Immunoprofiling of T cell responses in melanoma patients
undergoing CPI therapy
342
LeeAnn Talarico1, Daniel Grubaugh2, Zheng Yan1, Aula Alami1, Jean-Luc Bodmer1,
Darren E. Higgins2, F. Stephen Hodi3, and Jessica Baker Flechtner1
1Genocea
Biosciences, Inc., Cambridge, MA, USA
2Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA
3Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA
Introduction
Methods: The
• Successful treatment of melanoma patients with checkpoint inhibitors
(CPI) has reinforced the importance of T cells in anti-tumor efficacy
TM
ATLAS
Platform
Figure 2. ATLASTM Technology WorkFlow
Figure 1. cLLO Facilitates MHC class I
presentation by MDDC
• Despite significant progress, CPI therapy is effective in only 40-50%
of treated patients, with substantial toxicity. It is therefore imperative to
understand the profile of T cell responses to determine if predictors of
responsiveness can be identified for those patients who respond to
immunotherapy
• ATLASTM is a T cell antigen discovery platform with which putative
antigens are expressed as individual clones that can be processed by
any patient’s antigen presenting cells and presented as peptide
epitopes in the context of their own MHC class I or II molecules. After
addition of the patient’s own CD4+ or CD8+ T cells, a readout of
activation can be measured for each tumor-associated antigen (TAA)
• This is a proof-of-concept study to determine if ATLAS™ technology
could be applied to characterize and profile T cell responses to TAA of
diverse human patients undergoing CPI therapy
Patient Characteristics
Cohort
Patient
ID
Cancer
CPI Therapy
T cell
Subset
Screened
Responder
CAL001
Melanoma
pembrolizumab
CD4+, CD8+
CAL003
Melanoma
pembrolizumab
CD8+
CAL009
Melanoma
pembrolizumab
CD4+, CD8+
CAL011
Melanoma
pembrolizumab
CD4+, CD8+
CAL012
Melanoma
ipilimumab + GM-CSF
CD4+, CD8+
CAL013
Melanoma
pembrolizumab
CD4+, CD8+
CD4+,
Progressor
Partial
Results
Figure 3. Multiple TAA are Recognized in Patients
Who Received CPI
CD8+
CAL002
Melanoma
pembrolizumab
CAL005
Melanoma
pembrolizumab
None
CAL014
Melanoma
pembrolizumab
CD4+, CD8+
CAL010
Melanoma
ipilimumab + GM-CSF
CD4+, CD8+
• 23 full-length TAA genes (labelled as Un001023) were obtained from the DNA Resource
Core at Harvard Medical School, recloned into
the ATLAS™ expression vector, and sequence
verified. Each TAA was recombinantly expressed
in E. coli.
• Blood samples were collected from ten
consented patients who had previously
undergone CPI therapy.
• Peripheral blood mononuclear cells (PBMC)
were enriched by density gradient centrifugation.
CD4+ and CD8+ T cells were sorted and nonspecifically expanded, and monocytes were
differentiated into dendritic cells (MDDC).
• Library clones were screened in replicates using
5,000 MDDC and 80,000 T cells, at an E.
coli:MDDC ratio of 100:1.
• Assay supernatants were harvested at 24hr and
stored at -80°C.
• Supernatant cytokines were analyzed using
either IFN-g ELISA or a Meso Scale Discovery VPLEX Proinflammatory Panel 1 (human) Kit.
Figure 4. Differential Cytokine Induction Profiles According to CPI Response
A
B
C
D
Cohort determination based upon imaging scans.
Responder= no progression, Progressor = progression, Partial = Inconclusive
Conclusions
• Memory T cell responses can be measured in
multiple, HLA-diverse patients who had
received CPI therapy, without the need to
derive cell lines or predict epitopes
• Both CD4+ and CD8+ T cell responses to
multiple TAA were detected
• Differential responses detected between CPI
Responders and Progressors
• The study was not powered to establish
significance of observations
IFN-g ELISA data for n=9 patients with each replicate shown, color
coded by patient. The responses are reported as ratios of the IFN-g
concentration measured to the TAA, divided by the average response
for the irrelevant antigen clones (GFP) in the same screen. Responses
have been further corrected for monotonous trends by quantile
normalization. The shaded area represents 95% of the data (defined as
background) corresponding to 6 standard deviations around the
geometrical mean response (GMR = 1.0).
Figure 5. Unique and Common CD4+ and CD8+
Responses with Greater Breadth in Responders
• Future steps include:
o Expansion of library to >100 TAA
o Screening of T cells from a much larger
group of patients
o Analyses will include responder and nonresponder comparisons:
• Specificity, frequency and breadth of
responses
• Relationship between CD4+ and CD8+ T
cell responses
• Polyfunctionality
Acknowledgements
• We are grateful to the patients who consented to participate in this
study
• Funding provided by the Ludwig Trust
• The authors would like to thank Biao Liu, an employee of Genocea
Biosciences, for data analysis
• The authors would also like to thank Janice Russell and Jennifer Wald
for sample coordination
MSD IFN-g values for Responders (5) and Progressors (2) used to
calculate frequency with which CPI-treated patients responded to each
clone, by CD4+ or CD8+ T cell subsets; data shown as percent of
patients whose IFN-g response was ≥ twice the GFP negative control.
Un022 and 023 were not in the CD8 library, while Un013 was not in the
CD4 library.
Supernatants from Progressors (2) and Responders (5) who received with pembrolizumab were analyzed using the
MSD V-PLEX Proinflammatory panel 1 (human) kit to determine the levels of IFN-g, IL-10, IL-12p70, IL-13, IL-1b, IL-2,
IL-4, IL-6, IL-8, and TNF-a secreted in response to each TAA. The natural log of the mean cytokine concentration from
two duplicate wells was determined. This value was then transformed by the mean of the distribution to produce the
value represented in each plot for the cytokine concentrations secreted from A) CD4+ and B) CD8+ T cells from
Progressors () and Responders ().
To further characterize differences between Responders and Progressors, cytokine responses to each TAA were divided
by responses to the negative control protein (GFP). For each TAA, the mean distribution of each ratio was determined
by cohort. Student’s t-tests were used to determine if there were statistical differences between the mean distribution of
cytokines in Responders and Progressors. If the differences were statistically different (p<0.05), the mean value was
plotted for C) CD4+ and D) CD8+ T cells as a cumulative value for the cytokine responses to that TAA.
www.postersession.com