Ligand-targeting drug delivery systems have made significant strides for disease treatments with numerous clinical approvals in this era of precision medicine. Herein, we report a class of small molecule-based immune checkpoint-targeting maytansinoid conjugates. From the ligand targeting ability, pharmacokinetics profiling, in vivo anti-pancreatic cancer, triple-negative breast cancer, and sorafenib-resistant liver cancer efficacies with quantitative mRNA analysis of treated-tumor tissues, we demonstrated that conjugate 40a not only induced lasting regression of tumor growth, but it also rejuvenated the once immunosuppressive tumor microenvironment to an "inflamed hot tumor" with significant elevation of gene expressions that were not accessible in the vehicle-treated tumor. In turn, the immune checkpoint-targeting small molecule drug conjugate from this work represents a new pharmacodelivery strategy that can be expanded with combination therapy with existing immune-oncology treatment options.
Ligand-targeting drug delivery systems have made significant strides for disease treatments with numerous clinical approvals in this era of precision medicine. Herein, we report a class of small molecule-based immune checkpoint-targeting maytansinoid conjugates. From the ligand targeting ability, pharmacokinetics profiling, in vivo anti-pancreatic cancer, triple-negative breast cancer, and sorafenib-resistant liver cancer efficacies with quantitative mRNA analysis of treated-tumor tissues, we demonstrated that conjugate 40a not only induced lasting regression of tumor growth, but it also rejuvenated the once immunosuppressive tumor microenvironment to an "inflamed hot tumor" with significant elevation of gene expressions that were not accessible in the vehicle-treated tumor. In turn, the immune checkpoint-targeting small molecule drug conjugate from this work represents a new pharmacodelivery strategy that can be expanded with combination therapy with existing immune-oncology treatment options.
Ligand-targeted therapeutic conjugates
have circumvented the pharmacokinetic
limitation of the conventional chemotherapeutic agents and facilitated
selective association with disease biomarkers to allow site-specific
dose escalation.[1−3] To reduce the collateral toxicity to normal cells
and improve the therapeutic index of cancer chemotherapy, small molecule
ligands and monoclonal antibodies have been used as a delivery moiety
of cytotoxic compounds in the forms of small molecule drug conjugates
(SMDCs) and antibody-drug conjugates (ADCs). Spurred by the eight
marketing authorizations of ADCs for cancer therapy since 2017, ADCs
have become one of the fastest-growing drug classes in oncology. However,
limited penetration into solid tumor masses, high cost of goods, and
premature drug release of the ADCs have motivated the SMDC development
for pharmacodelivery applications as several are under clinical investigations.[4,5] A comparative analysis between chemically defined, cell-surface
antigen carbonic anhydrase IX (CAIX)-targeting ADCs and SMDCs has
suggested that SMDC technology allowed efficient targeting and accumulation
in the tumor mass to mediate potent antitumor effects dosing with
the same drug molar ratio to that of ADCs.[6] Concurrently, cancer immunotherapy, eradicating tumor cells via
enhancing patient’s immunity, has been deployed for mono- or
combination therapeutic options in treating malignant tumors.[7] Indeed, various types of immunotherapy, including
immune checkpoint inhibitors,[8] T cell transfer
therapy,[9] and chimeric antigen receptor
T-cell immunotherapy (CAR-T)[10,11] have been successfully
used for treatment. Yet, clinical evidence has provided insight into
the poor prognosis associated with the degree of immune activation
effects of the tumors.[12−14] In turn, developing new immunostimulatory agents
is thus essential to compensate for such deficiencies in cancer treatment.
Here, we devise a new therapeutic module of the immuno-SMDC (iSMDC) that targets an immune checkpoint
ligand in the tumor microenvironment (TME) and simultaneously delivers
a cytotoxic compound in the form of chemically defined SMDCs.Externalized phosphatidylserine (PS) on the tumor cells[15,16] or tumor-derived exosomes in the TME have become a cancer diagnostic
biomarker. Its inherent immunosuppressive properties have also propelled
the development of several PS-targeting agents under clinical investigations.[17−20] In addition to the conventional apoptotic function of PS,[21,22] PS exposure on the surface of the tumor cells, through binding to
TIM and TAM family proteins and Stabilin 1 or 2 receptors,[23−25] readily suppressed immune activation of dendritic cells, macrophages,
and T-cells.[26] PS-targeting molecules,
including antibodies,[27−29] liposomes,[30] ADCs,[31] targeting peptide carrying paclitaxel,[32] and SMDCs,[33−35] have shown sound antitumor
effects in preclinical mouse models and undergone evaluations in clinical
trials. Herein, the development of iSMDCs was based
on zinc(II) bis-dipicolylamine (Zn-DPA) and its derivative conjugating
to maytansinoid with a hydrophilic linker. Leveraging specific interaction
between the coordinated zinc ions within Zn-DPA and the anionic phosphate
moieties, Zn-DPA has been employed in numerous biomedical applications.[36−41] This particular strategy offers a multitude of antitumor growth
properties: (1) highly potent maytansinoid can readily initiate apoptosis
of tumor, (2) newly triggered PS externalization from tumor cell death
can provide an additional homing signal to recruit circulating conjugates,
and (3) binding of Zn-DPA to PS on the tumor cells can modulate PS
binding to receptors of the immune cells and lead to the rejuvenation
of the functionalities of the immune cells in the TME.Moreover,
since PS overexpression was found in many solid tumors,
we have profiled the new maytansinoid iSMDC against
the growth of pancreatic cancer, triple-negative breast cancer, and
liver cancer. We assessed the PS-targeting ability and structure-activity
relationship study via linker modifications with subsequent pharmacokinetics
profiling to improve the stability and tolerability of the conjugates in vivo. We observed eradication of small or shrinkage of
large tumor xenografts at well-tolerated doses. We then demonstrated
that this maytansinoid conjugate could elicit immune cell infiltration
and rejuvenate the “cold” tumor microenvironment to
the inflamed “hot” tumor. The current study underscores
the importance of the interplay between targeting an immune checkpoint
ligand and delivery of highly potent cytotoxic in a chemically defined
small organic molecule drug conjugate. Notably, it presents a new
pharmacodelivery platform that, with the growing list of new small
organic molecules identified as immune checkpoint inhibitors/binders,
harnesses the potent activities of chemotherapy and compliments the
immunostimulatory effects of cancer therapy.
Results and Discussion
Synthesis of the Zn-DPA Derivative and Zn-DPA Maytansinoid Conjugates
To evaluate the impacts among the targeting ligand, linker stabilities
on the pharmacokinetic profiles, and in vivo activities
of the conjugates, the Zn-DPA maytansinoid conjugates were designed
and synthesized through (1) a variety of hydrophilic linkers containing
ethylene glycol units, (2) installing steric hindrance with methyl
groups on the adjacent carbon next to the sulfur group, (3) employing
two different maytansinoids used in the clinical trials for ADCs,
DM1 (compound 14) and DM4 (compound 16),
and (4) incorporating a modified PS-targeting Zn-DPA analog. Recent
studies have demonstrated that even in the absence of ligand internalization,
some ADCs or SMDCs could achieve potent efficacies through efficient
payload release within the tumor mass, either mediating by different
extracellular proteases or by reduction of disulfide linkers.[6,42−46] Modifications around the dipicolylamine group were shown to increase
binding affinity and selectivity toward PS by taking advantage of
additional secondary noncovalent interactions in hydrogen bonding
and hydrophobic insertion into the membrane.[47,48] For the synthesis of the DPA derivative, we installed a hydrophobic
alkyl chain to one side of the dipicolylamine unit (Scheme S1, compound 11). In Scheme S1, synthesis of the DPA derivative started with the
coupling of hexanoic acid to methyl 6-aminopicolinate, which was then
followed by the reduction of ester with NaBH4 to furnish
compound 3. Oxidation of compound 3 with
MnO2 proceeded smoothly to allow the formation of aldehyde
intermediate 4, and the overall yield for these three
steps was 70%. Reductive amination between 2-picolylamine and intermediate 4 was performed to obtain the alkyl-modified picolylamine
precursor 5. Treatment of 5-hydroxyisophthalate 6 with lithium aluminum hydride in dry THF at 50 °C furnished
the fully reduced 3,5-bis(hydroxymethyl)phenol 7 in good
yield at 97%. Alkylation of 7 with 2-(4-bromobutyl)isoindoline-1,3-dione
was performed in the presence of potassium carbonate to afford the
corresponding diol compound 8 in 44% yield. Following
the MnO2 oxidation of diol 8, the resulting
dicarbaldehyde intermediate 9 was then coupled with picolylamine
precursor 5, and crude product 10 was then
treated with hydrazine to furnish the Zn-DPA derivative 11 (49% yield in three steps). To probe the release of the payload,
we chose to employ two different maytansinoids, DM1 (compound 14) and DM4 (compound 16), that have been investigated
in the clinical trials for ADCs. We synthesized the caged maytansinoid
precursors by introducing steric hindrance with dimethyl groups on
either side of the adjacent carbon next to the disulfide linkage (Scheme S2 and Figure ). In Scheme S2, maytansinoid precursors 15 and 17, differing
in methyl group substituents on adjacent carbons next to the sulfur,
were synthesized through disulfide exchange with 3-(pyridin-2-yldisulfaneyl)propanoic
acid 13 in moderate yields. In addition, maytansionoid
precursor 19 was obtained by first reacting payload 14 with 2,2’-dithiobis(5-nitropyridine) followed by
the treatment of intermediate 18 with 4-mercapto-4-methylpentanoic
acid in THF and potassium phosphate buffer (50 mM, pH 7.5). Next,
the approaches to accessing different maytansinoid conjugates linking
to DPA 24 or the modified DPA derivative 11 are outlined in Scheme and 2, respectively. The key step
is the conjugation of different linkers between the targeting DPA
moiety and the maytansinoid-containing precursors. Activation of propanoic
acid in intermediate 13 with EDCl and HOBt followed by
the addition of DPA 24 gave DPA-linker 25a in 52% yield. Payload 14 (DM1) was stirred with 25a in CH2Cl2, and the resulting conjugate 26a was obtained in 92% yield. Disulfide containing benzoic
acid 21 was activated with EDCI and HOBt followed by
coupling with DPA 24 to afford 25b in 84%
yield. Conjugate 26b was obtained via disulfide exchange
between payload 14 and intermediate 25b.
To increase the solubility and stability of the eventual conjugates,
we have incorporated small-unit linear ethylene glycol to bridge the
derivatives of the targeting ligand and the cytotoxic payload. Large-unit
pegylation could increase the solubility of the conjugate, yet the
resulting steric interference might modulate the targeting ligand’s
binding affinity or hinder cargo release.[49] Reductive amination between biphenyl-4-carboxaldehyde and DPA 24 gave 25c in 69% yield, which was then coupled
with PEG-containing linker 23 (Scheme S2) to provide intermediate 28. Removal of the
Boc protecting group in 28 with TFA allowed the conjugation
with maytansinoid precursor 15 to furnish conjugate 29 (47% in two steps). In Scheme , conjugate 32 was synthesized
by coupling the activated intermediate 13 and the DPA
derivative 11 followed by disulfide exchange with payload 14. PEG-containing linker 23 was first activated
by EDCI and HOBt, and DPA derivative 11 in CH2Cl2 was coupled to provide intermediate 34 in 77% yield. TFA deprotection of the Boc group in 34 followed by the conjugation with maytansinoid precursor 15 has led to the synthesis of conjugate 35 (74% in two
steps). Conjugates 39a and 39b were synthesized
via common intermediate 37a, obtained by reductive amination
of biphenyl-4-carboxaldehyde and DPA derivative 11. Intermediate 38a, employing the PEG-containing linker 23,
was coupled with maytansinoid precursor 15 or 19 to provide conjugate 39a or 39b, respectively.
Alternatively, the LiOH hydrolyzed product with a 1-(4-chlorophenyl)cyclohexanecarbonyl
chloride functional group in 38b_acid was then reacted
with PEG linker group f (Scheme ) to give compound 41 in 73 % yield. After
TFA deprotection of intermediate 41, maytansinoid precursor 17 was introduced by the forming amide bond in conjugate 42. Formation of the resulting Zn-DPA conjugates 27a, 27b, 30, 33, 36, 40a, 40b, and 43 was carried
out by incubating each of the DPA-maytansinoid conjugates 26a, 26b, 29, 32, 35, 39a, 39b, and 42 with two
equivalents of Zn(NO3)2 at room temperature,
respectively. In Figure S1, comparative
spectroscopic analysis and structural characterizations among conjugate 40a and its key intermediates 39a, 38a, and 15 were carried out to demonstrate the formation of the drug
conjugate. Moreover, the chemical shifts at the dipicolylamine region
after the complex formation between zinc and dipicolylamine were also
observed (Figure S1). In addition, we synthesized
imaging probe Zn11-794 (Scheme S3) to address the tumor-targeting ability of the new DPA derivative 11in vivo. Taken together, modular constructions
between different targeting ligands (DPA 24 and its derivative 11), linker fragments, and drug payload precursors have allowed
the synthesis of a collection of conjugates (Figure A) for structure–activity and property
relationship investigation.
Figure 1
Design of small-molecule maytansinoid conjugates
with active targeting
of phosphatidylserine (PS) at tumor tissue and linkers that allow in vivo circulation stability. With the controlled release
of the cytotoxic maytansinoid in the tumor microenvironment, activation
of the apoptotic pathway can lead to the amplification of the homing
signal in situ as the very exposure of PS shall facilitate
recruitment of circulating conjugates that result in the enhancement
of anti-tumor activities.
Scheme 1
Synthetic Procedures for Zinc Dipicolylamine Maytansinoid
Conjugates 27a, 27b, and 30
Properties of ZnDPA maytansinoid conjugates. (A) Chemical
structures
of the newly synthesized conjugates. (B) Cytotoxic effects on MIA
PaCa-2 human pancreatic cells, HCC 1806 human triple-negative breast
cancer cell, and Detroit 551 normal skin fibroblast. After a 72 h
incubation of conjugates or parent cytotoxics with the cells, the
ability to reduce tetrazolium compound by the viable cells was determined.
(C) In vivo pharmacokinetic profiles of each conjugate
in male ICR mice (n = 3) at 5 mg/kg with intravenous
administration, where a = payload 14 and b = payload 16. CL, clearance; Vss, apparent volume of distribution at steady
state; AUC, area of drug concentration under the curve; mpk, mg/kg.
Design of small-molecule maytansinoid conjugates
with active targeting
of phosphatidylserine (PS) at tumor tissue and linkers that allow in vivo circulation stability. With the controlled release
of the cytotoxic maytansinoid in the tumor microenvironment, activation
of the apoptotic pathway can lead to the amplification of the homing
signal in situ as the very exposure of PS shall facilitate
recruitment of circulating conjugates that result in the enhancement
of anti-tumor activities.Properties of ZnDPA maytansinoid conjugates. (A) Chemical
structures
of the newly synthesized conjugates. (B) Cytotoxic effects on MIA
PaCa-2 human pancreatic cells, HCC 1806 human triple-negative breast
cancer cell, and Detroit 551 normal skin fibroblast. After a 72 h
incubation of conjugates or parent cytotoxics with the cells, the
ability to reduce tetrazolium compound by the viable cells was determined.
(C) In vivo pharmacokinetic profiles of each conjugate
in male ICR mice (n = 3) at 5 mg/kg with intravenous
administration, where a = payload 14 and b = payload 16. CL, clearance; Vss, apparent volume of distribution at steady
state; AUC, area of drug concentration under the curve; mpk, mg/kg.
Synthetic Procedures for Zinc Dipicolylamine Maytansinoid
Conjugates 27a, 27b, and 30
Cytotoxicities and In Vivo Pharmacokinetic
Profiles of the Conjugates
We then examined cytotoxicities
of the newly synthesized conjugates (Figure A) against MIA PaCa-2 (pancreatic), HCC1806
(triple-negative breast cancer) cancer cell lines, and a normal fibroblast
Detroit 551 (Figure B). The IC50 of maytansinoids 14 and 16 inhibited MIA PaCa-2 and HCC1806 cancer cell growth, ranging
from 70 to 4 nM. In general, the conjugates exhibited significantly
less cytotoxicities toward normal Detroit 551 cells. In particular,
conjugates 40a, 40b, and 43 harnessed prodrug properties against these cancer cell lines relative
to the parent maytansinoids, suggesting that linker modifications
could improve the stability and shield their cytotoxic properties in vitro (Figure B). Compared to 36, the addition of the biphenyl
group in conjugate 40a has resulted in better stability
and prodrug properties. Premature release and inadequate delivery
of the cytotoxic cargo could increase off-target organ distribution
and toxicities. To address the longevity of the intact conjugate during in vivo systemic circulation, single intravenous dose pharmacokinetic
studies (Figure C)
showed a reduction of clearance (CL) rate and volume distribution Vss (0.2∼0.5) of conjugates 40a, 40b, and 43, suggesting that pegylation
of the linkers increased stability and preferential systemic distributions
were in circulation. Notably, structure–property relationship
studies among conjugates 30, 36, and 40a have demonstrated that alkyl-chain modifications in ZnDPA
analog 11 and biphenyl moiety addition in the linker
region could result in a slower CL (mL/min/kg) rate (0.9 for 40avs 2.5 for 30 and 6.0 for 36) and a 5-fold decrease in volume distribution (Vss) to achieve an intact conjugate AUC of 105,599
(ng/mL hr) for 40a (Figure C). For conjugates 40b and 43, steric hindrance introduced by the dimethyl group on the
adjacent carbon next to the sulfur group of the maytansinoids has
resulted in ∼20% loss of AUC. Furthermore, 40a was highly potent against ovarian, skin, and oral cancer cell lines
(Figure S2). Taken together, we identified 40a with improved pharmacokinetic profiles and harnessed prodrug
properties among the designed Zn-DPA maytansinoid conjugates.
Tumor Targeting Ability of the Zn-DPA Derivative and Systemic
Stability of Conjugate 40a
As the Zn-DPA motif
was shown to play an essential role in PS recognition,[34] we next addressed the association properties
of analog 11 with PS-containing liposomes. By using uniform-sized
liposomes with 100 % DOPC as a non-specific binding control, a surface
plasmon resonance (SPR) assay with PS-coated liposomes (DOPC/ DOPS
(3:1,v/v)) was carried out according to the reported procedures.[35] In comparison to the Zn-DPA compound 24, Zn-DPA derivative 11 exhibited a relative improvement
of the PS-association property (Figure S3) that resulted from a 3-fold improvement of dissociation koff (0.00358 s–1) over compound 24 (0.0113 s–1). This result suggested that
a favorable hydrophobic interaction through alkyl-chain addition to
the dipicolylamine moiety in 11 had provided stronger
complexation in PS-containing liposomes. Indeed, Zn11-794, through conjugation between analog 11 and a near-infrared
dye794 (Figure A),
showed in vivo HCC1806 tumor targeting ability and
lasting tumor site accumulation for up to 3 days (Figure B). This data also demonstrated
PS expression in the HCC1806 tumors. Next, we showed that conjugate 40a, harboring analog 11 as the PS-targeting
moiety, was stable in plasma incubation (Figure C). However, 40a was readily
cleaved with the tumor homogenates (Figure C). Treatment of glutathione transferase
inhibitor ethacrynic acid significantly rescued the abundance of intact
conjugate 40a in the tumor homogenate, suggesting that
the tumor homogenate could facilitate cleavage of the disulfide bond
in 40a (Figure C). Since PS lacks an internalization mechanism, we reason
that the disulfide linkage can be readily cleaved in the tumor microenvironment
to release maytansinoids in vivo. Indeed, in HCC1806
tumor-bearing mice, 40a not only exhibited great plasma
stability with negligible detection of 14 (Figure D), but a significant time-dependent
increase of 14 released from 40a in the
tumor mass was also readily observed (Figure E). In all, these results demonstrated that
conjugate 40a could target and accumulate in tumor sites
with limited exposure of 14 in plasma and allow controlled
release of payload 14 in the tumor mass (Figure F).
Figure 3
In vivo targeting ability, plasma stability, and in vivo biodistribution of modified zinc dipicolylamine
conjugates in subcutaneous HCC1806 tumor xenografts. (A) Chemical
structures of dye_794, modified ZnDPA conjugated with dye_794: Zn11-794, cytotoxic payload 14, and conjugate 40a. (B) In vivo detection
of PS-expression in the HCC1806 tumor xenograft model. Representative
IVIS images of new Zn11-794 fluorescence probe in mice.
Targeted with significant and lasting tumor site accumulation of Zn11-794 were observed up to 72 h with a single intravenous
dose of the conjugate at 2 mg/kg. (C) Time- and tumor homogenate-dependent
cleavage of conjugate 40a was determined. The addition
of glutathione S-transferase (GST) inhibitor, ethacrynic
acid, has rescued the cleavage of 40a. (D) Comparison
of plasma (n = 3) stability of intact conjugate 40a (black bar) and the payload 14 released from 40a (red) in HCC1806 bearing mice after a single intravenous
dose (2 mg/kg) of conjugate 40a. (E) With samples collected
at indicated time points, the amount of maytansinoid 14 via direct injection (black bar) or released from 40a (red bar) in the collected tumor tissues was determined by LC/MS/MS.
(F) Plasma and tumor distribution with AUC comparisons between injection
of untargeted 14 and targeted delivery of 14 in the form of conjugate 40a.
In vivo targeting ability, plasma stability, and in vivo biodistribution of modified zinc dipicolylamine
conjugates in subcutaneous HCC1806 tumor xenografts. (A) Chemical
structures of dye_794, modified ZnDPA conjugated with dye_794: Zn11-794, cytotoxic payload 14, and conjugate 40a. (B) In vivo detection
of PS-expression in the HCC1806 tumor xenograft model. Representative
IVIS images of new Zn11-794 fluorescence probe in mice.
Targeted with significant and lasting tumor site accumulation of Zn11-794 were observed up to 72 h with a single intravenous
dose of the conjugate at 2 mg/kg. (C) Time- and tumor homogenate-dependent
cleavage of conjugate 40a was determined. The addition
of glutathione S-transferase (GST) inhibitor, ethacrynic
acid, has rescued the cleavage of 40a. (D) Comparison
of plasma (n = 3) stability of intact conjugate 40a (black bar) and the payload 14 released from 40a (red) in HCC1806 bearing mice after a single intravenous
dose (2 mg/kg) of conjugate 40a. (E) With samples collected
at indicated time points, the amount of maytansinoid 14 via direct injection (black bar) or released from 40a (red bar) in the collected tumor tissues was determined by LC/MS/MS.
(F) Plasma and tumor distribution with AUC comparisons between injection
of untargeted 14 and targeted delivery of 14 in the form of conjugate 40a.
In Vivo Antitumor activities of Conjugate 40a
With the determined chemical stability of conjugate 40a and slow clearance rate during blood circulation, we evaluated
the in vivo antitumor activities (Figure ) in MIA PaCa-2 and HCC1806
human xenografts models. In the first set of efficacy investigations,
although conjugates 27a, 27b, and 33 possessed low circulation exposures (Figure C) and still exhibited antitumor activities,
significant body weight loss during treatment was readily observed
(Figure S4). This data suggested that the
uncontrolled or premature releases of maytansinoids might contribute
to the undesired systemic toxicities. To circumvent this issue, we
leveraged those respective modifications in the linker and targeting
ligands of conjugates 36 and 40a and observed
potent antitumor efficacies without apparent body weight loss. These
conjugates were dosed intravenously at 1 mg/kg twice weekly for two
weeks (Figure ). Comparative
study based on equimolar doses of cytotoxic maytansinoid in 14 (0.3 mg/kg) and in conjugates 36 (1 mg/kg)
and 40a (1 mg/kg) was performed. The data showed that
conjugate 40a’s improved systemic exposure of
the intact conjugate could provide potent and lasting anti-MIA PaCa-2
activity in vivo (Figure A). Concurrently, we have carried out a 28-day
repeat dose pilot toxicity study of 40a in rats and showed
that treatment of 40a did not alter organ weights (liver
and kidneys). In general, hematologic (leukocyte, neutrophil, lymphocyte,
and platelet) parameters were normal, except for a reduced erythrocyte
count. Biochemical parameters, such as GOT and GPT, were slightly
increased (not significant). In addition, BUN and creatinine levels
were normal for the 40a treatment group in this pilot
toxicity study (Figure S5). We envision
that optimizing the dosing regimen (amount and frequency) during the
treatment might further expand the therapeutic window of this class
of conjugates. In addition, potent antitumor efficacy was also observed
against HCC1806 triple-negative breast cancer (TNBC) xenografts, indicating
the benefit of targeted delivery and controlled release of the maytansinoid 14 in the form of SMDC (Figure B). Gratifyingly, potent efficacies and shrinkage of
larger (400–800 mm3) HCC1806 tumors were observed
when dosing at 2.5 mg/kg once per week for three weeks (Figure C). This study has provided
another aspect of TNBC treatment, where lessening tumor burden could
facilitate surgical removal procedures.
Figure 4
In vivo antitumor efficacies. The treatment regimen
was presented as the amount in mg/kg and the weekly dosage frequency.
The amount of the payload 14 deployed for each treatment
was calculated from the percentage of 14 in the total
dose of conjugate or drug used in the corresponding treatment. (A)
Comparisons of anti-MIA PaCa-2 pancreatic cancer activities and body
weight changes between conjugate 36, 40a, and cytotoxic payload 14 (at equivalent doses of 14), when administered intravenously at a time point (twice
per week), are illustrated with red arrows. (B) Comparisons of anti-HCC1806
triple-negative breast cancer activities and body weight changes between
conjugate 40a and cytotoxic payload 14 when
administered intravenously at a time point (twice per week) are illustrated
with red arrows. (C) Treatment and shrinkage of large (450–850
mm3) HCC1806 triple-negative breast cancer tumor with weekly
doses of conjugate 40a at 2.5 mg/kg.
In vivo antitumor efficacies. The treatment regimen
was presented as the amount in mg/kg and the weekly dosage frequency.
The amount of the payload 14 deployed for each treatment
was calculated from the percentage of 14 in the total
dose of conjugate or drug used in the corresponding treatment. (A)
Comparisons of anti-MIA PaCa-2 pancreatic cancer activities and body
weight changes between conjugate 36, 40a, and cytotoxic payload 14 (at equivalent doses of 14), when administered intravenously at a time point (twice
per week), are illustrated with red arrows. (B) Comparisons of anti-HCC1806
triple-negative breast cancer activities and body weight changes between
conjugate 40a and cytotoxic payload 14 when
administered intravenously at a time point (twice per week) are illustrated
with red arrows. (C) Treatment and shrinkage of large (450–850
mm3) HCC1806 triple-negative breast cancer tumor with weekly
doses of conjugate 40a at 2.5 mg/kg.In the second set of the efficacy tests, conjugate 40a was evaluated in an immunocompetent, sorafenib-resistant
hepatocellular
carcinoma (HCC) model[50] with a relevant
expression of tumor-associated profiles in the TME. In this particular
animal model, the bioluminescence reporter and luciferase activity
provided noninvasive monitoring of tumor burden and progression at
the liver (Figure A). Notably, conjugate 40a elicited potent activity
against HCC tumor growth with only 1 mg/kg injected twice weekly for
two weeks (Figure B). Indeed, conjugate 40a-treated livers showed a significant
decrease in tumor burden examined by bioluminescence (Figure B), resulting in the marked
reduction of total liver weight at 14 days after treatment (Figure C,D). In addition,
the reduction of Ki67 expression in conjugate 40a-treated
tumors was observed (Figure E), suggesting that the treatment with 40a could
significantly diminish the proliferation of liver cancer cells and
inhibit tumor growth. Overall, we have showed that conjugate 40a not only exerted potent antipancreatic cancer and anti-triple-negative
breast cancer activities but also significantly decreased HCC tumor
burden in a sorafenib-resistant model.
Figure 5
In vivo anti-liver cancer efficacies of conjugate 40a. (A)
Experimental scheme for HCC induction and treatment
regimen. (B) Bioluminescence detection between mice treated with the
vehicle and conjugate 40a via intravenous administration
at 1 mg/kg with the indicated regimen. (C) Representative images and
(D) total liver weights of the vehicle (day 14)- or conjugate 40a-treated mice livers harvested at the indicated time post
drug administration. (E) Ki-67 staining of vehicle- or conjugate 40a-treated tumor tissues at the indicated time point. A significant
reduction of Ki-67 staining (yellow arrows) in the 40a-treated tumor was observed on a scale of 100 or 20 μm.
In vivo anti-liver cancer efficacies of conjugate 40a. (A)
Experimental scheme for HCC induction and treatment
regimen. (B) Bioluminescence detection between mice treated with the
vehicle and conjugate 40a via intravenous administration
at 1 mg/kg with the indicated regimen. (C) Representative images and
(D) total liver weights of the vehicle (day 14)- or conjugate 40a-treated mice livers harvested at the indicated time post
drug administration. (E) Ki-67 staining of vehicle- or conjugate 40a-treated tumor tissues at the indicated time point. A significant
reduction of Ki-67 staining (yellow arrows) in the 40a-treated tumor was observed on a scale of 100 or 20 μm.
Profiling of Immunogenic TME and Gene Expression Induced by
Conjugate 40a
Limited tumor-infiltrating immune
cells in immunosuppressive TME can modulate the treatment’s
outcome. Many solid tumors are characterized as “cold tumors”
with low proinflammatory cytokines and T-cell infiltration.[51] On the other hand, “hot tumors”
might potentiate clinical response rates of (PD-L)1/PD-1 immunotherapy
and therapeutic strategies that can sensitize cold tumors into hot
tumors have been investigated.[52,53] To address the influence
of conjugate 40a treatment on the immune milieu of the
HCC TME, we first analyzed conjugate 40a-treated tumors
by immunohistochemical (IHC) staining and found increased infiltration
of the Gr-1+ monocytes and polymorphonuclear granulocytes
and F4/80+ macrophages that formed inflammatory foci in
the tumor modules (Figure A). Moreover, on days 3, 7, and 14, a significant increase
of cytotoxic CD8+ T-cell infiltration was readily located
in the 40a-treated tumor (Figure B). These results showed that treatment of
conjugate 40a increased the permeation of multifaceted
immune cells in the TME, thereby turning the HCC microenvironment
into the ″hot″ status. Next, we profiled the landscape
of immunogenic gene expression induced by conjugate 40a with isolation of the total RNA from vehicle- or conjugate 40a-treated tumor tissues. In particular, absolute copies
of 700 inflammation-related mRNAs were measured. An 18-gene set of
tumor inflammation signature (TIS), associated with antigen presentation,
T cell/NK cell abundance, interferon activity, and T-cell exhaustion,
was validated to be positively correlated to anti-PD-1 blockade responsiveness
in a clinical setting.[54] Significant increases
in gene expression of stimulatory factors for inflamed tumors,[53] such as tumor-cell-derived chemokine CC ligand
5 (CCL5) and chemokine (C-X-C motif) ligand 10 (CXCL10), were identified
with the treatment of conjugate 40a (Figure A). Dendritic cells (DCs) were
shown to produce CXCL10 to recruit CXCR3-expressing CD8+ T cells to tumors, while CCL5 could provide homing signals for circulating
T cells to infiltrate the tumor.[55] In addition,
as a critical regulator for inflammatory TME and T cells’ cytotoxicity,
gene expression of signal transducers and activators of transcription
1 (STAT1) was significantly elevated in the 40a-treated
tumor. Gene expression of an initiator of inflammation and chemoattractant
CCL2 was significantly elevated in the conjugate 40a-treated
tumors (Figure A).
As the efficacy of immunotherapy correlates with the infiltration
of cytotoxic T cells, recruitment of circulating T cells and regulated
stimulations by CCL2 allowed in situ activation in
the TME. This quantitative comparative gene set analysis has shown
striking elevations of T-cell functions, macrophage functions, NK
cell functions, chemokine and receptor functions, and the inflammation
score in conjugate 40a-treated tumor tissues (Figure B). A previous study
has demonstrated that the maytansine-bearing antibody-drug conjugate
induced immunogenic cell death of tumor cells, apoptosis, necrosis,
and triggered the release or expression of danger-associated molecular
patterns (DAMPs).[56] These DAMPs were shown
to activate innate immune cells effectively, trigger the release of
cytokine and chemokines by the innate immune cells, and therefore
change the immune milieu of the tumor microenvironment.[57,58] We therefore postulated that conjugate 40a could induce
cell death of HCC cells in vivo and triggered the
release of DAMPs, which subsequently activated local inflammation,
including the recruitment and activation of macrophages and NK cells.
The primary activation of innate immune cells by DAMPs further triggered
the release of cytokines and chemokines, which increased the second
wave of accumulation of immune cells, including CD8+ T
cells in the tumor microenvironment. This finding provided insight
into 40a treatment in potentiating a “cold”
TME to an immune-inflamed “hot” tumor state and expanding
the combination treatment scope with other immunotherapeutics.
Figure 6
(A) Liver sections
of vehicle- or conjugate 40a-treated
tissues with hematoxylin-and-eosin (H&E) staining. In addition,
F4/80 and Gr-1 immunohistochemical staining. T: tumor region, L: adjacent
liver tissue. (B) Immunohistochemical analysis (yellow arrows) of
CD8α positive cells in vehicle- or conjugate 40a-treated tissues. Scale bars: 100 or 20 μm.
Figure 7
(A) Quantitative analysis of conjugate 40a-induced
immunogenic gene expression in the TME. (B) Enhancement of T cell,
macrophage, NK cell, chemokine & receptor, interferon, and inflammation
functions in the TME by conjugate 40a, n = 3.
(A) Liver sections
of vehicle- or conjugate 40a-treated
tissues with hematoxylin-and-eosin (H&E) staining. In addition,
F4/80 and Gr-1 immunohistochemical staining. T: tumor region, L: adjacent
liver tissue. (B) Immunohistochemical analysis (yellow arrows) of
CD8α positive cells in vehicle- or conjugate 40a-treated tissues. Scale bars: 100 or 20 μm.(A) Quantitative analysis of conjugate 40a-induced
immunogenic gene expression in the TME. (B) Enhancement of T cell,
macrophage, NK cell, chemokine & receptor, interferon, and inflammation
functions in the TME by conjugate 40a, n = 3.
conclusions
The SMDC is an emerging modality for the
selective delivery of
drug payloads. Digital and experimental analyses with “-omics”
platforms generated a myriad of new disease-specific or -associated
antigens. Targeted delivery via the chemically defined SMDC is largely
underexplored as limited small organic ligands have been studied in vivo systemically when linking to conventional chemotherapeutics.
This work demonstrated the design and evaluation of a pharmacokinetically
optimizable and chemically defined SMDC that targets an immune checkpoint
antigen in the TME. By employing ultratoxic maytansinoid payloads
and leveraging an in situ amplification of the homing
signal effect, conjugate 40a effectively shrank the growth
of many solid tumors. Moreover, CD8+ T cell infiltration significantly
increased in the conjugate 40a-treated tumor mass that
sensitized tumors from the intrinsic immune-suppressive TME. A quantitative
study on tumor inflammation-related mRNA expression revealed inductions
of key gene expressions, such as STAT1, CXCL10, CCL5, and CCL2, and
rejuvenation of TME with enhancement in T cell, macrophage, NK cell,
chemokine, and cytokine functions. The current study thus established
an immune checkpoint targeting conjugate enabling penetration of multifaceted
immune cells into the tumor mass and potentiating new therapeutic
strategies combined with immune checkpoint blockade treatment. In
the current study, the synergy between the complementarity of a targeting
moiety, the longevity of the conjugate with stable linkers, and the
drug pharmacology is not only essential to developing effective ligand-targeted
cancer therapeutics; it also offers important features for further
development of a “theranostic” targeting phosphatidylserine
immune checkpoint. Moreover, since revamping the TME immunity by turning
it into a “hot tumor” leads to the liberation of antigens
that are not initially accessible, this work can be expanded for combination
therapy with existing treatment options.
Experimental Section
Synthesis: General
All materials used were commercially
obtained and used as supplied unless otherwise noted. Reactions were
performed under argon or nitrogen and monitored by analytical thin
layer chromatography with glass-backed plates (5 × 10 cm) precoated
with 60 F254 silica gel (supplied by Merck & Co., Inc., Whitehouse
Station in Readington Township, NJ). Flame-dried glassware was cooled
and used for reactions requiring anhydrous conditions under an argon
or nitrogen atmosphere. The resulting chromatograms were visualized
by an ultraviolet lamp (λ = 254 nm) followed by dipping in an
ethanol solution of vanillin (5% w/v) containing sulfuric acid (3%
v/v) or phosphomolybdic acid (2.5% w/v) after charring with a heat
gun. Solvents used for reactions, including THF, diethyl ether (ether),
DMF, toluene, dichloromethane, and pyridine, were dried and distilled
under an argon or nitrogen atmosphere before use. Using silica gel
60 of 230–400 mesh size supplied by Merck with eluent systems
given in volume/volume ratios, flash chromatography was routinely
used to separate and purify product mixtures. 1H and 13C NMR spectra were collected from a Varian Mercury-300 (300
MHz), a Varian Mercury-400 (400 MHz), a Bruker Avance Neo AV4400,
an AV4600, and a DMX-600 (600 MHz), with reporting of chemical shift
values in ppm relative to the TMS in delta (δ) units. Multiplicities
were denoted as s (singlet), br s (broad singlet), d (doublet), t
(triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets),
and m (multiplet). Coupling constants (J) were reported
in Hertz. With an Agilent 1100 MSD mass spectrometer, electrospray
mass spectra (ESMS) were recorded as m/z values,
and for obtaining HRMS, a Bruker (Impact HD) Autoflex Max TOF/TOF
(MALDI) was used. All test compounds with >95% purity were determined
by an Agilent 1100 series HPLC system using a C18 column (Thermo Golden,
4.6 mm × 250 mm) with detailed conditions described in the Supporting Information. IUPAC nomenclature of
compounds was determined with ACD/Name Pro software.
General Procedure: Formation of Conjugates with Incubation of
Zn(NO3)2
To a stirred solution of conjugate
precursors (1 equiv.) in CH2Cl2 was added Zn(NO3)2 (2 equiv.) in MeOH at room temperature. The
mixture was sonicated for 5 min, and then the mixture was concentrated
under reduced pressure to furnish the eventual conjugates, which were
HPLC-assayed to confirm the purity of >95% for animal studies.
To a solution of compound 24 (2.00 g, 3.403 mmol) in MeOH (34 mL) at room temperature, biphenyl-4-carboxaldehyde
(1.24 g, 6.806 mmol) was added. The reaction solution was slowly warmed
to 70 °C and stirred for 20 h. The solution was cooled down to
0 °C, and then sodium borohydride (0.52 g, 13.611 mmol) was slowly
added. The reaction was slowly warmed to room temperature and stirred
for 4 h, and then saturated NH4Cl(aq) was poured
into the reaction mixture. After MeOH was removed, the residue was
extracted with CH2Cl2 (50 mL × 2). The
combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by
flash chromatography with 5% MeOH in CH2Cl2 to
yield the compound 25c (1.78 g, 69%). 1H NMR
(600 MHz, CDCl3) δ 8.51–8.49 (m, 4H, CH-Py./DPA),
7.62–7.54 (m, 13H), 7.44–7.41 (m, 2H), 7.41–7.38
(m, 2H), 7.35–7.31 (m, 1H), 7.13–7.10 (m, 4H, CH-Py./DPA),
7.06 (t, J = 1.5 Hz, 1H), 6.86 (d, J = 1.4 Hz, 2H), 3.97 (t, J = 6.4 Hz, 2H), 3.85 (s,
2H), 3.80 (s, 8H, CH2-αPh./DPA), 3.65 (s, 4H, CH2-αPh./DPA), 2.76–2.72 (m, 2H), 1.87–1.83
(m, 2H), 1.75–1.69 (m, 2H). 13C NMR (151 MHz, CDCl3) δ 159.9, 159.3, 149.1 (CH), 141.1, 140.7, 140.0, 139.6,
136.5 (CH), 128.9 (CH), 128.7 (CH), 127.2 (CH), 127.1 (CH), 122.8
(CH), 122.0 (CH), 121.5 (CH), 113.6 (CH), 67.8 (CH2), 60.2
(CH2), 58.7 (CH2), 53.8 (CH2), 49.3
(CH2), 27.3 (CH2), 26.9 (CH2). HRMS
(ESI): calc. for C49H51N7NaO+: 776.4047, found: 776.4062.
MIA PaCa2
cells or HCC1806 cells were grown in RPMI 1640 medium, and Detroit
551 cells were grown in Dulbecco’s modified Eagle’s
medium. Growth media of Detroit551 were supplemented with the following:
10% fetal bovine serum, 50 U/mL of streptomycin and penicillin, and
1% nonessential amino acids. The MTS assay was performed to examine
cell viability. With cells (2500–3000 cells/well) in flat-bottom
96-well plates for 24 h growth, to the medium was then added the serially
diluted compound and the cells were further incubated for 72 h. At
the end of the 72 h incubation period, media were removed and a 100
μL mixture solution including MTS and PMS was added. Incubation
of the cells for 1.5 h at 37 °C in a humidified incubator with
5% CO2 was carried out to convert the tetrazolium salt
into formazan by the viable cells. The conversion to formazan was
measured by absorbance (490 nm) using a BioTek PowerWave-X Absorbance
microplate reader. The collected data were normalized using DMSO-treated
controls (100% viability) and background controls (0% viability) to
verify growth inhibition, while the IC50 value was calculated
as the amount of compound that resulted in a 50% reduction in cell
viability in comparison with DMSO-treated controls using GraphPad
Prism version 4 software (San Diego, CA, USA).
Biacore SPR binding assay
Phospholipids, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-[phospho-l-serine] (DOPS) were obtained
from Avanti Polar Lipids (Alabaster, AL) in chloroform solutions.
These stock solutions were combined to the indicated ratios. To a
round-bottom flask, a 0.4 mL aliquot of lipid solution at concentration
of 10 mg/mL was added and evaporated under a N2 gas stream
to furnish a thin lipid film. Rehydration of lipid films was carried
out in PBS buffer for at least 1 h at room temperature. Resulting
suspensions were extruded through a 100 nm polycarbonate filter using
an Avanti MiniExtruder following the manufacturer’s instructions.
Zeta potential (ZP) and liposomes’ size distributions were
recorded by dynamic light scattering (DLS) and microelectrophoresis
using a Zetasizer Nano ZS instrument. Then, using a Biacore T200 biosensor
equipped with an L1 sensor chip (GE Healthcare), binding kinetics
between the conjugates and liposome were recorded at 25 °C. Preconditioning
of new sensor chips was performed with running buffer (5% DMSO in
phosphate-buffered saline (PBS) with final pH 7.4) and two consecutive
30 s pulses of 2:3 v/v 50 mM HCl/isopropanol at a flow rate of 30
μL/min. A fresh liposome capture plate was prepared for each
binding cycle. In PBS buffer, liposomes were diluted to 0.5–1
mM and captured to saturation (30–150 s) across isolated flow
cells at 2–5 μL/min. In a single injection, conjugates
were first diluted with running buffer and injected over lipid surfaces.
At flow rate of 30 μL/min, association and dissociation phases
were examined for 60s. At the end of each binding cycle, the surface
was regenerated by injecting 2:3 v/v 50 mM HCl/isopropanol and equilibrated
with running buffer before the next injection of the test compound.
By subtracting SPR signals from a reference flow cell (DOPC immobilized
surface), unspecific binding was removed. Using the bivalent analyte
model, sensograms were fit globally with BIAcore T200 evaluation software
3.0.
Pharmacokinetic Studies of Conjugates
Six-week-old
male ICR mice, from the Biolasco Taiwan, were divided into groups
of three and dosed at 5 mg/kg intravenously. Blood samples were drawn
from each animal at time points of 0.003, 0.083, 0.25, 0.5, 1, 2,
4, 6, 8, and 24 h and stored on ice (0–4 oC). With
centrifugation (3000 rpm for 15 min at 4 °C in a Beckman Model
Allegra 6R centrifuge), plasma was separated from the blood and stored
in frozen conditions (−20 °C). In addition, mice bearing
HCC1806 tumor were i.v. administered with cytotoxic payload 14 of 0.6 mg/kg and conjugate 40a of 2 mg/kg
(in 10% DMA/20% Cremophor EL/70% (5% dextrose)) when the mean tumor
volume was approximately at the range of 500–900 mm3. The mice were sacrificed, and the blood samples of 0.5 mL each
and tumor samples were collected at 0.5, 2, 6, 24, 72, and 168 h after
administration. Each time point group included 4 mice. Mouse blood
samples were collected in EDTA tubes and centrifuged at 13,000 rpm
for 5 min at 4°C for plasma collection. Plasma and the harvested
tumor samples were stored at −80°C until use. Fifty microliters
of mouse plasma or the sample of tumor homogenated in ddH2O with dilution ratio of 1:3 (w/v) by MiniBeadbeater-16 (BioSpec
Products Inc., OK, USA) was mixed with 100 μL of acetonitrile
containing 250 ng/mL BPR0L187. The mixture was vortexed for 30 s and
then centrifuged at 15000g for 20 min. The supernatant
was transferred to a clean tube, and 15 μL of the supernatant
was injected onto LC/MS/MS. Plasma samples were analyzed by liquid
chromatography tandem mass spectrometry (LC/MS/MS). The chromatographic
system Agilent 1200 series LC system and an Agilent ZORBAX Eclipse
XDB-C8 column (5 μm, 3.0 × 150 mm) interfaced
to an MDS Sciex API4000 tandem mass spectrometer equipped with an
ESI in the positive scanning mode at 600 °C was used. Data acquisition
was collected via multiple reactions monitoring (MRM). A gradient
system was employed for the separation of analyte and IS. Mobile phase
A was 10 mM ammonium acetate aqueous solution containing 0.1% formic
acid. Mobile phase B was acetonitrile. The gradient profile was as
follows: 0.0–1.1 min, 50% B; 1.2–3.7 min, 55%B–90%B;
3.8–5.0 min, 90%B–50%B. The flow rate was 1.5 mL/min.
The autosampler was programmed to inject 15 μL of the sample
every 5 min.
IVIS Imaging of HCC1806 Tumor with Zn11-794
HCC1806
tumor-bearing mice were used when the mean tumor volume reached around
500–700 mm3. Tumor volume in mm3 was
calculated by the following formula: volume = (length × width2)/2 and measured with a digital caliper. Untargeted Dye 794
or Zn11-794 (in 10% DMA/20% Cremophor EL/70% (5% dextrose)) was i.v.
administered at 2 mg/kg. All treated mice were imaged by using an
IVIS spectrum system at 24, 48, and 72 h. Briefly, the mice were anesthetized
by 2.5% isoflurane inhalation and placed on the stage of IVIS apparatus
with imaging conditions set as follows: excitation filter, 745 nm;
emission filter, 820 nm; exposure time, auto; bin, 8 (medium); f/stop,
2; field of view, 22.7 cm. Using Living Image 4.5 software (PerkinElmer,
Alameda, CA, USA), fluorescence intensity was quantified and the image
was processed.
Animal Studies
Cancer cells, suspending in phenol red
free medium/DPBS, were mixed with Matrigel (356237, BD Biosciences,
San Jose CA, USA) in a 1:1 ratio. Human pancreatic cancer MIA PaCa-2
(1 × 106 cells) or triple-negative breast cancer HCC1806
(1 × 106cells) cells were subcutaneously inoculated
to left flanks of male nude mice 6 weeks old (Biolasco, Taiwan) or
female nude mice 6 weeks old by using a 1 mL syringe (needle 24G ×
1 in., 0.55 × 25 mm; TERUMO). Tumor dimensions were measured
twice a week with an electronic caliper (FOW54-200-777, PRO-MAX, Newton,
Massachusetts, USA), and the volume of the subcutaneously growing
tumor in mm3 was calculated by the following formula: volume
= (length × width[2])/2. Conjugates
were formulated 10% DMA/20% Cremophor EL/70% injectable solution of
5% dextrose (D5W) for treating MIA PaCa-2 and HCC1806 xenograft tumors.
The MIA PaCa-2 or HCC1806 tumor-bearing mice were grouped, and conjugates
were administered when the mean tumor volume was approximately at
200–250 mm3 or 600–700 mm3 (large
tumor) with dose regimens: conjugate 40a of 1 mg/kg or
2 mg/kg and cytotoxic payload 14 of 0.3 mg/kg at twice
(day 1 and day 4) a week for 2 weeks. For the studies with large tumors,
a weekly dose of conjugate 40a of 2.5 mg/kg was used.
Body weight of the mice and tumor volume were measured twice weekly.In addition, a previously reported oncogene-induced, sorafenib-resistant
HCC mouse model[50] was used in the current
study to examine the antitumor activities of synthesized compounds.
Male C57BL/6j mice 4–5 weeks old were purchased from the National
Laboratory Animal Center (Taipei, Taiwan) and kept in the laboratory
animal center (LAC) of NHRI. The mice received 2 μg of pCMV(CAT)T7-SB100
(Addgene #34879), 10 μg of pT/Caggs-NRASV12 (Addgene #20205),
and 10 μg of pKT2/CLP-AKT-LUC plasmids through hydrodynamic
injection and were monitored for tumor growth weekly using IVIS until
the development of HCC in the liver. The mouse number used in each
experiment was indicated in the figures/legends. The animal study
was reviewed and approved by the NHRI IACUC (Institutional Animal
Care and Use Committee). HCC-bearing mice with the total flux from
IVIS imaging above 1 × 109 photons/s were used for
treatment of conjugates. Indicated conjugate 40a was
dosed intravenously at 1 mg/kg with a frequency of twice (day 1 and
day 4) a week for 2 weeks. Repetitive IVIS imaging was performed to
track tumor progression, and tumor tissues of the treated HCC-bearing
mice were collected at day 3, day 7, and day 14 post conjugate administrations
for histological examination and RNA extraction. The tumor tissues
of vehicle-treated HCC-bearing mice were collected at day 14 post
treatment to serve as control samples.
Repeat-Dose Toxicity Study
Male SD rats 8 weeks old
(n = 5 per group) were i.v. bolus-administered with
control vehicle (2.5 mL/kg) and 1 mg/kg 40a once a week
for 4 weeks (days 1, 8, 15, and 22). The animal body weights were
measured daily during the study period. At the end of the study on
day 29, all the animals were euthanized with 100% CO2 and
sacrificed for organ harvest followed by organ weight measurements
and for blood sample collection followed by hematology and serum chemistry
assays. Hematology and serum chemistry parameters were determined
using a HEMAVET 950 automated analyzer (Drew Scientific, Santa Clara,
CA, USA) and a FUJI DRI-CHEM NX500 automated analyzer (FUJIFILM, Tokyo,
Japan), respectively.
Immunohistochemical Staining
Paraffin-embedded liver/tumor
tissue sections were deparaffinized, rehydrated, underwent heat-induced
antigen retrieval, and then incubated with primary Abs. The primary
Abs types used for detection of Ki-67, F4/80, Gr-1, and CD8alpha were
SP6 (Abcam), BM8 (Biolegend), RB6F8C5 (Biolegend), and D4W2Z (Cell
Signaling Technology), respectively. ImmPRESS anti-rat Ig, ImmPRESS
anti-rabbit Ig, polymer detection kits, DAB peroxidase substrate kit,
(Vector laboratories) liquid permanent red substrate (Dako), and Hematoxylin
Gill II (Leica) were used for detection and visualization. The images
were captured using an automatic digital slide scanner Pannoramic
MIDI with a Plan-Apochromat 20×/0.8 objective (3D HISTECH) by
the Pathology Core Laboratory of NHRI.
Nanostring Analysis
Total RNA was extracted from conjugate 40a-treated or vehicle-treated tumor tissues of HCC-bearing
mice with the RNA-easy kit (QIAGEN). The concentration (absorbance
at 260 nm) and purity (A260/280 and A260/230 ratios) of the extracted
RNA were measured by spectrophotometry, and the integrity of the RNA
was further determined by a 2100 Bioanalyzer system (Agilent Technologies).
The RNA, hybridized with barcoded probes (NanoString Technologies)
provided in the nCounter Mouse PanCancer Immune Profiling panel kit,
was then used for measurement of the mRNA expression of 770 genes
related to immune responses. Nanostring nSolver 4.0 and nCounter advanced
analysis 2.0 software (NanoString Technologies) were used for data
processing, immune cell profiling, and pathway scoring according to
developer’s instructions.
Ethical Approval
Animals used in this study were maintained
and treated according to the animal protocols (NHRI-IACUC-106076-A,
NHRI-IACUC-107045-A, NHRI-IACUC-107130-A, NHRI-IACUC-109063, and NHRI-IACUC-108091-A)
that were approved by NHRI IACUC.
Statistical Analysis
GraphPad Prism 7 (GraphPad Software,
La Jolla, USA) and Student’s t test were used
for statistical analysis.
Authors: Vivek Verma; Rajeev K Shrimali; Shamim Ahmad; Winjie Dai; Hua Wang; Sumin Lu; Rahul Nandre; Pankaj Gaur; Jose Lopez; Moshe Sade-Feldman; Keren Yizhak; Stacey L Bjorgaard; Keith T Flaherty; Jennifer A Wargo; Genevieve M Boland; Ryan J Sullivan; Gad Getz; Scott A Hammond; Ming Tan; Jingjing Qi; Phillip Wong; Taha Merghoub; Jedd Wolchok; Nir Hacohen; John E Janik; Mikayel Mkrtichyan; Seema Gupta; Samir N Khleif Journal: Nat Immunol Date: 2019-07-29 Impact factor: 25.606
Authors: Olga Schweigert; Christin Dewitz; Katja Möller-Hackbarth; Ahmad Trad; Christoph Garbers; Stefan Rose-John; Jürgen Scheller Journal: Biochim Biophys Acta Date: 2013-11-25
Authors: Adam J Plaunt; Kara M Harmatys; William R Wolter; Mark A Suckow; Bradley D Smith Journal: Bioconjug Chem Date: 2014-03-13 Impact factor: 4.774
Figure 1
Scheme 1
Scheme 2
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7