Recent progressing approaches to improve the efficiency of CAR-T cell immunotherapy against solid tumors. 1. Promoting CAR-T cell trafficking and infiltration at tumor sites through approaches like engineering CAR-T cells to express the enzyme heparanase to degrade tumor extracellular matrix, or incorporating chemokine receptor genes into CAR-T cells to guide them to tumor locations. 2. Preventing tumor immune escape by blocking immune checkpoint molecules like PD-1 and CTLA-4 that inhibit CAR-T cells, or knocking out the adenosine 2A receptor to reduce CAR-T cell dysfunction. 3. Developing bi-specific CAR-T cells targeting two different tumor antigens to reduce immune escape, and

1. RECENT PROGRESSING APPROACHES TO IMPROVE EFFICIENCY OF CAR-T CELL
IMMUNOTHERAPY AGAINST SOLID TUMORS
Topic Of Review Report presentation
Sushma Ahirwar, M.Sc. Biotechnology
BSBE Department IIT-Indore
Presented By-
Sushma Ahirwar, Roll no. 2103171011
M.Sc. Biotechnology BSBE Department IIT-I

2. Discussion points
 Introduction
 CAR-T Cell Overview
 Challenges of CAR-T cell therapy against solid tumors
 Recent progressing approaches to increase CAR-T efficacy against solid tumors-
1. Prevent tumor immune escape
2. Promoting CAR-T trafficking and infiltration
3. Reducing CAR-T therapy toxicity issues-
 Conclusion and future perspectives
 Acknowledgment
 References

3. Phase 3 escape
Stages of cancer progression
Phase 1 elimination Phase 2 equilibrium
Images Reference – Kuby Immunology book, Chapter 19 Cancer and immune system.

4. Cytotoxic T
cell
Target cell
TCR
CD 8
MHC 1 Antigen
B 7 CD28
Signal 1
Signal 2
Cytotoxic T cell
Target cell
TCR
CD 8
MHC 1 Antigen
B 7 CD28
MHC 1
Signal 1
Signal 2
Signal 3
Granzyme
and
perforin
Perforin
Channel
Granzyme
triggers
apoptosis
of target
cells
Overview of Cytotoxic T cell’s activity So what happens in case of cancer cells which cause
immune escape ?
Progressive
loss of class
1 MHC
Tumor cell
immune escape
Class 1 MHC

5. Limitations of conventional therapy in
cancer treatment-
* Adverse side effects on body.
* Drug resistance of cancer stem cells
due to elevated ABC transporter.
* Tumor heterogeneity a big challenge
etc.
Emergence of cancer immunotherapy
For specific and targeted killing of tumor
* By using mAb against tumor antigen
* Bi-specific antibodies
* Adoptive T cell therapies etc.

7. CAR-T cell (Chimeric antigen receptor-T cell)
 Genetically engineered T cells derived from patient’s own body.
 CAR-T cells have capability to identify tumor antigen in MHC independent manner by its scFv domain.
Linker
single chain variable
fragment
Hinge
Extra cellular domain
Intra cellular domain
Transmembrane domain
Stimulatory region ( CD 3ζ )
Co-Stimulatory region ( CD28)
Cell membrane
CAR-T receptor complex
Reference- Sadelain, Riviere & Brentjens, Nat Rev Cancer, 2003 Sadelain, AACR Education Program, 2014

8. Historical perspective of generation of ‘chimeric antigen receptor T cell design’
Image REFERENCE- https://doi.org/10.2174/1389201019666180418095526

9. 2014-
FDA designate CARs a ‘Breakthrough
therapy’ for cancer.
2017-
FDA approved CD-19 CAR-T cell
immunotherapy for treating blood
malignancies such as ALL ( acute
lymphoblastic leukaemia).

10. Challenges Associated with CAR-T cell immunotherapy against solid tumors

11. Tumor Marker Type of tumor
Her-2/neu Breast
Progesterone receptors Breast
PSA (Prostate specific antigen) Prostate
CA15-3, BR 27.29 Breast
CA 125 Ovarian
Breast
CA 72.4, CA 19-9, CEA Gastric Gastric
NSE, CYFA 21.1 Lung Lung
CA 125 Ovarian
mesothelin mesothelioma and ovarian cancer
Alpha-fetoprotein (AFP) Liver cancer and germ cell tumors
List for some solid tumor markers
Reference-https://www.cancer.gov/about-cancer/diagnosis-staging/diagnosis/tumor-markers-list

12. A. Immunofluorescence staining . HPSE is stained with
red fluorescent dye (Alexa Fluor 555)….
Abbreviations - M- monocyte, FI-T- Freshly isolated T
cells, LTE –T- Long time expanded T cell
Recent progressing approaches to improve the efficiency of CAR-T cell immunotherapy against solid tumors
1. Promoting trafficking and infiltration of CAR-T cells at the tumor site-
1.1 HPSE CAR-T cells- Degrade ECM
 Long time in vitro–cultured T lymphocytes lack
expression of the enzyme heparanase (HPSE)(fig A),
which degrades heparan sulfate proteoglycans, the
main components of ECM.
 Therefore engineered CAR-T cells to express HPSE
(HPSE-CAR-T) and showed their improved capacity to
degrade the ECM, which promoted tumor T cell
infiltration and antitumor activity (Fig B and C)
B. Flow cytometry analysis of CD3+ T
cells detected within the tumor samples
C. Immunohistochemical analysis showing CD3+ T cell infiltration in NB
tumor CHLA-255 cells implanted in the kidney of mice infused with either
CAR+ or CAR(I)HPSE+ LTE-T cells.
Reference- Caruana et al., http://www.nuatre.com/articles/nm.3833

13. 1.2 Incorporation of chemokine receptor gene in CAR-T cell-
 Chemokines like CXCL1,CXCL2 etc. are involved in
the development and metastasis of HCC
(hepatocellular carcinoma), these chemokines can
be utilized as therapeutic targets and combined
with CAR-T cell therapy. HCC express high level of
CXCR2 ligands.
Fig (B, C, D) -enhanced migration and infiltration of
CXCR2 CAR-T cells to tumors.
 Mice were inoculated with HuH-7 tumor cells, and
then treated with Control T, Mock CAR-T, or CXCR2
CAR-T cells.
 Tumors were resected and tumor-infiltrated human
T cells were detected by flow cytometry and
immunohistochemistry (IHC) on day 3, 5, and 7 after
T-cell treatment.
Reference- (Liu et al.) , https://doi.org/10.1002/eji.201948457

14. 1.3 - CLTX CAR-T cell
 Chlorotoxin (CLTX) is venom derived from the scorpion (Leiurus quinquestriatus)
 It bind effectively with GBM and reduce the GBM metastasis
 Do not found to be cytotoxic to kill tumor so still need to be improve.
A) CLTX binds broadly to freshly-dispersed primary GBM cells and to cultured tumor
lines Freshly dispersed viable (DAPI-) patient brain tumor (PBT) GBM cells (CD45- CD31-)
were immunostained for expression of IL13Rα2, HER2, and EGFR, or binding by CLTX-
Cy5.5. Percentages of stained cells (blue) above isotype control (grey) are indicated in
each histogram.
B
B) Percent total target cell killing of different PBT-TS cells co-cultured
with mock, CLTX-CAR, or IL13Rα2-CAR T cells
Reference- (Wang et al.), https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500824/

15. 2. Approaches to prevent the immune escape of tumor cells-
2.1 PD-1 and CTLA-4 checkpoint inhibition /gene knock out-
Fig. Tumor regressions of patients who received concurrent
nivolumab and ipilimumab
Nivolumab- PD-1 inhibitory mAb
Ipilimumab- CTLA-4 inhibitory mAb
Panel A- 1 mg/kg nivolumab + 3
mg/kg ipilimumab
Panel B- 0.3 mg/kg nivolumab + 3
mg/kg ipilimumab
 CTLA-4 -cytotoxic T lymphocyte associated protein 4
 PD-1 -programmed cell death 1
 CTLA-4 is expressed by activated and regulatory T
cells (Tregs), and exerts competitive binding for
stimulatory CD28 ligands (CD80/CD86).
 PD-1 is expressed by activated and exhausted T cells,
and binding to its ligands PD-L1 and PDL-2 (On
tumor cell surface) directly inhibits TCR signalling.
 So these checkpoints cause dysfunction of CAR-T
cells when cultured with tumor cell lines.
 Hence PD 1 and CTLA-4 checkpoint blocade/gene
knockout in CAR-T cells may proved as a novel
approach.
Reference- Wolchok et al. 2013, DOI 10.1056/NEJMoa1302369

16. 2.2 – Adenosine 2A (A2A) receptor gene knockout –
P4 CAR-T cells were cocultured with CRL5826 (lung carcinoma)in the
presence or absence of various doses of 2-chloroadenosine (CADO),
(adenosine analog),
graph A -Tumor cell killing by P4 CAR-T cells was inhibited in the presence of
CADO in a dose-dependent manner .
graph B-cytokine IFN-γ secretion of P4 CAR-T cells were reduced in the
presence of CADO as well .These results confirmed that CADO could inhibit
the tumor cell killing capacity and the cytokine release of CAR-T cells.
Adenosine
Adenosine 2A
receptor
CAR -T cell
Regulatory T cells
CD39
CD 73
CAR-T
dysfunction
Fig. -schema illustrating inhibition of T effector cells by the
interaction between adenosine produced by T-regs and A2A
References- (Li et al.), https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781731/

17. Graph C- Increment in tumor cell killing after culturing of CRL5826 with AKO (A2A receptor knockout P4 CAR T
cells) in the presence of different dose concentration of CADO
graph D -and also increase in the level of IFN-γ has been seen.
C D
References- (Li et al.), https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781731/

18. 2.3 Development of bi-specific CAR-T cells-
the number of residual CAPAN1 tumor cells after a 72-
hour coculture with NT, CAR-MUC1, CAR-PSCA, or
bispecific CAR (CAR-MUC1+ CAR-PSCA)
number of residual CAPAN1 tumor cells after a 72-hour
coculture with NT, CAR-MUC1, CAR-PSCA, or bispecific CAR
(CAR-MUC1+ CAR-PSCA)
Reference- Anurathapan et al., https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945803/, DOI 10.1038/mt.2013.262

19. 3. Approaches to reduce toxicity issues of CAR-T therapy-
3.1 Identification of highly expressed tumor-associated antigen and tumor specific antigen-
3.2 Induction of negative feedback signaling in CAR-T cells (inhibitory CAR-T cells- iCAR-T cell).
3.3 Suicide gene incorporation in CAR-T cells.
first suicide gene used is herpes simplex virus (HSV) thymidine kinase, it make the cell susceptible to
ganciclovir. Ganciclovir is a competitive inhibitor of dGTP ( deoxyguanosine triphosphate), it's binding with
DNA inhibits the DNA replication and ultimately cause apoptosis of that cell due to inhibition of DNA
replication (Bonifant et al.)

20. Conclusion and future perspectives
 To improve CAR-T cell immunotherapy, researchers have identified various approaches such as checkpoint receptor
inhibition, bispecific CAR-T cell, HPSE CAR, chemokine-receptor containing CAR (CXCR2-CAR), chlorotoxin-CAR, etc.
 Sometimes a single approach doesn’t work against a particular solid tumor, so the combination of two approaches
may prove effective for treating a particular solid tumor type.
 Need to study a tumor microenvironment and T cells’ antitumor activity pathways in detail, that we can use a tool to
modify CAR-T cells for improving its therapeutic efficiency against solid tumors.
 Identification of tumor-specific and tumor-associated antigens to reduce on-target/off-tumor cytotoxicity
 CLTX-CAR-T cell is still a new approach, its other aspects need to study more such as its safety, efficacy, exact
receptor for CLTX binding, and CLTX cytotoxicity, etc.

21. THANK YOU !