-human CD38 antibody daratumumab (mAb38) (Darzalex, Janssen
Biotech, USA), mouse anti-human CD19 antibody clone 4G7 (mAb19)
(Bio X cell, USA), mouse anti-human CD19 clone B3D (mAb19B)
(ExBio, Czech Republic) and polyclonal immunoglobulins Flebogamma
(Ab) (Grifols, Spain) were purified from excipients (e.g. glukose, NaCl,
glycin) before conjugation by filtration using an Amicon®Ultra cen-
trifugal filter units 30 K and reaction ITH buffer as a solvent. All other
chemicals and solvents were of analytical grade. The solvents were
dried and purified by conventional procedures and distilled before
[
11,12]. Nevertheless, some other CD molecules, such as CD19, CD22,
CD38 and CD79b, remains highly interesting targets for the advanced
B-NHL treatment within future development [13,14].
To improve anticancer drug efficiency a variety of drug delivery
systems (DDS) have been studied. In June 2019, FDA approved ac-
celerated approval for the Antibody Drug Conjugate (ADC) polatu-
zumab-vedotin (anti-CD79b mAb conjugated with mitotic toxin
monomethyl auristatin E) for the therapy of R/R diffuse large B-cell
lymphoma (DLBCL), the most prevalent type of lymphoma in the
western hemisphere [15,16]. ADCs, however, rely on small cytotoxic
molecules with potent systemic toxicity, e.g. anti-mitotic agents, and
their anti-lymphoma activity is mediated by the mAb-mediated targeted
delivery of the toxins to the lymphoma cells. Apart from ADCs, also
other nano-sized DDS can help to overcome insolubility of hydrophobic
drugs, prolong the time of circulation in the bloodstream, minimize the
side-toxicity and increase drug concentration in the tumor tissue thanks
to the enhanced permeability and retention (EPR) effect [17]. More-
over, passive accumulation of DDS in the tumor tissue can be enhanced
by various targeting moieties including monoclonal antibodies and
their fragments, saccharides, lectins, (oligo)peptides etc. [18,19]. Syn-
thetic biocompatible polymers could be used in these actively targeted
DDS instead of the linker (used in the concept of ADC for controlled
release of carried drugs) with multiple binding sites for the drug at-
tachment and thus scale up the loading capacity of ADC up to ten times
used.
3,3′-[4,4′-Azobis(4-cyano-4-methyl-1-oxo-butane-4,1-diyl)]bis
(thiazolidine-2-thione) (ABIK-TT) was prepared as described previously
[31].
2.2. Synthesis of monomers
N-(2-hydroxypropyl)methacrylamide (HPMA) was synthesized by
reaction of methacryloyl chloride with 1-aminopropan-2-ol in di-
chloromethane in the presents of sodium carbonate as described pre-
viously [32]. N-(tert-butoxycarbonyl)-N′-(6-methacrylamidohexanoyl)
hydrazine (Ma-εAh-NHNH-BOC) was synthesized by two-step synth-
eses. First, methacryloyl chloride was reacted with 6-aminohexanoic
acid in the presence of NaOH and afterward formed 6-methacrylami-
dohexanoyoic acid was reacted with tert-butyl carbazate using DCC
[33].
[
20].
The most frequently used techniques for polymer attachment to
antibodies [21,22] are based on the aminolytic reaction between amino
groups of mAb with aminoreactive groups of synthetic polymers. Un-
fortunately, the involvement of amino group of mAb, either from lysine
residues or N-terminal, often leads to reduction of binding activity of
the mAb. Recently, for the mAb-polymer construct formation the re-
action of the thiol groups introduced to the antibody with the mal-
eimides presented in the synthetic polymer structure, so called Michael
addition, has been studied widely [23,24]. Actively targeted hybrid
polymer-mAb system containing therapeutic anti-CD20 mAb [20,25]
combined with biocompatible polymer based on N-(2-hydroxypropyl)
methacrylamide (HPMA) with attached doxorubicin via enzymatically
cleavable oligopeptide spacer (GFLG) or pH-labile hydrazone bond was
described [26,27]. The polymer-mAb systems with a star-like structure,
in which several polymer grafts are attached to the central monoclonal
mAb, enable a much higher loading capacity of carried drug when
compared to the ADC [24,28]. It was shown that HPMA copolymer-
bound doxorubicin has considerably reduced non-specific toxicity in-
cluding hepatotoxicity, nephrotoxicity, cardiotoxicity and myelotoxi-
city [29]. Even within the compassionate use of polymer-pirarubicin
conjugate in human reduced cardiotoxicity was proved [30].
2.3. Synthesis of polymer precursors
Semitelechelic polymer precursor PDOX containing main chain-end
maleimide (MI) group and Dox connected via hydrazone bond to the
side chain of polymer was prepared as described previously [34].
Briefly: semitelechelic copolymer P* containing main chain-end TT
group and BOC-protected hydrazide groups in the side chains was
prepared by free radical copolymerization of HPMA (840 mg,
5.86 mmol) and Ma-εAh-NHNH-BOC (157 mg, 0.5 mmol) monomers
initiated by ABIK-TT (320 mg, 0.66 mmol) in DMSO (6 mL) under inert
atmosphere in polymerization ampule [35]. Yield of the polymerization
was 81% (667 mg). Content of TT groups was determined by using UV-
VIS spectrophotometry on Specord 205 (Analytik Jena, Jena, Germany,
−1
−1
ε
305 = 10,700 L·mol ·cm in methanol [36]). The MI reactive group
was introduced to semitelechelic polymer precursor P* by the amino-
lytic reaction of N-(2-aminoethyl)maleimide with the TT group [37].
The MI group content in the polymer precursors was determined by a
modified Ellman's assay as the difference between cysteine concentra-
tions before and after reaction with the MI groups of the polymer [38].
The yield of MI group introduction reached 69%. Hydrazide groups of
polymer precursor P were deprotected by using mixture of TFA:-
TIPS:distilled water in ratio 38:1:1 and characterized by using TNBSA
Here we designed, synthesized and tested physico-chemical prop-
erties and in vitro and in vivo anti-lymphoma efficacy of precisely de-
signed and synthesized HPMA-based copolymers targeted with anti-
CD20 mAb rituximab, two anti-CD19 experimental antibodies and anti-
CD38 mAb daratumumab in experimental therapy of CD20-negative
patient-derived lymphoma xenografts derived from patients after
failure of rituximab-based front-line therapies.
−1
−1
(ε500 = 17,200 L·mol ·cm ) [39]. Dox·HCl was connected via hy-
drazone bond in methanol with acetic acid as described previously
forming polymer precursor PDOX with Dox [40]. The yield of Dox at-
tachment reached 95%. Polymer precursors P*, P, PDOX were char-
acterized using HPLC system, see Table 1, (Shimadzu, Kyoto, Japan)
equipped with column SuperSW TSK3000 (Tosho Bioscience, Grie-
sheim, Germany) in combination with multi-angle light scattering de-
tector Dawn Helieos-II(Wyatt Technology Co., Santa Barbara, USA) and
2
. Material and methods
.1. Materials
-Aminopropan-2-ol, methacryloyl chlorid, 6-aminohexanoic acid,
2
1
tert-butyl carbazate, N,N′-dicyclohexylcarbodiimide (DCC), 4,4′-Azobis
161

O. Lidický, et al.
Journal of Controlled Release 328 (2020) 160–170
Table 1
standard. The final concentration of the conjugates in incubation media
Characterization of polymer precursors.
was equivalent to 0.5 mM DOX.
−
1
End group1 Dox (wt
%)
M
n
/MUV2
Polymer precursor
M
w
(g·mol
)
Đ
notation
2.6. In vitro methods – cell lines
⁎
P
25,600
28,500
57,000
1.64 TT
1.51 MI
1.93 MI
n.a.
n.a.
9.2
1.36
1.01
P
3
UPF4D cell line and VFN-D2 patient-derived xenograft (PDX) were
P
DOX
-
both established at the Institute of Pathological Physiology, Charles
University, from a malignant peritoneal effusion of a patient with
DLBCL at second lymphoma relapse after failure of R-CHOP-based in-
duction and salvage therapy containing rituximab, cisplatin and high-
dose cytarabine. UPF4D and VFN-D2 thus represent “sister” models (a
cell line and a PDX model) derived in parallel from one patient. Despite
the fact that the primary lymphoma cells were CD20-negative, UPF4D
1
2
End groups located in the end of main polymer chain.
Functionality of main chain end groups determined from the molecular
weight determined from SEC (M ) divided by the molecular weight obtained
n
from the content of main chain end groups (MUV).
3
Functionality was not determined due to the overlap of the DOX spectra
with the modified Ellman's assay.
+
−
cell line has a variable expression of CD20 with CD20 and CD20
Table 2
side-populations (Figure SI1). CD20-negative side population was used
for in vitro experiments. SU-DHL-5 and RAJI are CD20-positive lym-
phoma cell lines purchased from German Collection of Microorganisms
and Cell Cultures, Braunschweig, Germany. Cell lines were cultivated in
RPMI-1640 medium (Thermo Scientific, Prague, Czech Republic) sup-
plemented with heat-inactivated 10% FBS for SU-DHL-5 or 15% FBS for
UPF4D20- cells and penicillin (100 U/mL) and streptomycin (100 μg/
mL).
Characterization of the APDCs.
APDC
Targeting unit
M
w
(g·mol−1)
Đ
Dox
wt
Content
of mAb
(wt%)
Rh (nm)
(
%
)
mAb19-PDOX
CD19
490,000
340,000
420,000
460,000
390,000
227,000
236,000
1.4 5.0
1.1 5.0
1.4 5.0
1.4 4.8
1.4 4.9
1.1 n.a.
1.1 n.a.
52
47
43
53
47
48
48
11.0
11.1
10.4
10.9
8.8
mAb19B-PDOX CD19
mAb20-PDOX
mAb38-PDOX
Ab-PDOX
CD20
CD38
Nonspecific
CD20
mAb20-P
9.8
mAb38-P
CD38
9.7
2.7. Cell cytotoxicity – IC50
4
For the in vitro cell viability assay 10 cells were seeded in 100 μL of
refractive index detector Optilab®-rEX (Wyatt Technology Co., Santa
media per well in 96-well flat-bottom plates (TPP, Sigma-Aldrich,
Prague, Czech Republic) 24 h before adding the conjugates or control.
Concentrations of the samples ranged from 0.02–10 μg/mL Dox eq. for
antibody-polymer conjugates and from 0.015–5 μg/mL for free drug
Dox. The cells were incubated with polymer conjugates or free drugs for
72 h. Then, 10 μL of the Alamar Blue® cell viability reagent (Thermo
Scientific, Prague, Czech Republic) was added to each well and in-
cubated for 4 h at 37 °C. The active component of the Alamar Blue re-
agent, resazurin, was reduced to the highly fluorescent compound re-
sorufin by viable cells. The fluorescence of resorufin was detected on a
Synergy Neo plate reader (Bio-Tek, Prague, Czech Republic) at an ex-
citation wavelength of 560 nm and at an emission wavelength of
590 nm [34]. Untreated cells were used as controls. Each concentration
was measured in triplicate in four independent experiments. IC was
Barbara, USA).
2.4. Synthesis of antibody polymer drug conjugates (APDC)
APDC (Table 2) were prepared by reaction of MI main chain end
group of polymer precursor PDOX and free thiol groups of monoclonal
IgG antibodies (anti-CD19, anti-CD 20, anti-CD 38) or serum im-
munoglobulin (Flebogama) reduced by DTT as previously described
[
24]. Briefly: solution of polymer precursor
PDOX (53.2 mg,
c ~ 100 mg/mL) in phosphate buffer (pH 7.2; 0.1 M NaCl, 1 mM EDTA,
bubbled with argon) was added to solution of anti-CD38 mAb reduced
by DTT (71.1 mg, c ~ 5 mg/mL) in the same buffer. Semitelechelic
polymers containing MI end groups reacted with the SH groups in the
mAb via thiol-ene chemistry to form covalent thioester bonds. Un-
reacted SH groups were blocked by the ethylenmaleimid addition after
the conjugation reaction. The mAb-polymer conjugate was desalted by
chromatography on a G-25 column and lyophilized. Final APDC were
characterized for molecular weights and hydrodynamic size using a
Shimadzu HPLC system equipped with UV detector, refractive index
5
0
calculated from measured fluorescence as the concentration of the drug
in which the viable cells were reduced by half.
2.8. Conjugate cell surface binding efficiency
(
Optilab®-rEX, Wyatt Technology Co., Santa Barbara, USA) and multi-
The binding efficiency of the conjugates was evaluated using flow
5
angle light scattering (DAWN EOS detector, Wyatt Technology Co.,
USA) using 0.3 M acetate buffer (pH 6.5) and a Superose™6 column. The
Ab content was estimated by amino acid analysis after OPA derivati-
zation (C-18 chromolith® colum; HPLC Shimadzu, Japan). The Dox
content was estimated by UV-VIS spectrophotometry using the extinc-
tion coefficient determined for hydrazone bound Dox [41]. The amount
of free thiol groups after the reduction was determined by using Ell-
man's reagent [42].
cytometry. Cells were washed with 0.5% BSA-PBS, and 2 × 10 cells in
50 μL of 0.5% BSA-PBS were incubated with antibodies or antibody
polymer drug conjugates in concentrations 1, 10 or 100 μg/mL of an-
tibodies or antibodies eq. for 30 min. The cells were then washed with
0.5% BSA-PBS and incubated for 30–40 min with 5 μL of APC-labeled
mouse anti-human CD38 (HIT2), CD19 (4G7) or CD20 (2H7) antibody
(Exbio, Prague, Czech Republic). Afterward, the cells were washed with
0.5% BSA-PBS and resuspended in 0.5 ml of 0.5% BSA-PBS with 1 μg/
ml Sytox Blue reagent (Thermo Scientific, Prague, Czech Republic) for
viability counts. Samples were measured using BD FACSVerse (Becton
Dickinson, Franklin Lakes, NJ, USA). The binding efficacies of the mAb-
targeted conjugates or mAb alone were calculated as the differences
between the fluorescent intensities of the mAb-APC-labeled cells and
the mAb-APC-marked cells after treatment with the mAb-targeted
polymer conjugates or mAb alone. Significant differences were per-
formed using GraphPad Prism (La Jolla, CA, USA, 5.5) and one-way
Analysis of variance (ANOVA) followed by Tukey's range test.
2.5. In vitro drug release from the polymer-dox conjugates
All APDC were incubated at 37 °C in 0.1 M phosphate/0.05 M NaCl
buffers adjusted to pH 5.0, 6.5 and 7.4, mimicking the pH of the in-
tracellular environment, extracellular tumor environment and blood-
stream, respectively. The amount of total released Dox was determined
using extraction of released DOX into the organic phase followed by
HPLC analysis, as described previously [15], using the free Dox as
162

O. Lidický, et al.
Journal of Controlled Release 328 (2020) 160–170
2
.9. 51Cr release assay
Griffols) to prevent non-specific bounding for 10 min at room tem-
perature and washed again. After then the samples were stained with
antibodies for 30 min and twice washed. Following fluorochrome-con-
jugated mAbs were used: CD19 PE (clone HIB19, BD Biosciences), CD20
(clone 2H7, BD Biosciences), CD38 (clone HIT2, BD Biosciences).
Samples were analyzed by a FACSCanto (Becton Dickinson, San Jose,
CA, USA). FCM results were processed with Kaluza software, version
2.1 (Beckman Coulter). Isotype-matched negative controls were used in
all the assays to distinguish positive from negative cells. For surface
antigen quantification we have used QuantiBRITE™ PE Quantification
Kit (BD Bioscience) according to manufacturer instructions. This Kit
contains test tubes with a mix of 4 types of beads, which differs by
amount of surface PE signals (low, med-low, med-high, high). Counting
geometric means we have performed a comparison of PE cell signals of
used PDX-models and cell lines to beads with known amount of surface
antigens, which also served as an interassay control.
Raji, SUDHL-5 and UPF4D were exposed in vitro to Rituximab
(
10 μg/mL), Daratumumab (10 μg/mL), to antibody-polymer con-
jugates containing antibody in the concentration 10 μg/mL, e.g.
mAb20-PDOX (20 μg/mL), mAb38-PDOX (20 μg/mL), P (20 μg/mL) or
DMSO and incubated at 37 °C and 5% CO for 24 h. Subsequently,
2
6
51
5
× 10 viable cells were labeled with Cr at 37 °C in 5% CO for 2 h.
2
5
1
Cr-labeled cells were then placed in 96-well plates at a cell con-
5
4
centration of 10 cells/well (CDC assay) or 10 cells/well (ADCC assay).
Cells were then exposed to antibodies and human serum (CDC, 1:4
dilution) or peripheral blood mononuclear cells (PBMCs) (ADCC, 40:1
5
1
effector: target ratio) for 6 h at 37 °C in 5% CO
2
.
Cr release was
measured as previously described [43]. PBMCs were obtained from
healthy donors (IRB approved protocol CIC-016) and isolated by His-
topaque-1077 ultracentrifugation of peripheral whole blood. Pooled
human serum was used as the source of complement for CDC assays.
2
.14. Statistical analysis
2.10. In vivo methods – immunodeficient mice
To assess the practical significance of treatment effectiveness, we
NOD·Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice (referred to as NSG mice)
plotted charts with growth curves indicating group mean tumor sizes
accompanied by expert opinion on the differences observed. To assess
the statistical significance of treatment effectiveness, on the other hand,
we calculated differences – for particular (available) time points (i.e.
days) – between mean tumor sizes in the control groups and groups
receiving mAb therapy as well as those between the latter groups and
the groups treated with targeted polymers (e.g. mAb19 vs. mAb). For
the daily time series of these differences, statistical hypothesis tests of
linear trend slopes equality to zero were carried out. The Bonferroni
correction was used to smooth the significance level for multiple si-
multaneous hypothesis tests.
were purchased from The Jackson Laboratory (Bar Harbor, ME, USA).
All animals were housed and maintained in a pathogen-free environ-
ment in individually ventilated cages and provided with sterilized food
and water. The experimental design was approved by the institutional
animal care and use committee (MSMT−32441/2018-7).
2.11. Patient-derived lymphoma xenografts (PDX)
Three PDX models, designated VFN-D2, VFN-B2 and VFN-M5, were
used in the current project. Similar to the sister cell line UPF4D, VFN-
D2 PDX cells have variable expression of CD20 both in course of serial
re-transplantations (from primary to secondary mice), and within one
biopsy suggesting posttranslational defect in CD20 expression (Figure
SI2) due to a clonal selective pressure [11]. Several mechanisms were
suggested but exact mechanism remains unclear [44,45]. We believe
the loss of CD20 expression in UPF4D and VFN-D2 cells are direct
consequence of previous rituximab-based therapy, but analysis of par-
ticular molecular mechanism responsible for the loss of CD20 are be-
yond scope of the current study. VFN-B2 was derived from patient with
the first relapse of CD20-negative Burkitt lymphoma after failure of
front-line rituximab-based intensified immunochemotherapy. VFN-M5
was derived from patient with so far untreated CD20-positive mantle
cell lymphoma (MCL). All three PDX models are CD19- and CD38-po-
sitive (Figure SI1).
Data was analyzed using the statistic calculation. Each model was
covering different time periods with different numbers of known data
points. For the purpose of assessing the statistical significance of
treatment effectiveness, we made an assumption that the calculated
differences (between mean tumor sizes in the control groups and groups
receiving mAb therapy as well as those between the latter groups and
the groups treated with targeted polymers) were generated by a process
which includes a deterministic linear trend in the form of.
y =
+
t +
t
t
0
1
where y
t
denotes the data-generating stochastic process of the
analyzed differences, t = 1, 2, …T is a time variable, T signifies the
length of the experiment (not the same for all experiments) in days and
β is the Gaussian IID white noise. Having concurrently performed 15
t
statistical hypothesis tests about the zero value of β , the Bonferroni
1
2.12. Experimental therapy of lymphoma-bearing mice
correction of 10% and 1% simultaneous significance levels was utilized,
resulting in individual significance levels of 0.6667% and 0.0667%,
respectively. The results are shown in Table 4.
6
NSG mice were subcutaneously inoculated with 5 × 10 of PDX
cells. Therapy was initiated when all mice developed palpable tumors.
At day 1 (D1) the mice were stratified so that all cohorts contained
animals with comparable calculated tumor volumes. Single dose
therapy of APDC (5 mg/kg Dox eq.) was administered intra-venously
via tail vein on D1. Tumor growth was recorded daily using three
perpendicular dimensions (in millimeters) with a digital caliper. Tumor
volumes were calculated using the following formula: π/6 × length ×
width × height. Observation was terminated (and experimental mice
from the cohort euthanized) when grown subcutaneously tumors ex-
ceeded 2 cm in the largest diameter.
3. Results and discussion
We have recently shown that one-point attachment of synthetic
polymer precursors to the mAb leads to a highly defined APDC suitable
for the treatment of various malignancies [25,46]. Here, we focused on
the therapeutic potential of stimuli-sensitive actively targeted bioma-
terials composed from central mAb decorated with biocompatible
polymer chains carrying classic chemotherapeutic drug doxorubicin,
see Scheme 1. We investigated in detail in vivo therapeutic potential of
the prepared constructs targeted by various mAbs on a panel of three
PDX lymphoma models derived from patients with aggressive B-NHL.
Two PDX models were established from patients with treatment-re-
fractory B-NHL, who failed after rituximab-based therapies. One PDX
models was derived from a patient with so far untreated B-NHL and
served as a positive control for anti-CD20-based approaches. Apart from
widely tested CD19 targeting with two different CD19 antibodies we
2.13. Characterization of the cell surface antigens density
The PDX cells (VFN-B2, VFN-M5, VFN-D2) and cell lines (UPF4D,
SU-DHL5) were washed in staining buffer containing phosphate saline
buffer (PBS) with sodium azide and 0,5% bovine serum albumin, in-
cubated in PBS with 0,5% human immunoglobulin (Flebogamma,
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Journal of Controlled Release 328 (2020) 160–170
Scheme 1. Overall description of the mode of the action of designed and developed APDC.
also studied a potential of CD38-targeting with the commercially
available therapeutic monoclonal antibody daratumumab. In addition
to the fact that daratumumab is approved for the therapy of multiple
myeloma, our recent pivotal data suggest the downregulation of CD38
antigen (plausibly by internalization of the antibody-antigen com-
plexes) upon treatment of PDX bearing mice with the naked dar-
atumumab antibody [47–49].
protected hydrazide groups (Fig. 1).
The polymerization was carried out to obtain the polymer precursor
with molar mass under the renal threshold limit for HPMA-based
⁎
P
polymers ~50–70,000 g/mol [50] so the polymer chains could be ex-
cluded from body by glomerular filtration after fulfilling their role in
the organism and degradation of the whole APDC construct. In the
second step, TT end groups were substituted with MI reactive groups
and hydrazide protecting groups were removed to give polymer pre-
⁎
cursor P with similar physico-chemical properties to P . The function-
3.1. Synthesis of polymer precursors
ality of maleimide functional groups decreased to the value close to 1
what fulfils the semitelechelic character of the polymer enabling the
one point grafting of polymer precursor to the mAb. MI groups of
Polymer precursors were prepared using the free radical poly-
merization initiated by TT functionalized azo-initiator to reproducibly
prepare semitelechelic copolymer of HPMA and monomer with
51
polymer P, used as a control in Cr release assay determining CDC and
Fig. 1. Scheme of the synthesis of the semitelechelic HPMA based copolymer precursor containing hydrazone bound Dox - PDOX and the maleimide terminal group.
164

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Journal of Controlled Release 328 (2020) 160–170
ADCC, were modified with access of cysteine prior the experiment.
Finally, anthracycline drug Dox was connected by hydrazone bond.
Polymer precursor PDOX contained 9.2 wt% of drug a sufficient amount
for the follow-up synthetic and evaluation steps (Table 1). The higher
molecular weight of the polymer precursor PDOX could be ascribed
unreacted polymer precursor, up to 12%. We suppose that the precursor
would be eliminated from the body in a short time due to the renal
filtration and would not affect the in vivo experiments. After the APDC
construct formation the size in the solution of all there APDCs increase
in comparison to free mAb, Rh ~ 5 nm, or polymer precursor, Rh = 4.2.
The hydrodynamic radius of APDCs increased approximately two times
in contrast to free mAb, thus showing the possibility for prolonged
circulation of all formed APDCs leading to the much profound si-
multaneous passive and active targeting ability into the lymphomas.
Dispersity of all APDCs was below 1.5 thus showing their high
uniformity. In the case of mAb19B-PDOX, mAb20-P and mAb38-P the
dispersity dropped to 1.1 showing the potential of the method to syn-
thesize the well-defined APDC. Unreacted thiol groups on mAb were
blocked by adding the N-ethylmaleimide to avoid any possible cross-
linking of the synthesized APDC. All the APDC showed proper physico-
chemical properties which nominate them as suitable candidates for
further studies.
partly to the inconsistency of M
w
determination based on anthracy-
clines absorption spectrum which is partly overlapping with used laser
in chromatography [51] and partly to the cross-reaction of maleimides
with hydrazide groups or amino group presented in the structure of the
doxorubicin. Nevertheless, the PDOX had the M
w
below the renal
threshold and increased M could even enhance the time of circulation
w
of the APDC. We can summarize that all the polymer precursors are
useable for the further APDC synthesis.
3.2. APDC synthesis and characterization
Five comparable APDC were synthesized, see Table 2, using the
same protocol employing “Michael addition”. The free thiol groups of
mAb, introduced by mild reduction using dithiothreitol, were reacted
with main-chain end located maleimide group of the polymer pre-
cursors P or PDOX. Mild reduction of mAb led in all used mAb to for-
mation of 8–12 thiol groups per mAb which was a sufficient amount of
reactive groups for the conjugation reaction. As published elsewhere
3.3. Drug release study
Stimuli sensitive nanomedicines should enable the drug activation
upon the external stimuli from the target tissue or cells. Moreover,
stability during the circulation is prerequisite for the elimination of
desired effects of the treatment and increase the efficacy of the target
tissue accumulation. Stability of prepared APDC containing pH-sensi-
tive hydrazone bond in environment modelling blood stream (pH 7.4)
and drug release kinetics from the polymer carriers at model of the
extracellular tumor environment (pH 6.5) and of tumor cells (pH 5.0)
were measured, see Fig. 3. All the APDC were highly stable in the PBS
buffer (pH 7.4) mimicking the condition of the blood stream with up to
10–18% release of drug within one day. Similar results were obtained
also in plasma proving the APDC stability after the injection and during
the delivery to the lymphoma mass. On the other hand, fast drug release
with 60–75% in 5 h in the buffer mimicking the intracellular condition
of the tumor cells, pH 5.0, was observed and the pH-sensitive behavior
of APDC was unambiguously validated. Indeed, even in the model of
extracellular tumor environment, pH 6.5, accelerated drug release
showing around 55–64% of drug released within 24 h was found. There
were slight differences between the APDCs, which were not significant
for the overall pH-responsiveness of APDCs. We can summarize that all
[
34] and determined by GPC, electrophoresis and binding ability assay,
the mild reduction of disulfide bridges does not influenced the overall
structure and activity of mAb. Within the following click reaction, be-
tween sulfhydryl groups of reduced mAb and main-chain end MI groups
of polymers, all APDC formed star-like structures employing the one
point attachment strategy for linking several polymer chains to mAb,
see Fig. 2. In sum, for all APDC we have not found any significance of
the mAb degradation to fragments during the synthetic steps.
All APDC contained after the reaction approximately 45–50% of
mAb in the conjugate. The molecular weight was comparable for all
APDC, a small increase of the molecular weight for mAb19-PDOX and
mAb38-PDOX could be escribed to the partial cross-linking of two mAb
by polymer containing two MI functional groups. Nevertheless, the
increase of molecular weight was very small and did not cause sig-
nificant increase of dispersity. All APDC showed similar content of the
drug (~ 5 wt%), which was sufficient for the following biological ex-
periments. APDC contained after the reaction small content of
Fig. 2. Schematic description of APDC composed of the central targeting antibody decorated with polymer drug carriers with attached drug via hydrazone bond.
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Journal of Controlled Release 328 (2020) 160–170
DHL-5 was not possible due to the lack of the CD38 epitopes. We can
summarize that the binding ability of the prepared APDCs was found
comparable with the original mAb in higher concentration tested,
where the saturation of the CD epitopes on the cell should occur.
Indeed, the binding ability was partially decreased and affected at
lower concentration in the both cell lines UPF4D and SU-DHL-5,
proving the influence of the modification of mAb into its binging
ability. Nevertheless, even at low concentration all APDC tested were
able to bind to the epitopes on the cells. We thus confirmed the ability
of the conjugates, antibodies decorated with polymer, to recognize their
targeting CD molecules and proved their binding to the cell surface with
high efficiency comparable to the unmodified antibodies in their higher
concentrations.
3.5. In vitro cytotoxicity
The in vitro cytotoxic activity of the conjugates mAb19-PDOX
,
mAb20-PDOX, mAb38-PDOX, mAb19B-PDOX, Ab-PDOX, PDOX and free
DOX was measured using Alamar Blue proliferating assay. All mAb
targeted constructs containing hydrazone bound Dox showed compar-
able cytotoxicity without significant differences. This could be as-
scribed to the release of the part of the drug already in the extracellular
space during the experiment. Cytotoxic effect could be caused by both,
the drug released inside the cell after the internalization of APDCs to
the cells and also by Dox released from the APDCs in the extracellular
space and penetrate throw the cell membrane independently on the
APDC targeting abilities. SU-DHL-5 cell line showed higher sensitivity
to the anthracycline drugs than UPF4D cell line. PDOX alone showed
lower toxicity on less sensitive UPF4D cell line in comparison to all
mAb containing constructs, showing the mAb-triggered targeting ben-
efit already in vitro. No significant difference in cytotoxicity was found
between polymer alone and targeted systems on SU-DHL-5 cell line. We
hypothesize that the higher effectivity of polymer alone is also con-
nected with higher sensitivity of the SU-DHL-5 cell line to doxorubicin,
which is partly released from all the pH-responsive APDCs in the
medium within the 3 day experiment. All the APDCs containing specific
monoclonal antibodies were more cytotoxic than the Ab-PDOX con-
taining nonspecific antibody, thus proving the benefit of the specific
active targeting on cytotoxicity. Expectably, the toxicity of free Dox was
significantly higher in contrast to all the polymer-based systems as was
several times described previously. This phenomenon is based on the
rapid internalization of free drug in comparison to the much slower
process of endocytosis of polymer-based nanomedicines (Table 3).
Fig. 3. Release of Dox from the conjugates mAb19-PDOX (□), mAb20-PDOX
(
○), mAb38-PDOX (◊) and mAb-PDOX (∆) at 37 °C in various pH modelling
blood stream condition – pH 7.4, dashed line in a); extracellular environment –
pH 6.5, full line in a); and intracellular environment in lysosomes – pH 5.0, full
line in b); n = 3.
the APDC have the same and highly favourable pH-sensitive release
profile of the drug, highly valuable for the drug activation in hypoxic
lymphoma tissue or lymphoma cells.
3.4. In vitro study of APDC binding efficiency
Grafting of the mAb structure with polymers has potential risk of
affecting its effectiveness and binding ability to their targeting epitopes
on cell membrane. Nevertheless, single-point attachment of the
polymer precursor to the thiol groups of reduced antibody avoids the
risk of modifying the hypervariable region of the antibody and it is most
likely attached to the hinge area. To evaluate the possible decrease of
binding ability, we evaluated in detail the binding efficiency of the
prepared APDC to their antigens. The expression of CD19, CD20 and
CD38 on UPF4D and SU-DHL-5 cell lines was evaluated using the flow
cytometry (Figure SI1). UPF4D cell line showed the expression of all
three CD molecules, while only CD19 and CD20 expression was found
3.6. CDC and ADCC determination
Due to the possible masking of the antibody Fc fragment after the
modification with polymer chains in APDC and connected depletion of
ADCC and/or CDC biological activity of mAb, we evaluated the ability
of the developed APDC to induce ADCC or CDC using standard Cr-re-
lease assay. Two drug free analog APDC constructs, mAb20-P and
mAb38-P, based on anti-CD20 and anti-CD38 mAb modified with
polymer precursor P were synthesized. ADCC and CDC activity of these
constructs was compared with the activity of unmodified mAb ritux-
imab (anti-CD20) and daratumumab (anti-CD38) and their mAb20-P
and mAb38-P constructs. Three cell lines were used for the Cr-release
assay: RAJI cell line expressing both CD20 and CD38; SUDHL5 cell line
expressing CD20, but not CD38; and UPF4D, which expresses CD38, but
not CD20 (CD20-negative side-population was used for the Cr-release
experiments). UPF4D was derived in parallel from the same patient as
the PDX model VFN-D2 used for in vivo experiments (Fig. 5).
for SU-DHL-5 cell line. The binding efficacy of the mAb19-PDOX
,
mAb20-PDOX and mAb38-PDOX were compared to the binding efficacy
of original unmodified mAb mAb19, mAb20 and mAb38 using UPF4D
and SU-DHL-5 B-cell lines, Fig. 4. To evaluate the binding efficacy
precisely the experiment setup employing consequential binding of
APC-labeled antibodies to the appropriate epitope blocked by APDC or
mAb in the previous step was applied.
Any significant difference of binding activity was not detected for
the UPF4D cell line between mAb19, mAb38 and their APDCc at con-
centration 100 and 10 μg/mL. Decrease of binding activity of mAb19-
P
DOX and mAb38-PDOX was observed only at the lowest concentration
of 1 μg/mL. Indeed, significantly lower binding activity was observed
for both cell lines in the case of mAb20-PDOX at concentrations 10 and
1
μg/mL. Similarly as for UPF4D cell line the binding activity of
The Cr-release assays confirmed that the extent of the antibody-
triggered cell lysis correlated with the level of expression of the targeted
CD antigens. In addition, the functional assays confirmed the antici-
pated weakening or even abrogation of immune-mediated activities of
the tested monoclonal antibodies after their conjugation to polymer
mAb19-PDOX for SU-DHL-5 cell line was not decreased at concentra-
tions 100 and 10 μg/mL compared to mAb19. Again, the binding ac-
tivity was significantly decreased only at the lowest concentration of
mAb19-PDOX. Measuring the efficiency for CD38 biding ability for SU-
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O. Lidický, et al.
Journal of Controlled Release 328 (2020) 160–170
Fig. 4. Comparison of binding efficiency of APDC (mAb19-PDOX, mAb20-PDOX, mAb38-PDOX) and original antibodies (mAb19, mAb20, mAb38) to the cell surface
membrane epitopes. The binding activity was evaluated by flow cytometry for a) UPF4D and b) SU-DHL-5 cell lines. Significant differences are labeled with asterisks:
***P < 0.001, **P < 0.01, and *P < 0.05.
Table 3
chains. In summary, the modification of the mAb by several polymer
chains, using the orthogonal one-point maleimide–thiol coupling in the
hinge area of mAb, are most probably blocking sterically the structures
of the Fc fragments which are responsible for activating the CDC or
ADCC. The results suggest that the APDC constructs induce cell cyto-
toxicity mainly due to the genotoxic effect of the carried drug, i.e.
doxorubicin. In this case, the antibodies serve mainly as the targeting
moieties with largely suppressed immunological mode-of-action.
Cytotoxicity expressed as IC50
.
Cell line APDC
UPF4D
SU-DHL-5
IC50 (ng DOX eq./mL)
IC50 (ng DOX eq./mL)
mAb19-PDOX
mAb19B-PDOX
mAb20-PDOX
mAb38-PDOX
Ab-PDOX
289 ± 27
378 ± 161
262 ± 24
276 ± 63
453 ± 53
1041 ± 441
95 ± 24
84 ± 47
82 ± 23
89 ± 37
221 ± 69
107 ± 86
3.5 ± 1.7
P
DOX
DOX
3.7. In vivo anti-lymphoma activity on a panel of PDX lymphoma models
16 ±
4
In vivo experiments were implemented on three PDX models de-
rived from two patients after failure of CD20-based front-line therapies
(
VFN-D2, VFN-B2) and one so far untreated patient (VFN-M5), see
Fig. 5. Evaluation of cell lysis (%) due to the CDC or ADCC using Cr release assay. Comparison of the polymer-modified antibodies mAb20-P, mAb38-P, polymer P
concentration 20 μg/mL, in polymer-modified antibodies equal to 10 μg/mL) and unmodified antibodies mAb20 and mAb38 (concentration 10 μg/mL) normalized
to the Isotype control, cell lines SUDHL-5, Raji and UPF4D, n = 3.
(
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Journal of Controlled Release 328 (2020) 160–170
Fig. 6. In vivo activity of the APDC after administration single dose of the conjugates mAb19-PDOX, mAb19B-PDOX, mAb20-PDOX, mAb38-PDOX, Ab-PDOX compared
to the untreated control CTRL and the comparison of the tumor size after the autopsy done on three PDX models a) VFN-M5; b) VFN-D2; c) VFN-B2. VFN-B2 n = 6,
WFN-M5 n = 7, WFN-D2 n = 7.
Table 4
characterization in the SI. The anti-lymphoma efficacy of APDCs was
compared using single low dose, 5 mg/kg Dox equivalent. The treat-
ment was neither accompanied by signs of apparent toxicity, nor did it
impact weight of the experimental animals. The overall good tolerance
of the single injection-based therapy suggests potential for future dose
escalation with better anti-lymphoma efficacy.
Statistical analysis of in vivo efficacy.
Differences
VFN-M5
VFN-D2
VFN-B2
CTRL vs. Ab-PDOX
0.0000***
0.0313
0.1565
0.0007*
0.0014*
0.9523
Ab-PDOX vs. mAb19-PDOX
Ab-PDOX vs. mAb19B-PDOX
Ab-PDOX vs. mAb20-PDOX
Ab-PDOX vs. mAb38-PDOX
0.0001***
0.0003***
0.0019*
0.0001***
0.0000***
0.0000***
The in vivo data confirmed that APDCs targeted with mAbs
0.0008*
0.0000***
(
mAb19-PDOX, mAb19B-PDOX, mAb20-PDOX and mAb38-PDOX) were
0.0001***
significantly more efficient in suppressing lymphoma growth than non-
specific APDC (Ab-PDOX) (Fig. 6, Table 4). The partial effect of non-
specific APDC could be ascribed to the EPR effect-based accumulation
of the non-targeted nanotherapeutics. Anti-lymphoma activity of CD20-
and CD38-targeting APDCs was comparable in mice xenografted with
VFN-M5 PDX cells derived from so far untreated patient. In contrast,
P-values of partial t-tests about zero slope of mean tumor size differences
statistical significance: * 10% and *** 1% simultaneous significance level).
(
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Journal of Controlled Release 328 (2020) 160–170
treatment of mice xenografted with PDX cells derived from patients,
who failed after rituximab-based regimen (VFN-D2, VFN-B2) confirmed
significantly better anti-lymphoma efficacy of the APDC targeted with
anti-CD38 daratumumab compared to anti-CD20 rituximab and other
APDCs. Even with small single Dox eq. dose the anti-CD38 APDC in-
duced long-term control of growth of. The reason for the observed
lower efficacy of the tested conjugates targeted with CD19 mAbs re-
mains elusive and will be further studied.
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