2. Contents
4 Cancer Research: Current Limitations and Possible Solutions
New tools and tech help address the limitations of current methodologies and treatments.
6 Custom Morphology Marker Development for Enhanced ROI
Selection with NanoString® GeoMx® Digital Spatial Profiler
Novel tech enables users to select biologically relevant ROIs based on tissue-specific
morphology markers.
11 Rapid Evaluation of PBMC Counting and Viability
Study demonstrates use of an automated cell counter to count PBMCs isolated from whole
blood samples.
16 How Can Stem Cells Help with Cancer Treatment?
Regenerating cells after therapy, targeting cancer pathways, generating immune cells, and
being therapeutic carriers are among uses.
22 Antibody Drug Development: Navigating Immune Response and
Multivariate Logistics on the Targeted Treatment Frontier
Studying these revolutionary drugs requires a comprehensive and precise approach to
understand the multivariate dynamics.
27 GPCRs: From Signal Pathways to Targeted Drug Development
How to break through bottlenecks to obtain full-length GPCRs as antigens for drug development.
34 Blockade of PD-L1, an Immune Checkpoint Molecule, in a Murine
Model of Melanoma
Study describes the cellular response to PD-L1 blockade using monoclonal antibodies in
melanoma models.
2
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395 Oyster Point Blvd., Suite 300
South San Francisco, CA 94080
3
4. 4
Cancer Research: Current
Limitations and Possible Solutions
New tools and tech help address the limitations of current
methodologies and treatments.
A major drawback of cancer treatments like radio-
therapy,chemotherapy,andimmunotherapyisthat
they can kill healthy cells along with the cancerous
cells. Across all treatment modalities, it has consis-
tently proven challenging to target cancerous cells
while not negatively affecting healthy ones. Much
work is being done to resolve these issues as well
as others that impact patient outcomes including
tumor recurrence and therapy resistance. Even be-
fore there is treatment, though, there need to be
robust ways to detect cancer, and current detec-
tion methods have their challenges.
Innovative cancer research is being conducted at
academic and medical institutions, as well as at
biopharmaceutical companies and in the life sci-
ence tool provider community. Within these pro-
grams, researchers and clinicians are developing
novel ways to detect, prevent, and treat cancer.The
chapters in this eBook will showcase some of the
new ideas, techniques, and tools that are helping
to advance cancer research by overcoming some of
the limitations of current methodologies.
Analytical techniques that allow for simultaneous
high-plex and spatial analysis of biological sam-
ples are in high demand as such techniques would
help in better identifying cancer. While immuno-
histochemistry provides spatial analysis, only one
biomarker at a time can be stained. Techniques
such as RNA-Seq provide high-plex gene expres-
sion analysis but do not provide spatial analysis.
A new solution is the NanoString® GeoMx® Digital
Spatial Profiler (DSP), which provides high-plex
and spatial analysis, tagging protein or RNA targets
with fluorescently labeled antibodies. The anti-
bodies act as morphology markers, with the mark-
ers being used to determine regions of interest to
analyze. While this development isexciting, a cur-
rent challenge is the lack of available morphology
markers. To this end, Canopy Biosciences® is devel-
oping a set of qualified and validated morphology
markers to address several cancer types, starting
with markers for T cells and macrophages.
The use of stem cells to treat cancer is also an area
of great interest. Sino Biological offers several
products to pair with various stem cell types, such
as hematopoietic stem cells, cancer stem cells, and
embryonic stem cells, to either treat or identify can-
cers. Radiotherapy or chemotherapy oftentimes
affects both cancerous and healthy cells, with the
latter undergoing slow cell growth or even cell
death. Hematopoietic stem cells may be combined
with colony-stimulating factors for infusion into
leukemia patients so that the hematopoietic stem
cells differentiate into new blood cells, replacing
5. 5
healthy cells that were killed due to radiotherapy
or chemotherapy.
Surface markers such as CD105, HER2/ERBB2,
CD166, CD44, Lgr5, and EpCAM may be used to
identify cancer stem cells in tumors, with such
identification being helpful in developing strate-
gies for treating tumors. Incubation of embryonic
stem cells with NK cell or T cell-initiating cytokines,
such as stem cell factor, IL-3, IL-7, IL-15, and FMS-
like tyrosine kinase receptor-3 ligand, leads to dif-
ferentiation of immune cells.
Stem cells are not the only cell types of inter-
est in cancer research. Leukocytes are also of in-
terest since cancer often affects their numbers.
A challenge with obtaining accurate leukocyte
counts is the existence of anucleated red blood
cells and platelets in whole blood samples. Logos
Biosystems’LUNA-FX7™ Automated Cell Counter is
being used to address this challenge. The counter
may be used to identify fluorescently stained leu-
kocytes in whole blood samples and, if peripheral
blood mononuclear cells are isolated from whole
blood, the counter may identify such cells while
determining their concentration and viability. Cell
counting is accurate to an R-square value of at least
0.98, and cell counting is possible even with whole
blood dilutions as low as 1:640.
Raybiotech’s focus is on developing immunoassays
for drug antibodies and protein-based drugs that
target malignant cells. Drug antibodies, accounting
for one-fifth of all new drug approvals, may target
specific malignant tumors, be massed produced,
and operate via several pathways, including block-
ing pathways, flagging cells for cell death, and
delivering drugs via endocytosis. Protein-based
drugs typically operate by interacting directly with
the cancerous cell, recruiting other molecules to
the cancerous cell, or improving the function of
adjacent cells. Raybiotech has immunoassays for,
among other purposes, drug antibody detection,
drug response detection, and drug antibody bind-
ing site detection.
ACROBiosystems is developing G protein coupled
receptors (GPCRs) for antibody-based drug thera-
pies. GPCRs, which aid in regulating physiological
processes, are transmembrane proteins that have
been linked to a number of diseases, including
cancer. GPCRs are a highly studied drug target,
accounting for 27% of the global market share of
therapeutic drugs. In addition, antibodies that tar-
get GPCRs have decided advantages over small
molecule drugs, some of which have slower clear-
ance rate in the body, longer action time, and less
frequent administration. The company currently
has several full-length GPCRs, either launched or in
development, for use with conditions such as lym-
phoma (CCR4), pancreatic cancer (CXCR4), colorec-
tal cancer (EGFR, LGR5) and myeloma (GPRC5D).
BioLegend has done research to show the effect of
blocking PD-L1 ligand on melanoma. PD-L1 is the
ligand of the PD-1 receptor, an immune checkpoint
molecule often found on tumor cells. As immune
checkpoint molecules limit immune response,
their expression on tumor cells causes suppression
of immune response. PD-L1 binding ultimately
causes T cell death. Blocking the PD-L1 ligand from
binding to the PD-1 receptor with an anti-PD-L1
antibody (GoInVivo™ anti-PD-L1 antibody clone)
resulted in activation of T cells, as shown by ele-
vated expression of common T cell markers like
CD25 and CD69. Using BioLegend’s LEGENDplex™
multiplex platform, blocking PD-L1 binding also re-
sulted in increased production of Th cytokines and
chemokines with a simultaneous limiting of pro-in-
flammatory cytokine production. There was also
a reduction in tumor growth with PD-L1 binding
blockage, as well as increased levels of CD8+
T cells.
6. 6
Custom Morphology Marker
Development for Enhanced ROI
Selection with NanoString®
GeoMx® Digital Spatial Profiler
Novel tech enables users to select biologically relevant ROIs based
on tissue-specific morphology markers.
Introduction
Molecular and cellular profiling of the tissue mi-
croenvironment is critical to understanding the
heterogeneity in biological samples and requires
technology capable of providing information
about the spatial context of cells. Yet, researchers
experience barriers when selecting the right tech-
nique—one that allows for both high-plex and
spatial analysis simultaneously.
Immunohistochemistry (IHC) is an important
method in basic clinical work for the exploration of
biomarkers, as it allows for confirmation of target
protein expression in the context of the microen-
vironment. Although IHC offers spatial context, it is
limited to staining or a single biomarker at a time.
Bulk molecular profiling techniques such as RNA-
Seq sacrifice spatial context but enable high-plex
gene expression analysis.
NanoString® GeoMx® Digital Spatial Profiler (DSP)
offers a solution to these issues and is capable of
high-plex spatial resolution of protein or RNA tar-
gets using fluorescently labeled antibodies as mor-
phology markers to guide the selection of regions
Figure 1. NanoString employs fluorescently labeled an-
tibodies as morphology markers to guide the selection
of ROIs. Tonsil tissue stained with DNA dye (blue), Pan-
Ck(green),andCD45(red)markers.
7.
8. 8
of interest (ROI). GeoMx DSP builds on the tenets
of traditional histology and enables users to select
biologically relevant ROIs based on tissue-specific
morphology markers.
Morphology Markers Enable
Biologically Relevant ROI Selection
Complex transcriptional and proteomic pathways
are responsible for creating highly organized tissue
structures, while a major hallmark of disease is the
breakdown of this organization. Therefore, identify-
ing specific structures is a critical first step in making
meaningfulanalyses.GeoMxDSPemploysmorphol-
ogy markers to select and separate ROIs into mean-
ingful groups to compare RNA profiles. Morphology
markers are fluorescently labeled antibodies target-
ing proteins that inform the presence of biological
structures, cell types, and compartments. Through
conscious selection of biologically relevant ROIs,
powerful transcriptome analyses can be performed.
However, the increased adoption of GeoMx DSP
has revealed a severe limitation to this approach—
namely, the lack of morphology markers available.
Although the Whole Transcriptome Atlas (WTA)
panel profiles the expression of >18,000 RNA tar-
gets, researchers are limited to off-the-shelf kits
targeting a select few morphology markers that
distinguish epithelial from immune cells. The Solid
Tumor TME morphology kit, for example, contains
a DNA stain and two markers, Pan-Ck and CD45,
that are commonly used to profile tumor and im-
mune compartments. Many researchers have ex-
pressed the desire for more targeted morphology
resolution—specific to additional cell types and
novel biomarkers.
To address this limitation, Canopy Biosciences® is
developing a portfolio of qualified and validated
morphology markers, focusing on critical targets
that will enable researchers to select ROIs with
greater precision. The catalog will be expanded
to cover morphology markers for a variety of can-
cer types and the most relevant markers in immu-
no-oncology. Further development will include
expansion into neuroscience applications. New
markers will be available to augment the current
off-the-shelf kits or create fully custom sets based
on study goals.
Morphology Marker Qualification
Marker development involves sourcing commer-
cially available antibodies—labeled with specific
fluorescent tags—and evaluating their suitabil-
ity for staining tissues with conditions compat-
ible with subsequent transcriptomic analysis.
Qualifying custom markers for GeoMx DSP is crit-
ical since the processing protocol for GeoMx RNA
assays includes a significant Proteinase K treatment
that can reduce the presence of specific epitopes
of certain antibody clones.
For this catalog, markers were tested following
the “qualified” and “verified” approaches in the
“Morphology Marker Guidelines” whitepaper pre-
sented by NanoString. Qualified markers demon-
strate expected staining patterns typical in a single
tissue,whileverifiedmarkershaveundergonemore
extensive testing on multiple tissues and were suc-
cessfully used in ROI segmentation. Our initial test-
ing of immuno-oncology antibodies resulted in a
mix of both qualified and verified markers.
Furthermore, target-specific positive tissue stain-
ing was verified by an experienced pathologist.
Antibody dilution was optimized to assess specific-
ity and reduce background fluorescence. Canopy
Biosciences uses a decade’s worth of expertise in
immunohistochemistry assay development and
9. 9
histopathology knowledge in the development of
this catalog to validate markers that will augment
the off-the-shelf kits currently available.
Selecting Fluorescently
Labeled Antibodies
GeoMx DSP uses standard filter sets for fluores-
cence imaging, commonly referred to as the FITC,
Cy3, Texas Red, and Cy5 channels. Antibodies are
conjugated to a fluorophore compatible with one
of these filter sets to enable visualization of the tis-
sue structures based on target specificity. However,
off-the-shelf marker kits make use of only three
channels for the DNA stain (FITC) and two morphol-
ogy markers (Cy3 and Texas Red), leaving the Cy5
channel open for custom marker development.
Immuno-oncology Markers Are the
First Added
The initial priority of the project was the addition
of a set of qualified immuno-oncology markers,
including markers for T cells (CD3) and macro-
phages (CD68), among others. These were some of
Figure 2. Wavelengths of GeoMx DSP fluorescence imaging channels for morphology marker conjugation. (Source: ThermoFisher
FluorescenceSpectraViewer)
Figure3.SegmentationofCD68andCD3inbreastcan-
cer samples. Analysis of Cancer Transcriptome Atlas
data shows enrichment of macrophages in CD68 seg-
ments(leftpanel),TcellsintheCD3segment(rightpan-
el), and lack of both cell types in the Pan-CK segment
(centerpanel).
10. 10
the first markers requested by researchers for cus-
tom addition to the Solid Tumor TME Morphology
Kitandarevalidatedintonsil,breastcancer,andcol-
orectal cancer tissues by Canopy Biosciences. Both
CD3 and CD68 differentiate alternate immune cell
phenotypes, enabling more accurate ROI selection
than is possible with a single tumor and immune
cell marker. Clients can now determine whether
gene expression of a signaling pathway correlates
with T cell or macrophage-rich tissue regions. The
pan-leukocyte marker (CD45) in the Solid Tumor
TME kit cannot distinguish between immune cell
subtypes, so custom marker development is nec-
essary for further cellular resolution.
Summary
Molecular and cellular profiling of the tissue micro-
environment is critical to understanding the het-
erogeneity in biological samples and requires tech-
nology capable of providing information about the
spatial context of cells. Here, we demonstrate how
the NanoString® GeoMx® Digital Spatial Profiler
(DSP) enables high-plex spatial resolution of pro-
tein or RNA targets using fluorescently labeled
antibodies as morphology markers, to assist with
ROI-guided analyses. Through accurate selection
of biologically relevant ROIs, transcriptional and
proteomic pathways can be deeply analyzed.
Although several off-the-shelf marker kits exist,
they make use of only three channels, leaving one
channel open for custom marker development.
Canopy Biosciences utilizes this open channel and
expertise in assay development and histopatholo-
gy knowledge to develop custom markers to aug-
ment the currently available kits. New morphology
markers are rigorously validated for precise local-
ization in multiple tissue types, starting with those
most relevant to current immuno-oncology re-
search. Canopy Biosciences’Validated Morphology
Marker Catalog will enable a broader audience of
researchers to take advantage of spatially resolved,
high-plex transcriptomic profiling.
Figure 4. Qualified morphology marker testing of custom markers. A) CD3 morphology marker validation on breast cancer tissue,
DNAstaininblueandCD3inred.B)CD68morphologymarkervalidationinDLBCLtissue,DNAstaininblueandCD68inyellow.
Additional Resource
ROI Selection Markers for GeoMx® Assays
11. 11
Rapid Evaluation of PBMC
Counting and Viability
Study demonstrates use of an automated cell counter to count
PBMCs isolated from whole blood samples.
Introduction
Peripheral Blood Mononuclear Cells (PBMCs) are
a vital source material used in myriad research
applications, including single-cell sequencing to
vaccine development and toxicological studies.
Furthermore, PBMC derivatives such as T cells, B
cells, NK cells, and stem cells are based on cell ther-
apies, including CAR-T cell therapies and regener-
ative medicines. Therefore, accurately measuring
numbers and viability of PBMCs after collection,
isolation, or expansion are essential to making
experimental or manufacturing decisions about
downstream processes. Yet, directly obtaining
leukocyte counts in whole blood using traditional
counting techniques is complicated by the pres-
ence of mature, anucleated RBCs and platelets.
Advantageously, the use of nucleic acid stains like
Acridine Orange and Propidium Iodide (AO/PI) al-
low the nucleated leukocytes to be differentiat-
ed and accurately counted within a whole blood
Figure 1. Diluted whole blood and PBMC-enriched
buffy coat stained using AO/PI fluorescent dye. The mi-
croscopic overlay images of the whole blood stained
with AO/PI (A,C), and PBMCs from enriched buffy
coat stained with AO/PI (B,D) were acquired using the
CELENA® X High Content Imaging System with a 20X
fluorite objective (www.logosbio.com). The yellow ar-
rows indicate nucleated cells in AO/PI positive stained
cells, while the red arrows indicate RBCs. The scale bar
is100µm.
12. 12
sample (Figure 1). Here, we exhibit the ability of
the new dual fluorescent LUNA-FX7™ Automated
Cell Counter to meet diverse cell counting needs
by demonstrating its use in counting leukocytes in
whole blood and the PBMCs isolated from whole
blood samples.
Material and Methods
One milliliter of the human peripheral blood sam-
ple was prepared, and the PBMC sample was ob-
tained by standard density gradient centrifuga-
tion technique using Histopaque-1083 (Sigma,
#10831)1. After final washing, the PBMCs enriched
in the buffy coat were resuspended in 100 µl PBS
or RPMI +10% FBS media. Cell counts were per-
formed in the LUNA-FX7™ with either the 2-channel
PhotonSlide™ (Cat# L12005) or LUNA™ 8-Channel
Slides (Cat# F72001) and used a modified default
protocol in the Fluorescence Cell Counting mode
(Table 1). Before loading the cells, cells were stained
at the standard ratio of the AO/PI reagent (Cat#
F23001); 18 µl of cells + 2 µl of AO/PI, and then 10
µl of the mix was loaded into a slide chamber.
Results
Evaluating the PBMC concentration and
viability with the LUNA-FX7™™
The separated PBMCs were analyzed by counting a
series of 2-fold dilutions using both the 8-channel
and 2-channel slides (Figure 2). The counts using
both slide types showed linearity with an R-square
value of 0.99 or above on the logarithmic scale of
concentrations over the serial dilutions. Not sur-
prisingly, with a greater volume of analysis, the 2-
channel slide showed slightly better consistency
than the 8-channel slide.
The easy counting of leukocytes in
whole blood
The leukocytes in whole blood were visualized by
fluorescence and also accurately enumerated with
the LUNA-FX7™. Among countless mature RBCs,
the fluorescence leukocytes were distinctively
counted from diluted whole blood (Figure 3).
Table1.TheoptimizedparametersettingsforPBMCorleukocytescountingoftheLUNA-FX7™onFluorescenceCellCountingmode
13. Better therapies start with better counts.
The LUNA-FX7
Fully Automated Cell Counter
TM
WBCs in whole blood PBMCs CAR-T cells
Incomparable accuracy and precision. Less than 1% CV.
Assured reliability through unique validation slides and Quality Control mode.
Higher throughput with multi-channel compatible 8-channel slides.
Fast and easy operation due to precise and reliable autofocus.
21 CFR Part 11 compliance through the CountWire™ software system.
Find out more at www.logosbio.com
14. 14
Conclusion
The enumeration of PBMCs was evaluated ade-
quately on the LUNA-FX7™ Automatic Cell Counter
with the 2-channel PhotonSlide™ and LUNA™
8-Channel Slides. With more slide options for
the LUNA-FX7™, LUNA™ 1-Channel Slides (Cat#
L72011) may be applied for a more comprehensive
concentration range by analyzing 47 image fields,
and LUNA™ 3- Channel Slides (Cat# L72021) are
beneficial for preset of triplicate analysis as count-
ing options. Indeed, leukocyte counting is much
more straightforward by loading the diluted blood
after AO/PI fluorescence staining. So, you can use
the LUNA-FX7™ to respect the archived records of
PBMCs along with leukocyte counts without inten-
sive labor.
Figure 2. The linearity of counting in serial dilutions of PBMCs. (A) Tagged (live or dead) fluorescent and brightfield overlay of several
dilutions. (B) Bar graph showing the results of 5 serial dilutions. (C) The logarithmic scale of the counts over the dilutions using both
2-channeland8-channelslidesdowntoconcentrationslessthan4.00E+04cells/ml.Thescalebarrepresents100µm.
15. 15
References
1. S Parasuraman, R Raveendran, and R Kesavan Blood
sample collection in small laboratory animals J
Pharmacol Pharmacother. 2010 Jul-Dec; 1(2): 87–93.
doi: 10.4103/0976-500X.72350
Figure 3. The Leukocyte count of whole blood on Fluorescent Cell Counting mode in the LUNA-FX7™. (A) Tagged leukocyte images of
fluorescent and brightfield overlay in serial dilutions of whole blood samples. (B) Bar graph displaying counting of 5 serial dilutions
from 1:40 to 1:640 of whole blood cells. (C) The linearity of counts appears across concentration on both 2-channel and 8-channel
slides.Thescalebarrepresents100µm.
Additional Resource
Automated Cell Evaluation for Single-Cell
RNA-seq Analysis
16. 16
How Can Stem Cells Help with
Cancer Treatment?
Regeneratingcellsaftertherapy,targetingcancerpathways,generating
immunecells,andbeingtherapeuticcarriersareamonguses.
Cancer is considered to be one of the most chal-
lenging diseases. There are several cancer treat-
ments available, such as surgery, radiotherapy,
chemotherapy, and immunotherapy, but they
often result in suboptimal efficacy, therapy resis-
tance, and tumor recurrence.1,2
Stem cells possess
unique characteristics, including self-renewal and
high-capacity of differentiation. Stem cells have
been widely studied and can be roughly catego-
rized into several groups: Embryonic stem cells
(ESCs), induced pluripotent stem cells (iPSCs), he-
matopoietic stem cells (HSCs), mesenchymal stem
cells (MSCs), neural stem cells (NSCs), and cancer
stem cells (CSCs). Recently, stem cells were shown
to have the potential to improve cancer treatment
by regenerating cells after heavy therapy, targeting
cancer pathways, generating immune cells, and
being therapeutic carriers (Figure 1).3
Stem Cell Transplantation
Hematopoietic stem cells (HSCs), located in bone
marrow, can form all mature blood cells in the body.
High-dose radiotherapy or chemotherapy effects
both cancerous and normal cells, causing slow cell
growth or even cell death.4
HSC transplantation is
now a standard treatment used in multiple myelo-
ma, leukemia, and lymphomas targets after rounds
of therapy to help patients recover. For example, in
leukemia patients, HSCs are infused to help prolif-
erate and differentiate into new blood cells by add-
ing colony-stimulating factors, which activate in-
tracellular signaling pathways in HSCs.5
Until now,
the infusion of HSCs is the only procedure of stem
cells that was approved by the FDA. However, the
occurrence of graft-versus-host-disease (GVHD)
when using allogeneic sources of HSCs remains a
challenge, which is often treated with immunosup-
pressive drugs.6-8
Targeting CSC Pathways for
Cancer Therapy
Cancer stem cells (CSCs) were first identified in
leukemia in 1994. These cells are generated by epi-
genetic mutations in normal stem cells or in pre-
cursor/progenitor cells and found within tumor
tissues. The failure of cancer treatment could be
attributed to the characteristics of CSCs. They can
generate tumors via self-renewal and differentia-
tion into multiple cellular subtypes as normal stem
cells. Although the proportion of CSCs in tumor
tissues is very low, they contribute to multiple tu-
mor malignancies, such as recurrence, metastasis,
heterogeneity, multidrug resistance, and radiation
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18. 18
resistance.9
Surface markers, such as CD166, CD44,
Endoglin/CD105, HER2/ERBB2, Lgr5, and EpCAM,
are often used to identify CSCs from highly het-
erogeneous cell types in tumors.10
The activities
of CSCs are regulated by pluripotent transcription
factors, many intracellular signal pathways, and ex-
tracellular factors, and these factors can be used as
drug targets for cancer treatment. Therefore, tar-
geting CSCs could provide a promise to treat var-
ious types of solid tumors.
Stem Cell Source for Production of
Immune Cells
Chimeric antigen receptor (CAR) T cells and natural
killer (NK) cells have been successfully applied for
anti-cancer immunotherapy. These cells are often
generated from the patient, but the quality and
quantity are hard to control in vivo. Outsourcing to
induced pluripotent stem cells (iPSCs) and embry-
onic stem cells (ESCs) could offer unlimited sources
and enable the expansion of this immunotherapy
to a larger number of patients.11
ESCs are isolated
from embryos, while iPSCs are induced from so-
matic cells in culture by overexpressing Yamanaka
factors: Oct3/4, Sox2, Klf4, c-Myc. Incubation of iP-
SCs and ESCs in growth medium containing NK cell
or T cell-initiating cytokines, for example, stem cell
factor, IL-3, IL-7, IL-15, and FMS-like tyrosine kinase
receptor-3 ligand promotes differentiation of im-
mune cells.
High Bio-activity Recombinant IL-15
Cell proliferation has been measured using MO7e
human megakaryocytic leukemic cells (Figure 2).
Figure1.Strategiesfortheapplicationofstemcelltherapyinthetreatmentofcancer.(Source:https://doi.org/10.3390/cells9030563)
19. 19
Stem Cells as Potential
Therapeutic Carriers
Tumors are considered chronic wound tissue and
their microenvironment, constituted by extracellu-
lar matrix (ECM) and secreted paracrine factors, can
attract the migration of mesenchymal stem cells
(MSCs).12
MSCs are multipotent adult stem cells
present in multiple tissues, including the umbili-
cal cord, bone marrow, and fat tissue. Several im-
mune-related cytokines are chemoattractants for
MSCs to prostate cancer, osteosarcoma, multiple
myeloma, and breast cancer cells, for example,
CXCL16, SDF-1, CCL-25, TNF-α, IL-1β, and IL-6.13
Thus, MSCs can be utilized as potential carriers de-
livering therapeutic agents in treating cancers.
Featured Recombinant
TNF-αα Protein
Sino Biological has developed bioactive recombi-
nant TNF-α cytokines from various species, includ-
ing human, mouse, cynomolgus monkey, rat, fer-
ret, and canine (Figure 3).
Featured Recombinant IL-1ββ Protein
Compared to the competing product, Sino
Biological’s IL-1β protein demonstrates a higher
bioactivity as shown below (Figure 4).
MSCs can release nano-sized exosomes, a type of
extracellular vesicles (EV) that contain various bio-
logical materials, to regulate cell-cell interaction.14
Anti-tumor mRNA, siRNAs, or small molecule drugs
were successfully packed into MSCs and target tu-
mor niche to enhance anti-tumor effects or silence
tumor-related genes.15
MSCs can also be utilized
to carry anti-cancer drugs via nanoparticles (NPs).
Figure2.
Figure3.
20. 20
MSCs-carried PLA NPs successfully targeted brain
tumors, and paclitaxel-loaded NPs inhibited lung
tumor growth in mice.16
Cancer cells could also be selectively killed by
oncolytic viruses (OVs), while the naked OVs are
easy to remove by immune cells. Stem cells, like
neural stem cells (NSCs), could be promising car-
riers to protect and deliver OVs to tumor sites.17
NSCs, originally present in the central nervous
system, can self-renew and generate new neu-
rons and glial cells. A previous study found OVs
carried by human NSCs in combination with
ionizing radiation and temozolomide could en-
hance cytotoxicity to glioma tumor cells in vitro
and increase the survival time of glioblastoma
multiforme (GBM)-bearing mice.18
Both MSCs and NSCs are easier to engineer to ex-
press different genes, which are responsible for the
conversion of a prodrug into cytotoxic metabolites
toward tumor cells.19
This gene therapy is called
“suicide gene therapy”, and two phase I clinical
trials have been completed. One used cytosine
deaminase (CD)-expressing NSCs to convert 5-flu-
orocytosine (5-FC) into tumor-toxic 5-fluorouracil
(5-FU) [NCT01172964, completed]. Another con-
verted ganciclovir from monophosphorylate to
triphosphate form, which is more cytotoxic, by
Herpes simplex virus thymidine kinase (HSV-TK)
expressing MSCs [EudraCT 2012-003741-15, com-
pleted]. However, the anti-tumor efficacy relies on
dose control, the number of stem cells localized
into the tumor microenvironment, and retention in
tumor sites.
In this mini-review, we introduced stem cell-related
cancer treatments. Although stem cell therapy has
achieved positive results, there are still some side
effects to consider, for example, transformation of
normal stem cells into cancer stem cells, the chron-
ic GVHD after allogeneic HSC transplantation, in-
creased immune response, etc. In summary, stem
cell technologies have high potential for tumor
treatments, but they still need further efforts to
overcome the challenges before advancing further.
References
1. Vanneman, M.; Dranoff, G. Combining
immunotherapy and targeted therapies in cancer
treatment. Nat. Rev. Cancer 2012, 12, 237–251.
Figure4.
21. 21
2. Wang X, Zhang H, Chen X. Drug resistance and
combating drug resistance in cancer. Cancer Drug
Resist. 2019;2:141-160.
3. Chu DT, Nguyen TT, Tien NLB, et al. Recent Progress
of Stem Cell Therapy in Cancer Treatment: Molecular
Mechanisms and Potential Applications. Cells.
2020;9(3):563. Published 2020 Feb 28.
4. Baskar R, Dai J, Wenlong N, Yeo R, Yeoh KW. Biological
response of cancer cells to radiation treatment. Front
Mol Biosci. 2014;1:24. Published 2014 Nov 17.
5. Hopman RK, DiPersio JF. Advances in stem cell
mobilization. Blood Rev. 2014;28(1):31-40.
6. Copelan, E.A. Hematopoietic Stem-Cell Transplantation.
N. Engl. J. Med. 2006, 354, 1813–1826.
7. Méndez-Ferrer, S.; Michurina, T.V.; Ferraro, F.; Mazloom,
A.R.; MacArthur, B.; Lira, S.A.; Scadden, D.T.; Ma’Ayan,
A.; Enikolopov, G.N.; Frenette, P.S. Mesenchymal and
hematopoietic stem cells form a unique bone marrow
niche. Nature 2010, 466, 829–834.
8. Lee, R.H.; Oh, J.Y.; Choi, H.; Bazhanov, N. Therapeutic
factors secreted by mesenchymal stromal cells and
tissue repair. J. Cell. Biochem. 2011, 112, 3073–3078.
9. Yu Z, Pestell TG, Lisanti MP, Pestell RG. Cancer stem
cells. Int J Biochem Cell Biol. 2012;44(12):2144-2151
10. Codd, A.S.; Kanaseki, T.; Torigo, T.; Tabi, Z. Cancer stem
cells as targets for immunotherapy. Immunology
2017, 153, 304–314
11. Iriguchi, S.; Kaneko, S. Toward the development of
true“off-the-shelf”synthetic T-cell immunotherapy.
Cancer Sci. 2019, 110, 16–22.
12. Aravindhan, S., Ejam, S.S., Lafta, M.H. et al.
Mesenchymal stem cells and cancer therapy: insights
into targeting the tumour vasculature. Cancer Cell Int.
2021, 21, 158.
13. Jung, Y.; Kim, J.K.; Shiozawa, Y.; Wang, J.; Mishra,
A.; Joseph, J.; Berry, J.E.; McGee, S.; Lee, E.; Sun, H.;
et al.Recruitment of mesenchymal stem cells into
prostate tumours promotes metastasis. Nat. Commun.
2013, 4,1795
14. Fuhrmann, G.; Serio, A.; Mazo, M.M.; Nair, R.; Stevens,
M.M. Active loading into extracellular vesicles
significantly improves the cellular uptake and
photodynamic effect of porphyrins. J. Control. Release
2015, 205, 35–44
15. Kooijmans, S.A.; Schiffelers, R.M.; Zarovni, N.; Vago, R.
Modulation of tissue tropism and biological activity
of exosomes and other extracellular vesicles: New
nanotools for cancer treatment. Pharmacol. Res. 2016,
111, 487–500.
16. Pascucci, L.; Coccè, V.; Bonomi, A.; Ami, D.; Ceccarelli,
P.; Ciusani, E.; Viganò, L.; Locatelli, A.; Sisto, F.; Doglia,
S.M.; et al. Paclitaxel is incorporated by mesenchymal
stromal cells and released in exosomes that inhibit in
vitro tumor growth: A new approach for drug delivery.
J. Control. Release 2014, 192, 262–270.
17. Marelli, G.; Howells, A.; Lemoine, N.R.; Wang, Y.
Oncolytic Viral Therapy and the Immune System: A
Double-Edged Sword against Cancer. Front. Immunol.
2018, 9, 866.
18. Duebgen, M.; Martinez-Quintanilla, J.; Tamura, K.;
Hingtgen, S.; Redjal, N.; Shah, K.; Wakimoto, H. Stem
Cells Loaded With Multimechanistic Oncolytic Herpes
Simplex Virus Variants for Brain Tumor Therapy. J. Natl.
Cancer Inst. 2014, 106
19. Sage, E.; Thakrar, R.M.; Janes, S.M. Genetically modified
mesenchymal stromal cells in cancer therapy.
Cytotherapy 2016, 18, 1435–1445.
Additional Resource
More Reagents for Cancer Research
22. 22
Antibody Drug Development:
Navigating Immune Response
and Multivariate Logistics on the
Targeted Treatment Frontier
Studying these revolutionary drugs requires a comprehensive and
precise approach to understand the multivariate dynamics.
Tremendous efforts and ongoing studies are de-
voted to minimizing symptoms while precisely
targeting malignant cells that ultimately turn into
cancer. One of the biggest breakthroughs to date
has been the employment of monoclonal antibod-
ies (mAbs) in targeted immunotherapy.These drug
antibodies (DAs) now account for one-fifth of all
new drug approvals. Their precision targeting has
been shown to increase cure effectiveness while
minimizing collateral damage caused by other
treatments such as chemotherapy.6
Drug antibod-
ies targeted to specific tumorigenic mutations or
molecular aberrations can be mass produced in
cell culture and theoretically employed to treat any
cancer. Antibodies can be chimeric, humanized, or
fully human and operate through three different
mechanisms leading to tumor death: block a path-
way, flag a cell for destruction, or deliver a drug via
receptor-mediated endocytosis.
Similarly, protein-based drugs are usually synthet-
ic versions of proteins that are produced naturally.
They modify cellular responses by either acting di-
rectly with the targets of interest, recruiting other
molecules to the site, or improving the function
of nearby cells. For example, interleukin 2 (IL-2) is
used in kidney and skin cancer treatment to inter-
fere with cancer growth, recruit immune cells, and
stimulate the production of immune cells that can
help destroy the cancer cells.
Both DAs and drug proteins can stimulate the pro-
duction of anti-drug protein antibodies that can
decrease therapeutic efficacy.1,2,7
Therefore, the
path to implementation of DA or protein-based
therapies must be navigated expertly in order to
maximize patient survival. To identify the most ef-
fective DA, it is important to understand and char-
acterize factors that affect individual pharmacoki-
netic variability post-injection and incidences of
23. Mouse PBMC populations shown with CD4/IFNγ dual staining (RayBright Red and RayBright Violet
450); before & after stimulation. Stained with kit cat# 137-00007; antibody cat# 135-08029 and
135-24013.
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24. 24
immune-related adverse events (IRAEs) associated
with foreign antibodies.1,2,7
While immunogenicity
in preclinical species cannot predict the incidence
of human ADAs, it does have utility in characteriz-
ing consequences of potential ADAs.3
Crucial data in optimizing mAB preclinical devel-
opment for precision cancer treatment include
monitoring production of neutralizing antibod-
ies, understanding factors that affect metabolism
and clearance rate, measuring the binding affinity
of each antibody to its target, finding the bound
epitope, and discovering biomarkers to indicate
treatment effectiveness or unintended down-
stream effects.5,7
It is also crucial to know how and
if any other health conditions impact any one of
these traits. For instance, pathogens such as viral
particles can bind to drug target receptors to facili-
tateinfectionorotherundesiredeffects.Identifying
receptor antagonists or other molecules that in-
hibit unwanted receptor activation is an ongoing
focus of drug discovery efforts.
When monitoring efficacy of and immunogen-
ic responses to an antibody therapeutic, the
researcher needs access to the most reliable,
Table 1. Multiple tools are in our cancer-fighting arsenal. A combination of singleplex and multiplex can be employed to unlock the
secretsoftargetedtherapy.
26. 26
Some, such as Tocilizumab, are even used in com-
bination to reduce symptoms that come with more
active forms of therapy such CAR-T,8
a bio-hack to
your immune system that bolsters it to better kill
uninvited cells, seen in Table 2. Regardless of the
mechanism of action, continued study on these
revolutionary drugs requires a comprehensive
and precise approach to understand the multivar-
iate dynamics. Paramount are immunoassays to
discover biomarkers, individualized immunologic
responses, and other potentially neutralizing par-
ticles in a real-body environment.
References
1. Baxi, Shrujal, et al.“Immune-related adverse events
for anti-PD-1 and anti-PD-L1 drugs: systematic review
and meta-analysis.”Bmj 360 (2018).
2. Eigentler, Thomas K., et al.“Diagnosis, monitoring
and management of immune-related adverse drug
reactions of anti-PD-1 antibody therapy.”Cancer
treatment reviews 45 (2016): 7-18.
3. FDA. Guidance for Industry - Immunogenicity
Assessment for Therapeutic Protein Products 2014
[Available from: https://www.fda.gov/downloads/
drugs/guidances/ ucm338856.pdf.]
4. Kwok, Gerry, et al.“Pembrolizumab (keytruda).”
Human vaccines & immunotherapeutics 12.11 (2016):
2777-2789.
5. Longoria, Teresa C., and Krishnansu S. Tewari.
“Evaluation of the pharmacokinetics and metabolism
of pembrolizumab in the treatment of melanoma.”
Expert opinion on drug metabolism & toxicology
12.10 (2016): 1247-1253.
6. Lu, RM., Hwang, YC., Liu, IJ. et al. Development of
therapeutic antibodies for the treatment of diseases.
J Biomed Sci 27, 1 (2020). https://doi.org/10.1186/
s12929-019-0592-z
7. Ovacik, Meric, and Kedan Lin.“Tutorial on monoclonal
antibody pharmacokinetics and its considerations in
early development.”Clinical and translational science
11.6 (2018): 540-552.
8. Si, Stephanie, and David T. Teachey.“Spotlight on
tocilizumab in the treatment of CAR-T-cell-induced
cytokine release syndrome: clinical evidence to
date.”Therapeutics and clinical risk management 16
(2020): 705.
Additional Resource
Paths to Optimize Antibody Therapeutics
27. 27
GPCRs: From Signal Pathways to
Targeted Drug Development
How to break through bottlenecks to obtain full-length GPCRs as anti-
gens for drug development.
Various proteins on the cell membrane are respon-
sible for cell protection, internal/external materi-
al transportation, and signal transmission. These
membrane proteins play diverse roles in cell biolo-
gy and have become a crucial target in drug devel-
opment. Over 50% of the currently approved drugs
target human membrane proteins. G protein-cou-
pled receptors (GPCRs) are the largest family of
membrane proteins.The primary function of GPCRs
is to transduce a wide and diverse array of extra-
cellular stimuli such as biogenic amines, peptides,
hormones, neurotransmitters, ions, odorants, and
photons into intracellular signals. This signaling, in
turn, regulates a myriad of physiological processes,
including cell metabolism, differentiation, growth,
neurotransmission, and sensory perception. GPCRs
are implicated in various diseases, including type
2 diabetes mellitus (T2DM), obesity, depression,
HIV, cancer, Alzheimer’s disease, etc. The crucial
role of GPCRs and the increased efforts in the drug
discovery field have led to GPCRs becoming the
most successful drug target class in the treatment
of various pathologies. This review discusses the
structure of GPCRs, signaling pathways, and the
development pattern of various stages of targeted
drugs. ACROBiosystems is focused on overcoming
the challenges of GPCR preparation and providing
full-length GPCR proteins to assist our customers,
and collaboratively researching and developing
antibody-drug and therapy strategies.
Structure of GPCRs
The common molecular structure of GPCRs con-
sists of seven transmembrane alpha helices, and
these domains divide the receptor into extracel-
lular N-terminus, intracellular C-terminus, three
extracellular loops, and three intracellular loops.
The extracellular ring contains two highly con-
served cysteine residues, which can stabilize the
spatial structure of the receptor by forming disul-
fide bonds. There is a G-protein binding site on the
intracellular loop. In the case of CCR5, the classical
GPCRs topology is shown in Figure 1.1
The GPCRs Signaling Pathways
The effectors of GPCR activation are the heterotri-
meric G-proteins Gα, Gβ, and Gγ. Activated GPCRs
act as GEFs (guanine-nucleotide-exchange fac-
tors) and exchange GDP for GTP in the Gα subunit,
which activates the protein. A GPCR is free of ligand
28. 28
(L) in its basal state. Gα binds to GDP and is associ-
ated with Gβγ. The heterotrimeric protein complex
might associate with the receptor at this point or
remain free in the membrane as pictured, but once
it encounters a ligand-bound GPCR, downstream
signaling is initiated (Figure 2). Upon ligand bind-
ing, the GPCR becomes activated and undergoes a
conformational change. The resulting GTP-bound
Gα separates from βγ and the active heterotrimeric
proteins. Currently, the GDP-αβγ complex, with the
participation of Mg2+
, exchanges GTP in the bound
GDP with the cytosol to form a GTP-αβγ complex.
Then the G protein is activated and separated from
the receptor and simultaneously disassembles into
two parts, GTP-α and βγ, which diffuse freely along
the cell membrane and directly interact with ef-
fector proteins, such as PLC or adenylate cyclase,
which results in effector activation and initiation
of a second-messenger cascade and completes the
extracellular transmission of signals into the cell.
The GTP in Gα is then hydrolyzed to GDP through
the activity of Gα and RGS proteins (not shown),
leading to Gα inactivation and reassociation of the
heterotrimeric protein complex. This process rep-
resents a full GPCR G-protein cycle.
Progress in drug development
targeting GPCRs
Changing the competitive landscape
GPCRs are the most intensively studied drug tar-
gets comprising approximately 27% of the global
market share of therapeutic drugs, with aggregat-
ed sales for 2011–2015 of $890 billion. In 2017,
survey data showed 481 therapeutic drugs target-
ing GPCRs, which compose 34% of FDA-approved
drugs and mediate their effects through at least
107 unique GPCRs. Approximately 320 drugs are
currently in clinical trials, of which 35% of these
drugs 64 potential novel GPCRs targets.3
Figure1.SchematicdiagramoftheclassicGPCRs1
Figure2.GPCRssignalpathway2
31. 31
In the field of drug development that targets
GPCRs, GPCRs antibodies have unique advantages
over small molecule drugs:
• The clearance rate in the body is lower,
action time is longer, and the corresponding
administration frequency is lower
• The selectivity of antibodies is significantly
higher than that of small molecules
• However, due to the blood-brain barrier,
antibody drugs cannot enter the central
nervous system. Therefore, for GPCRs that are
expressed in both the peripheral and central
nervous systems, only the peripheral part of
the drug needs to be designed; therapeutic
antibodies can be developed to make the
drug mainly distributed in the peripheral
area and reduce the toxic side effects on the
central nervous system.
Overview of Leading Projects
There are currently about 13 GPCRs targeted anti-
body projects under development that are active
worldwide, as shown in the table below. The target
is mainly concentrated in CCR4, CALCRL, CCR5,
GCGR, GPRC5D, GLP1R, C5AR, CB1, S1PR1, CCR8,
CCR7, GPR49, and AGTR1. The representative drug
is Leronlimab, a humanized IgG4 monoclonal an-
tibody targeting CCR5, an HIV viral entry inhibitor.
By masking CCR5, the HIV (R5) subtype is blocked
from entering healthy T cells, thereby protecting
these cells from viral infections.
ACROBiosystems assists
in the development of
targeted GPCRs antibodies
The first step in preparing antibodies is antigen im-
munity, and the first step in immunity is the prepa-
ration of antigens. Since GPCRs are seven-pass
transmembrane proteins, it is extremely difficult to
obtain biologically active soluble GPCRs antigens.
To obtain a full-length GPCRs antigen for drug devel-
opment, you need to break through two bottlenecks:
• How to increase the expression? Unlike
secretory proteins, the expression and
display of membrane proteins are limited
Table1.TargetedGPCRsdruginformationunderresearch(pharmacodia)
32. 32
by the membrane area. Many membrane
proteins involve functions related to material
transport and signaling; overexpression on
the cell membrane can cause irreversible
damage to cells. These characteristics of
membrane proteins greatly limit their
expression. Therefore, to obtain enough
full-length membrane proteins for immunity
and drug development, it is necessary to
design and optimize the expression interval,
expression system, culture conditions, etc.
Even so, the cost of obtaining milligram
membrane proteins is still much higher than
that of soluble proteins.
• How to maintain the uniformity and
activity of membrane proteins during
expression and purification? The
transmembrane domain of membrane
proteins is highly hydrophobic, so
unprotected exposure to water will lead
to nonspecific protein aggregation and
even denaturation. Therefore, in the
process of enrichment and purification of
membrane proteins, it is necessary always
to maintain the hydrophilic properties
of the surrounding environment of
membrane proteins. The most widely used
method involves removing membrane
proteins from the cell membrane of the
phospholipid bilayer through detergent4
and
forming micelles to maintain their natural
conformation and functional activity as
much as possible. Furthermore, there are
also methods such as Nanodisc,5
virus-like
particles (VLP),6
and polymer lipid particles
(PoLiPa).7
Therefore, to meet different application needs in
the drug development process that targets GPCRs,
ACROBiosystems has specially set up platform solu-
tions with VLP, Detergent, and Nanodisc to provide
full-length GPCRs proteins such as GPRC5D, CXCR4,
CCR5, and CCR8.
Table2.GPCRsfromACROBiosystems
33. 33
In addition to GPCRs, ACROBiosystems can pro-
vide a complete range of full-length multi-pass
transmembrane proteins, including four-pass
transmembrane protein CD20, Claudin18.2, and
five-pass transmembrane protein CD133. More
full-length proteins are under development.
References
1. Dong HF, Wigmore K, Carrington MN, Dean M,
Turpin JA, Howard OM. Variants of CCR5, which
are permissive for HIV-1 infection, show distinct
functional responses to CCL3, CCL4 and CCL5.
Genes Immun. 2005 Oct;6(7):609-19. doi: 10.1038/
sj.gene.6364247.
2. Hanlon CD, Andrew DJ. Outside-in signaling--a
brief review of GPCR signaling with a focus on
the Drosophila GPCR family. J Cell Sci. 2015 Oct
1;128(19):3533-42. doi: 10.1242/jcs.175158. Epub 2015
Sep 7..
3. Hauser AS, Attwood MM, Rask-Andersen M, Schiöth
HB, Gloriam DE. Trends in GPCR drug discovery: new
agents, targets and indications. Nat Rev Drug Discov.
2017 Dec;16(12):829-842. doi: 10.1038/nrd.2017.178.
4. Wiseman DN, Otchere A, Patel JH, Uddin R, Pollock
NL, Routledge SJ, Rothnie AJ, Slack C, Poyner DR,
Bill RM, Goddard AD. Expression and purification
of recombinant G protein-coupled receptors: A
review. Protein Expr Purif. 2020 Mar;167:105524. doi:
10.1016/j.pep.2019.105524. Epub 2019 Oct 31.
5. McLean MA, Gregory MC, Sligar SG. Nanodiscs: A
Controlled Bilayer Surface for the Study of Membrane
Proteins. Annu Rev Biophys. 2018 May 20;47:107-124.
doi: 10.1146/annurev-biophys-070816-033620.
6. Ho TT, Nguyen JT, Liu J, Stanczak P, Thompson AA,
Yan YG, Chen J, Allerston CK, Dillard CL, Xu H, Shoger
NJ, Cameron JS, Massari ME, Aertgeerts K. Method
for rapid optimization of recombinant GPCR protein
expression and stability using virus-like particles.
Protein Expr Purif. 2017 May;133:41-49. doi: 10.1016/j.
pep.2017.03.002.
7. Hothersall JD, Jones AY, Dafforn TR, Perrior T, Chapman
KL. Releasing the technical‘shackles’on GPCR drug
discovery: opportunities enabled by detergent-free
polymer lipid particle (PoLiPa) purification. Drug
Discov Today. 2020 Aug 21:S1359-6446(20)30337-8.
doi: 10.1016/j.drudis.2020.08.006.
Additional Resource
Discover more about GPCRs
34. 34
Blockade of PD-L1, an Immune
Checkpoint Molecule, in a
Murine Model of Melanoma
Study describes the cellular response to PD-L1 blockade using
monoclonal antibodies in melanoma models.
Highlights
Injection of anti-PD-L1 antibody in a mouse mela-
noma model:
• Increases levels of activated CD4+
splenic
T cells.
• Stimulates production of Th cytokines and
suppresses pro-inflammatory cytokines.
• Increases antigen-specific T cells and reduces
tumor growth.
Summary
Immune checkpoint molecules help to regulate
and limit the immune response. As tumor cells are
able to exploit these mechanisms, new immuno-
therapy treatments target immune checkpoints.
We used a monoclonal antibody to block PD-L1
binding in a murine model of melanoma and char-
acterized T cell subsets and cytokine profiles. We
found that treatment with an anti-PD-L1 antibody
resulted in activation of T cells, increased produc-
tion of Th cytokines and chemokines, increased
levels of tumor-specific T cells and caused a reduc-
tion in tumor growth.
Introduction
Cancer treatment is classically composed of a com-
bination of surgery, radiation, and chemothera-
py. More recently, immunotherapy-based strate-
gies have emerged as another treatment option.
Immunotherapy relies on using the body’s own
immune system to recognize and mount an attack
against cancer cells. Immunotherapy can be broad-
ly categorized into cell-based therapies in which
cells are engineered ex vivo before being reintro-
duced into the patient and soluble factor-based
35. BioLegend products are manufactured in an ISO 13485:2016-certified facility to ensure the highest quality standards.
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For 20 years, BioLegend scientists have supported researchers by developing reagents for flow cytometry, multiomics, and
cell bioprocessing that are critical to accelerating cancer research. Along with our expanding line of reagents, we provide
educational resources that help researchers understand and decipher the complexity of cancer.
We have thousands of scientist-crafted reagents
engineered to combat cancer
• MojoSort™ magnetic cell separation to purify and enrich
target cells
• GMP antibodies and recombinant proteins to activate cells
• Cell-Vive™ media for T/NK cell expansion
• Flow cytometry antibodies to immune profile cells
• Immunoassays that quantitate cancer biomarkers
Discover the difference at: biolegend.com/cancer
Explore our latest infographic and other helpful resources.
Cancer cells evolve new escape mechanisms. Our resources
keep you apprised of developing technologies, such as CAR-T
and CAR-NK cells. Explore our newest infographic to learn
about this advanced immunotherapy. On our website, you
can find additional educational content, including webinars,
blogs, protocols, and scientific posters.
Bring new cancer solutions to light.
Scan the QR code
to download the
infographic.
36. 36
therapies in which monoclonal antibodies or other
proteins are introduced into the patient to mount
an anti-tumor response.
One approach to developing immunothera-
py-based treatments is through the inhibition of
immune checkpoint molecules (reviewed in 1).
Immune checkpoints maintain immune homeosta-
sis and limit the immune response, in part, to pre-
vent autoimmunity. Tumor cells are able to exploit
this system to prevent an anti-tumor response by
expressing checkpoint markers typically found on
antigen presenting cells (APCs). Because immune
checkpoint molecules are often composed of re-
ceptors and ligands, they are able to be targeted
using monoclonal antibodies. Antibodies that can
block ligand-receptor binding allow T cells to re-
main in an activated state to mount an immune
response against the tumor cells.
One such immune checkpoint molecule that
has been studied for use in immunotherapies is
the PD-1 receptor and one of its ligands, PD-L1.2
Engagement of PD-L1 and PD-1 initiates down-
stream signaling pathways ultimately leading to T
cell death.3
As melanoma cells show elevated ex-
pression of PD-L1, the PD-L1 blockade may be par-
ticularly useful in the treatment of melanoma.4, 5
In
this report, we use a GoInVivo™ anti-mouse PD-L1
antibody in a murine model of melanoma and char-
acterize the T cells in the tumor microenvironment
and draining lymph nodes, study the antigen-spe-
cific T cell response, and phenotype the cytokine
profile in the serum.
Materials and Methods
In vivo injection of PD-L1 Antibody
BALB/cJ or C57BL/6 mice were injected with 106
B16F10 melanoma cells in the flank. Mice were
subsequently treated with either 100 or 200 µg of
GoInVivo™ anti-PD-L1 antibody clone 10F.9G2 (Cat.
No. 124328) or isotype control (Cat. No. 400666) ev-
ery 3 days for a total of 3 doses. Dosing schedule
was performed as indicated in the figure legend.
Tissue samples and serum were collected 14 or 24
days following initial dose. Spleen samples were
collected and made into a single cell suspension
for flow staining.
Flow cytometry staining
Cells were stained with anti-CD25 (Cat. No. 102033),
CD69 (Cat. No. 104530), CD152 (CTLA-4) (Cat. No.
106305), CD279 (PD-1) (Cat. No. 109111), CD278
(ICOS) (Cat. No. 313531), I-A/I-E (MHC II) (Cat. No.
107605), CD11c (Cat. No. 117317), CD80 (Cat. No.
104729), CD86 (Cat. No. 105011), MHC pentam-
er (ProImmune), and 7-AAD (Cat. No. 420403).
Cells were acquired with an LSR Fortessa™ (BD
Biosciences) and analyzed using FlowJo software.
Microscopy
Draining lymph nodes were isolated. Tissues were
sectioned in a microtome cryostat at 7 microme-
ters after freezing in OCT media. Sections were
stained with anti-CD4 (Cat. No. 100425), CD8 (Cat.
No. 100727), CD11c (Cat. No. 117346), and B220
(Cat. No. 103251), without fixation or antigen re-
trieval. Images were taken on an Olympus IX83 in-
verted microscope.
Cytokine and chemokine measurement
Cytokine and chemokine profiles were character-
ized using BioLegend’s LEGENDplex™ system (Cat.
Nos. 740740, 740134, 740007). A detailed protocol
has been published.6
37. 37
For more information regarding LEGENDplex™,
please visit: biolegend.com/legendplex
All reagents are from BioLegend, unless
otherwise noted.
Results
Effective blocking of PD-L1 using a mono-
clonal antibody in a melanoma model
To examine the efficacy of a PD-L1 blocking anti-
body in the treatment of a mouse cancer model,
melanoma, we injected mice with 106
B16F10 mel-
anoma cells in the flank. Mice were subsequently
treated with a GoInVivo™ anti-PD-L1 blocking anti-
body every three days (or as indicated). Following
treatment, tissues and serum were collected for im-
munophenotyping (Figure 1).
Anti-PD-L1 treatment increases T cell
activation status
Splenic CD4+
cells were harvested 14 days af-
ter treatment with anti-PD-L1 antibody or the
corresponding isotype control and stained for
common T cell activation markers. Mice that had
been treated with anti-PD-L1 showed an activat-
ed phenotype as measured by increased levels of
CD25, CD69, ICOS, and CD279 (PD-1) as compared
to mice treated with an isotype control (Figure 2).
Importantly, CTLA-4, another immune checkpoint
molecule which negatively regulates the immune
Figure1.Experimentallayoutoftreatmentof
anti-PD-L1antibodytreatmentinamelanomamodel.
Figure2.SplenicCD4+
Tcellstreatedwithanti-PD-L1
(200µgbyI.V.injectionondays0,3,6)demonstrated
anactivatedphenotype.Cellswerecollected14days
followingimplantation.
38. 38
response, was expressed at low levels and re-
mained unchanged in mice that had been injected
with anti-PD-L1.
Splenic Dendritic Cells Downregulate
CD80 following PD-L1 treatment
Next, we examined splenic dendritic cells to deter-
mine whether the increase in activated T cells cor-
related with alterations in the population of APCs.
For these experiments, we first gated on classical
dendritic cells that are MHC II high and CD11c high.
Dendritic cells (DCs) from animals that had been
treated with the anti-PD-L1 antibody showed lower
expression of CD80 (Figure 3). We hypothesize that
downregulation of CD80 may occur to allow for
CD86-CD28 interaction and less binding to CTLA-4.
Anti-PD-L1 treatment results in
elevated numbers of CD8+
DCs in
draining lymph node
To further characterize the APCs, we examined the
draining lymph nodes from animals that had been
injected with anti-PD-L1 and isotype controls. In
animals that received the anti-PD-L1 treatment,
there is an increased level of CD8a+
dendritic cells
in draining lymph nodes. CD8a+
dendritic cells are
APCs that have the ability to cross-present antigens
to CD8+
T cells, which is a common phenomenon
during tumor elimination.7
Anti-PD-L1 treatment in melanoma mod-
els alters the cytokine and chemokine
profile in serum
To more completely analyze the immune response
following anti-PD-L1 treatment, we used our
LEGENDplex™ multiplex platform to analyze the
chemokine and cytokine profile in serum (Figure
4). Notably, we found that animals treated with
anti-PD-L1 had increased content of cytokines
typically associated with a Th1 response, includ-
ing IFN-γ and TNF-α. Additionally, there was an in-
crease in cytokines associated with Th9 and Th22
phenotypes, IL-9 and IL-22, respectively. However,
this did not correspond with an overall increase in
soluble mediators, as they simultaneously limited
the production of other pro-inflammatory cyto-
kines including IL-12p70, IL-27, IL-33, and GM-CSF,
indicating that the cytokine response in these an-
imals was specific. Lastly, anti-PD-L1 treatment
resulted in the elevated production of import-
ant chemokines including MIP-3α, Eotaxin, MIG,
and IP-10. These chemokines may play a role in
anti-tumor immunity by mobilizing other cells.
Figure3.SplenicDCsshowdecreasedlevelsofCD80
(top).CellswerefirstgatedonclassicalDCs(MHCII
high,CD11chigh).IncreasedlevelsofCD8+
DCsin
thedraininglymphnodeofanimalstreatedwith
anti-PD-L1(bottom).TissuewasstainedwithCD4
(green),CD8(red),CD11c(cyan),andB220(blue).
39. 39
Increase in antigen-specific CD8+
tumor
infiltrating lymphocytes (TIL) following
anti-PD-L1 treatment
To examine antigen-specific T cells during tumor
development, we implanted a B16 melanoma cell
line that expresses the peptide SIYRYYGL, which is
derived from the Nucleoprotein of Vesicular sto-
matitis virus (498-505) and examined the tumor
24 days after implantation and antibody treat-
ment. Using an MHC pentamer loaded with the
SIY peptide, we were able to track CD8+
TILs. As
expected, animals that received the anti-PD-L1
treatment had expanded pools of antigen-spe-
cific TILs as compared to those that received the
isotype control (Figure 5). Finally, as a confirma-
tion of other published models,4,5
treatment with
Figure5.Animalstreatedwithanti-PD-L1(100µgI.P.
injectionondays7,13,16followingimplantationof
melanomacellline)showincreasedantigen-specific
TILsandareductionintotaltumorarea.
Figure 4. Cytokine profile in the serum from treated mice
shows elevation of Th cytokines and chemokines. Animals
treated with PD-L1 also showed a simultaneous decrease
of other pro-inflammatory cytokines.
40. 40
an anti-PD-L1 antibody significantly reduced the
tumor growth, as measured by tumor area, in this
melanoma model.
Conclusions
Immune checkpoint molecules make an attrac-
tive target for anti-cancer therapy, as inhibition
of these pathways results in an elevated immune
response, and they often rely on a ligand-recep-
tor interaction. Blockade of these pathways can
often be easily performed using a bioactive an-
tibody, like our GoInVivo™ functional antibodies.
As melanoma cells express PD-L1 to help evade
an anti-tumor response, we sought to under-
stand the specific cellular phenotype of the PD-
L1 blockade in a mouse melanoma model. As
expected, the blockade of PD-L1 resulted in ele-
vated activation of T cells as measured by expres-
sion of common T cell activation markers, includ-
ing CD25 and CD69. Interestingly, this activation
phenotype corresponds with a downregulation
of CD80, another T cell co-stimulatory molecule,
on APCs. We hypothesize that this downregula-
tion may occur to alter the interaction of other
immune checkpoint molecules, including reduc-
ing binding to CTLA-4 and to allow for more in-
teraction between CD86 and CD28. Using our
LEGENDplex™ multiplex platform, we surveyed
the cytokine and chemokine response in the se-
rum following anti-PD-L1 treatment. Interestingly,
anti-PD-L1 treatment increased certain cytokines
associated with a Th response; however, there was
not an overall increase in all soluble mediators, as
the treatment also caused simultaneous down-
regulation of other pro-inflammatory cytokines.
Importantly, treatment with anti-PD-L1 resulted
in increased levels of CD8+ tumor-specific T cells
which is also correlated with a reduction in tumor
size. Collectively, these results describe the cellu-
lar response to the blockade of PD-L1 in a mouse
melanoma model and suggest that anti-PD-L1
monoclonal antibodies may provide an effective
treatment for melanoma.
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