This document contains slides from a presentation by Keith L. Knutson on immune-based cancer therapies. It discusses various approaches to boosting immune responses against cancer, including vaccines, monoclonal antibodies, adoptive T cell therapies, and immune checkpoint blockade. Specific examples are provided of clinical trials investigating therapies targeting HER2 and folate receptor alpha for breast cancer and PD-1/PD-L1 blockade for melanoma and triple negative breast cancer. The slides also review the roles of different immune cells like CD4 and CD8 T cells, B cells, and dendritic cells in the adaptive immune response against cancer. Conflicts of interest for the presenter are also listed.

1. ©2014 MFMER | slide-1
Immune-based
Therapies for Cancer
Keith L. Knutson
Professor of Immunology
Director, Cancer Research, Mayo Clinic
Florida

2. ©2014 MFMER | slide-2
Conflict of Interest
• TapImmune, Inc.
• Cancer Vaccines – Jacksonville, FL
• Scientific Advisory Board (unpaid)
• Several Patent Licensing Agreements (Mayo)
• Kiromic, Inc.
• Cancer Vaccines – Lubbock, TX
• Scientific Advisory Board (Stock)
• Intelligent Immunity, Inc.
• Cancer Vaccines – Jacksonville, FL
• Scientific Advisory Board (Unpaid)
• Founder (Stockholder)
• Antigen Express, Inc.
• Cancer Vaccines – Cambridge, MA
• Scientific Advisory Board (Paid)

3. ©2014 MFMER | slide-3
The Burdens of Breast and Ovarian
Cancers
Ovarian cancer in the USA
2016: 22K new cases,
14K deaths
Breast cancer in the USA
2016: 247K new cases,
40K deaths

4. ©2014 MFMER | slide-4
Natural Host Immune Defenses Against TNBC
are Associated with Reduced Recurrence
Kreike et al. Breast Cancer Research 2007 9:R65
With infiltrating
lymphocytes
No infiltrating
lymphocytes
Probabilityofdisease-freesurvival

5. ©2014 MFMER | slide-5
textbookofbacteriology.com
Cells of the Immune System

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CD4 “helper” T cells
•Inflammation (macrophages and
neutrophils)
•Antibodies
•Induce/Enhance cytotoxic T cells
•Immune-surveillance
•Epitope-spreading
CD8 “cytolytic” T cells
•Tumor lysis
B cells
•Antibodies
•Signaling
•ADCC
•Complement
The adaptive immune response

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Differentiation of the adaptive immune
response

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Immuno-oncology
• Cancers evade immunity by activating a variety of
immune suppression mechanisms
– PD-1/PD-L1
– CTLA-4
– Tregs
• Approaches to boosting immune responses
– Vaccines
– Monoclonal antibodies
– Adoptive T cell therapies
– Immune checkpoint blockade

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Why do we vaccinate?

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Target neoantigen choices for a
cancer vaccine
• Microbial neoantigens
• Amino acid mutation neoantigens.
• Frameshift / fusion neoantigens
• Splicing variant neoantigens
• Nonmutated ‘self’ antigens (subdominant
neoantigens)

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MS Lawrence et al. Nature 000, 1-5 (2013) doi:10.1038/nature12213
Somatic mutation frequencies observed in
exomes from 3,083 tumor–normal pairs

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Overexpressed self proteins as a source of tumor
neoantigens
Normal Cell
Tumor Cell
MHC
Dominant Epitope Subdominant Epitope

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– Four helper T cell epitopes
w/GM-CSF
– One arm:
• 2+(FISH+) and 3+
– Three mos. following last
dose of trastuzumab
• Disease-free.
– Objectives
• Immunogenicity (i.e.
proof of principle)
• Safety
• Feasibility
CD4T cell
CD8T cell
Tumor/DC
Bcell
e.g.Folate receptor alphaHER-2/neu
Phase I Trial Design
DRB1*0101, DRB1*0301
DRB1*0401, DRB1*0404
DRB1*0405, DRB1*0701
DRB1*0802, DRB1*0901
DRB1*1101, DRB1*1201
DRB1*1302, DRB1*1501
DRB3*0101, DRB4*0101
DRB5*0101

14. ©2014 MFMER | slide-14
Generation of HER2-Specific T Cells with
Resected HER2 Breast Cancer
C D 1 p5 9 p8 8 p4 2 2 p8 8 5 E C D IC D TT
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
8 0 0
1 0 0 0
1 2 0 0
1 4 0 0
1 6 0 0
A n tig e n
Antigen-specificTcells
(permillionPBMC)
P re -Im m u n iz a tio n
P o s t-Im m u n iz a tio n
C ontrol A g

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Phase 1B DCIS – Degenerate
Subdominant Epitope Vaccines
-1 0 6 8 6 12 18 24
Registration
Consent
Week
SAFETY AND
IMMUNOGENICITY
Surgery
• Dose finding (n=9 arm)
• 500 mg/peptide
• 1000 mg/peptide
• 1500 mg/peptide
• Expansion (n=25)
• Immunogenicity
• Safety
2 4
VACCINATIONS
HER2 Expression 1+, 2+, or 3+
DOD BCRP BC151104

16. ©2014 MFMER | slide-16
FRa in Breast Cancer is Associated with Cancer
Recurrence
Hartmann et al. Int J Cancer 2007; 121:938-42
100
80
60
40
20
0
Weak FR
expression
Strong FR
expression
0 1 2 3 4
Years
Average Staining Intensity — 0-1.0 — 1.1-3
ProportionFreeofRecurrence(%)

17. ©2014 MFMER | slide-17
C y c D 1 F R 3 0 F R 5 6 F R 7 6 F R 1 1 3 F R 2 3 8 F R P ro te in T T
0
2 5 0
5 0 0
7 5 0
1 0 0 0
1 2 5 0
1 5 0 0
A n tig e n
Antigen-specificTcells
(permillionPBMC)
P re -Im m u n iz a tio n
P o s t-Im m u n iz a tio n
N S
p = 0 .0 0 4
p = 0 .0 0 8
p = 0 .0 0 0 0 4
p = 0 .0 0 0 0 9
p = 0 .0 0 1
p = 0 .0 0 5
p = 0 .0 1
C o n tro l A g
Generation of FRa-specific T cells

18. ©2014 MFMER | slide-18
BC141410: FRa Vaccination to Prevent
Progression of Triple Negative Breast
Cancer
Stages IIb/III
TNBC
Placebo
N=93
Vaccine
N=187
Conventional
Therapy
• Multicenter Phase II Trial to Test Whether Vaccine
Prevents Recurrence in Patients Diagnosed and
Treated for TNBC
FRa is preferentially overexpressed in TNBC

19. ©2014 MFMER | slide-19
IgG immunity to tumor antigens increasesfTreatment
with herceptin and chemotherapy
Taylor, et al., 2007, Clin Cancer Research
Knutson, et al., 2015, ASCO Annual meeting
Knutson, et al., 2015, Cancer Research, In Press
P re P o s t
0
1
2
3
4
P re P o s t
0 .0
0 .5
1 .0
1 .5
Antibodies(g/ml)
P re P o s t
0 .0
0 .5
1 .0
Antibodies(g/ml)
P re P o s t
0
1
2
3
4p = 0.01 p = 0.01
p < 0 .0 0 1
p = 0 .0 0 7
A B C
D
Antibodies(g/ml)
Antibodies(g/ml)
P re P o s t
0 .0
0 .1
0 .2
0 .3
0 .4
0 .5
P re P o s t
0
2
4
6
8
C E A T T
FE
Antibodies(g/ml)
Antibodies(g/ml)
N SN S
H E R 2 -IC D
H E R 2 -E C D F ra g
IG F B P 2 p 5 3

20. ©2014 MFMER | slide-20
Increases in HER2 antibody following treatment is
associated with improved survival
Knutson, et al., 2014, ASCO Annual meeting
Generation of antibody responses to HER2
is associated with improved progression
free and overall survival
Marker or Collection
Time
Cox Univariate
Hazard Ratio
(95% CI)
Score
p-value
PROGRESSION-FREE SURVIVAL
HER2-ICD 0.0042
No ---
Yes 0.506 (0.253-1.014)
HER2-ECD Frag 0.0007
No ---
Yes 0.383 (0.193-0.758)
OVERALL SURVIVAL
HER2-ICD 0.038
No ---
Yes 0.733 (0.367-1.465)
HER2-ECD Frag 0.026
No ---
Yes 0.643 (0.346-1.196)Knutson, et al., 2015, ASCO Annual meeting
Knutson, et al., 2015, Cancer Research, in press
NCI R01 CA152045

21. ©2014 MFMER | slide-21
Anticancer Research 2009
T cells are isolated from surgical
specimens.
Presumably, the T cells are enriched
in tumor killers.
Adoptive T cell therapy with tumor infiltrating
lymphocytes

22. ©2014 MFMER | slide-22
What do CD8 T cells do?

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Discovery Medicine
Adoptive T cell therapy with CAR T cells

24. ©2014 MFMER | slide-24
The Checkpoint Blockade Revolution
Lymphomation.org

25. ©2014 MFMER | slide-25
Immune checkpoint targets

26. ©2014 MFMER | slide-26

27. ©2014 MFMER | slide-27
PD-1 / PD-L1 Blockade in Melanoma
OncoLog, February 2014, Vol. 59, No. 2
A patient with ipilimumab-refractory melanoma is shown before (left), after one cycle, and after three cycles of
treatment with the anti–PD-1 antibody MK-3475 (10 mg/kg every 3 weeks).

28. ©2014 MFMER | slide-28
PD-1 / PD-L1 Blockade in Triple Negative Breast Cancer

29. ©2014 MFMER | slide-29
Combination therapy results in complete
regression and sustained progression free survival
~75% Complete Regression Rate
Karyampudi, et al. Cancer Res. 2014
TX

30. ©2014 MFMER | slide-30
PD-1 blockade leads to an enhanced production of IL-
10 by murine ovarian TIDCs.
Ig G a -P D -1 a -P D -L 1
0
4 0 0
8 0 0
1 2 0 0
IL-10(pg/mL)
* * *
* * *
n .s .
S e ru m A s c ite s
0
4 0 0
8 0 0
5 0 0 0
6 0 0 0
IL-10(pg/mL)
N T
a -P D -1
p < 0 .0 0 1
p < 0 .0 0 1
Lamichhane P. 2015, unpublished

31. ©2014 MFMER | slide-31
Conclusions
• The immune system is a complex system designed to
deal with many different diseases and infections.
• Cancers utilize sophisticated suppressive mechanisms to
evade immunity.
• Immune-based therapies are designed to boost adaptive
immunity to the extent of overcoming suppressive
mechanisms.
• Vaccines
• Monoclonal Antibody Therapy (Trastuzumab)
• Adoptive T Cell Therapy
• Immune Checkpoint Blockade

32. ©2014 MFMER | slide-32
Acknowledgements
Financial support
VGTI FL
K01 100764
R01 113861
R01 152045
Mayo Ovarian Cancer SPORE
Mayo Breast Cancer SPORE
Mayo Comp Cancer Center
Komen Foundation
Mayo CTSA
MOCA
VaxOnco
TapImmune
Andersen Foundation
Cancurables
National Breast Cancer Coalition
Department of Defense BCRP
Department of Defense OCRP
Cathy Andorfer, Ph.D.
Michael Asiedu, Ph.D.
Karla Ballman, Ph.D.
Marshall Behrens, B.Sc.
Matt Block, M.D., Ph.D.
Amy Degnim, M.D.
Haidong Dong, Ph.D.
Courtney Erskine, B.Sc.
Karin Goodman, R.N.
Lynn Hartmann, M.D.
Karen Hedin, Ph.D.
Timothy Hobday, M.D.
Jim Ingle, Ph.D.
Kimberly Kalli, Ph.D.
Scott Kaufmann, M.D., Ph.D
Michael Kline, Ph.D.
James Krempski, B.Sc.
Puru Lamichhane, Ph.D.
Matt Maurer
Toni Kay Mangskau
Sharon Mercill, Ph.D.
Manu Nair
Aziza Nassar, M.D.
Douglas Padley
Edith Perez, M.D.
Claudia Preston, M.D.
Danell Puglisi-Knutson, B.A.
Barath Shreeder
Vera Suman, Ph.D.
Jennifer Reiman, Ph.D.
Marta Santisteban, M.D., Ph.D.
Jean Stahl, R.N.
Dan Visscher, M.D.
Raphael Clynes, M.D. Ph.D. Columbia University
Martin Cannon, Ph.D. University of Arkansas
Nora Disis, M.D. UW
Mac Cheever, M.D. UW
Doug McNeel, M.D. Ph.D. Uwisc
Glynn Wilson, Ph.D. Tapimmune
Eric von Hofe, Ph.D. Antigen Express
Mayo VGTI FL
Lavakumar Karyampudi, Ph.D.
Patrick Yeramian, M.D. Ph.D.
Richard Jove, Ph.D.
Kathleen Kemp
Shaun White, M.A.
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