Scientists genetically engineered T cells to produce chimeric antigen receptors (CARs) that target MUC1, a protein overexpressed in 95% of triple negative breast cancer (TNBC) cells. The CAR consisted of an antibody that binds to MUC1 linked to T cell activating domains. When infused into mice with human TNBC tumors, the engineered MUC1-targeting CAR T cells reduced tumor burden by specifically lysing the cancer cells and releasing cytokines and chemokines. The CAR T cells showed long term efficacy in reducing tumors over 60 days, though tumors began progressing faster after that, possibly due to loss of the MUC1 target or increased immune suppression. The study demonstrated that CAR T cells

1. Genetically engineered
CAR T cells
in cancer treatment
Rajina Shakya
Lab of microbiology

2. Introduction
T-cells
 Type of lymphocyte
 Important role in
immune response such
as
◦ immunity power
◦ Clearance of pathogens
◦ Allergy
◦ Inflammation
◦ Homeostasis and
autoimmune pathology
◦ Pathogenesis of cancer
MEMORY
T CELL

3. T-cell in cancer
 Immune cells
continuously search
for tumor cell to target
and destroy defective
cells.
 TCR binds with
antigen and kill tumor
cells.
 But, some receptors
like PD1 blocks T
cells action.
 Some drugs
competitively bind
and inactivate these
receptors.
 More side-effects,
less effective, so
more effective
Inactivation of
T-cell by a receptor
Active T-cell by
blocking the receptor
Weak Strong

4. Chimeric antigen receptor T cells (CAR T)
 Genetically engineered T cells to produce an
artificial T-cell receptor for use in immunotherapy.
 new ability to target a specific protein.
Target
protein
Genetically modified T cells
CAR T cell

5. CAR T cell in cancer
Premise is to modify T cells to recognize and
destroy cancer cells.
Act as a "living drug" against cancer cells.
CAR T cell
Cancer
cell

6. Mechanism of CAR
T
 Combines both antigen-
binding and T-cell
activating functions into a
single receptor.
 Firstly, inactive virus is
inserted into T cells which
helps to develop chimeric
antigen receptor (CAR)
 CAR binds with antigen of
tumor and cause
cytotoxicity by increasing
cytokines and
interleukins.

7. Scientists harvest T cells from people, genetically alter them, then infuse the
resulting CAR-T cells into patients to attack their tumors.

8. Basic components of CAR T
receptor
EndodomainEctodomain
Spacer
(enhance
flexibility)
• More stable
than CD3
• Secondary signals
to activate
immune response

9. Advancement in CAR T
receptor
Primary transmitter
of T cell
activation signals
• T cell proliferation
• cytokine secretion
• resistance to apoptosis
• in vivo persistence. • exhibit improved
effector functions
• better in vivo
persistence

10. • CARs (aka TRUCKs or armored CARs)
• enhance T cell expansion
• Persistence
• anti‐tumoral activity include cytokines
such is IL-2, IL-5, IL-12 and co‐stimulatory ligands
Introduction of
4th generation CAR T cells

11. Mucin (mucus like)
Mucins line
the apical
surface of
epithelial
cells in the
lungs,
stomach,
intestines,
eyes and
several
other
organs.

12. MUC1 in tumor
 Mucin 1, cell surface
associated (MUC1)
or polymorphic epithelial
mucin (PEM) is
a mucin encoded by
the MUC1 gene in humans.
 Mucins protect the body from
infection by pathogen.
 But, overexpression of MUC1 is
often associated with various
cancer.
Breast cancer cell
Excess white mucus
Leads to
excess stretching of ducts

13. MUC1 prevent immune cell/drug
interaction with cancer cells
 The MUC1 protein : the
second most targetable
tumor antigen
 MUC1 is expressed on
95% of TNBC
 Prevents hydrophobic
chemotherapeutic drugs
from passing through.
 Glycosylation causes
binding to growth factors
 MUC1 prevents drug
interaction as well as
immune cell interaction
with the receptors of
cancer cells.
Highly hydrophobic glycosylated MUC1
Triple negative breast cancer

14. Structure of normal and tumor
MUC1
 TAB004 accumulated only in the tumor, but not in
any other organs.
 Thus, TAB004 detects tMUC1 in a highly specific
manner, sparing recognition of normal tissues.

15. Construction of Chimeric Antigen
Receptor and Retroviral Vector
Production
SFG based retroviral
backbone
plasmid encoding
GP2-293 cell
SFG
retroviral
plasmid
GP2-293 cell
GP2-293 cell
CD28
TAB004
CD3z
linker
MUC28z
Chimeric antigen receptor
Retroviral
solution

16. Activated with
Anti-CD3/CD28 beads
3 days
Anti-CD3/CD28 beads
removed
Activated T cells Retroviral
supernatant
Spinoculation
IL-7 and IL-15
treatment
in vitro in vivo
T cell transduction experiment
Transduced T cells
Blood from
patient
TNBC NSG mice
MUC28z
CAR T cell

17. MUC28z CAR expression in
activated T cells
Engineered receptor MUC 28z

18. Expression level of
tMUC1 in TNBC
TNBC lysis by MUC
28 CAR T cells
using MTT assay

19. Cell surface marker expressions
by MUC 28z CAR T cells in vitro
↑ ↓ ↑ ↑

20. MUC28z CAR T cells release
antigen-specific cytokines and
chemokines
TNBC
Large amt of cytokines & chemokines release
by MUC28z CAR T cells

21. MUC 28z CAR T cells reduce
HCC70 tumor burden in vivo
↑ ↑

22. MUC28z CAR T cells long term
efficacy in tumor reduction in
vivo
• Experiment carried out until 81 days
• But, tumor progress faster after ~60 days
• Possible reasons:
 A single CAR T cells injection may not
be sufficient
 tMUC1 is lost during remaining tumor
progression
 Increased PD1 expression blocks anti-
tumor immune response
tMUC1
expression
(no change)
↑tMUC1
???

23. Conclusion
 The antigen-specific production of Granzyme B, IFN-γ, IL-2, TNF-α,
along with several other cytokines confirmed the activated and lytic
phenotype of MUC28z CAR T cells
CD28
TAB004
CD3z
linker
MUC28z
CAR T cell
 Activation of T cells more efficiently
 Release of cytokines and chemokines
 Lysis of triple negative breast cancer cell lines

24. Thank you for your
attention
* Article: CAR T Cells Targeting the Tumor MUC1 Glycoprotein Reduce Triple-
Negative Breast Cancer Growth