hello this is Jasmine Trigg and I'm a scientist at Sartorius today I'll be discussing a poster
previously presented at the American Association of meteorologists conference and the post was
titled high throughput quantification of antibody dependent phagocytosis use in lifestyle analysis
phagocytosis a specific form of endocytosis is a critical component of both innate and
adaptive immune responses the uptake and clear clearance of viable tumor cells can
be promoted with monoclonal antibodies via antibody dependent cellular phag cytosis also
known as adcp all through the blockade of don't eat new signals such as cd47
these mechanisms hold immuno therapeutic promise and are studied extensively in drug development
so here we have developed and validated an in vitro assay for the high throughput
quantification of Phagocytosis using the ink site lifestyle analysis system
the ink sitebag cytosis assay combines Frodo for ink site reagents and integrated image-based
fluorescence measurements in a simple mix and read protocol the data that I'll be
showing today exemplify that liveso analysis is a powerful tool for quantitative morphological
and functional assessment of adcp and this is amenable to screening for therapeutic candidates
the introsite lifestyle analysis system is a fully automated phase contrast and multi-color
fluorescent system that resides inside a standard cell culture incubator for optimal cell viability
the system is designed to scan plates and flasks repeatedly over time in combination
with our software this offers fast flexible and Powerful control for continuous lifestyle analysis
and this comprises image acquisition processing and data visualization we also offer Suite of
reagents and consumables that are non-perturbing and these are cell labeling and Reporting reagents
so phagocytosis is a process by which certain living cells are able to engulf and digest other
cells or particles Frodo reagents for incusite phagocytosis use aph sensitive conjugated
fluorophore that has been Incorporated to enable lifestyle Imaging of phagocytic events the assay
principle shown here is based on a low level of presence of frodo-labeled material in media that's
around pH 7.4 which following engulfment and figure lysosome formation results in the increase
in the acidic environment and this leads to an increase in the fluorescence intensity within
cytoplasm of the cell using the ink site lives analysis system this increase in fluorescence
can be automatically Quantified over time with integrated software to segment the areas of high
intensity additionally they observed phagocytic signals can be confirmed using the ink site HD
phase contrast images and these allow for direct visualization of cell engulfment in real time
for example in the figure in the right here photo labeled apoptotic gercat cells were
co-cultured with j7748.1 mouse macrophages at a range of Target to effector ratios
phase and fluorescent images that 20x were acquired using the exercise system and the
phagocytosis was Quantified using the fluorescent segmentation mask which is
shown in the images here in blue when we look at the time course we can see that there's a
density dependent increase in red fluorescence following engulfing over the 24-hour time period
tumor cells can be targeted with prophetic monoclonal antibodies which
engage host immune cells and promote engulfment and clearance on the body
one strategy for this is the blockage of don't eat new signals such as cd47 and these are present on
the surface of tumor cells and enable evasion of phagocytosis to investigate these don't eat new
signals frodo-labeled ccrfcm tumor cells were treated with monoclonal antibodies targeting
cd47 or IGG ice type control and they were co-cultured with bone marrow drive to macrophages
if we look at the images here on the left these are phase and fluorescence images
for five microgram per ML anti-cd47 and these are acquired using the ink site lives analysis system
the images show an increase in fluorescence as these cells were engulfed a kinetic increase in
frescence was observed with monoclonal antibody mediated response being concentration dependent
to eliminate the possibility of anti-seed 47 treatment resulting in Target cell
apoptosis and inducing ephocytosis we also performed phagocytosis and cell health assays
here frodo-labeled ccrfcm cells were treated with antecedented seven or campothesium which
acted as our apoptotic control and co-cultured with j7748.1 mouse Mac
phages we did this in the presence of the inksi and xm5 dye and this is a cell Health reagent
we found that anti-city 47 treatment induced phagocytosis without inciting apoptosis
to investigate phagocytosis of viable tumor cells via antibody dependent cellulocytosis
we assessed two clinical monoclonal antibodies targeted against cd20 these were rituximal and
subtraxima which is a trucks made by similar and these are both the true approved treatments for
cd20 positive B cell cancers photo labeled Ramos target cells at a rate of densities from 100 to
12.5 K per well we're treated with anti-cv20 Moroccan antibodies for toximab or troxima
or an igg-1 ice type control and then they were co-cultured with bone marrow derived macrophages
if we look at the time course on the left here this shows that traxma-induced
um adcp response is both time and target cell density dependent the results in the middle
show an increase in fluorescence for truxma and rituximab compared to IGG control at all of the
target cell densities that we've studied lastly we talk some AB treatment showed a
concentration dependent increase in adcp using the target cell density of 50k cells per wow
we also assessed the effects of anti-cd-20 monoclonal antibodies in
inducing phagocytosis using primary Mac phages as the affect the cells
monocytes isolated from Whole Bloods were differentiated into macrophages using cytokines
frodo-labeled Ramos cells were treated with monoclonal antibodies targeting anti-cd-20 again
here we use rituximab or truxma or IGG ice type control and co-cultured these with the primary Mac
phages that we've differentiated so the macrophage differentiation itself was monitored and
Quantified using the integrated software of the ink site Advanced Label 3 classification analysis
and we can see the images of the classification masks here so on the left we have images for the
classification mask for monocytes which was on Day Zero with a pink outline and the right hand
image shows macrophages with a blue classification um mask outline on day seven looking at the time
course kinetic data this shows that rituximab and troxima induced adcp in a concentration
dependent manner with similar kinetic profiles being observed over the four hour time period
foreign next we investigated some native and engineered products map FC mutants
frodo-labeled vammal cells were treated with Native and engineered human with toxinant
Isotopes and to see the 20 igg-1 and cd20 igg-1 foots which is a non-fuco selected FC mutant and
anti-c20i21 and Q which is a non-glycosylated fc mutant and they were co-cultured with bone marrow
drive Mac fetus with a 1 to 2.5 k effects Target ratio if we look at the microplate graph on the
left here this shows the kinetic response for total red object area over four hours
we observed concentration dependent adcp response for and cd20 igd1 monoclonal antibody and the FC
mutated igg-1 foot but not for the igg-1 and Q or IGG control and the ec50 values for these were
shown in the table here these results align with previously observed effector functions
additionally we've studied adcp using adherent target cells that had varying
her2 antigen expression profiles so adcp was examined using her2 positive ae565 or
her2 low MTA adherent target cells cells were labeled with Frodo and then treated
with an anti-her2 monoclonal antibody and co-cultured with bone Margaret macrophages
if we look at the images Auntie her to induced an increase in fluorescence for au565 cells but
little to no fluorescence was up there for MDA mb23 cells the time course shows an adcp response
for au565 cells and reveals that this occurs in a time and concentration dependent manner
and to her to induce adcp for a565 cells but not for the MDA cells and this is consistent
with established correlations between her to expression and adcp response
so to conclude the photo reagents for ink site in combination with the site lifestyle analysis
system provides an image-based fluorescent solution for visualization and quantification of
thyocytosis our experimental findings demonstrate antibody mediated cell life cytosis across a
range of effector Target cell types and they substantiate the recognized prophagocytic effects
of monoclonal antibodies such as anti-cb47 anticed 20 and also anti-air2 taken together
these data exemplify that lifestyle analysis is a powerful tool for quantitative morphological and
functional assessment of adcp which is amenable to screening for therapeutic candidates thank you