Co esponding au ho : V. V. Rajesham
Copy igh © 2025 Au ho (s) e ain he copy igh o his a icle. This a icle is published unde he e ms o he C ea i e Commons A ibu ion Liscense 4.0.
A e iew on ca - cell he apy- adi ional s a egies o cance ea men
V. V. Rajesham *, K. Madhu i, M. Abhi am, B. Ashmi ha and T. Rama Rao
CMR College o Pha macy, Kandlakoya (V), Medchal (M &D), Hyde abad 501401, Telangana, India.
Wo ld Jou nal o Biology Pha macy and Heal h Sciences, 2025, 21(01), 114-125
Publica ion his o y: Recei ed on 22 Oc obe 2024; e ised on 12 Decembe 2024; accep ed on 14 Decembe 2024
A icle DOI: h ps://doi.o g/10.30574/wjbphs.2025.21.1.0970
Abs ac
Cance is he wo ld's op cause o dea h. Nume ous cy o oxic immuno he apies and adi ional ea men s ha e been
c ea ed and in oduced o he ma ke . The de elopmen o a iable immuno he apy ha a ge s cance s a bo h he
cellula and gene ic le els is necessa y due o he complica ed beha iou o umo s and he in ol emen o mul iple
gene ic and cellula a iables in umo igenesis and me as asis. A new he apeu ic T cell enginee ing echnique called
chime ic an igen ecep o (CAR) T cell ea men in ol es in i o al e ing pa ien blood-de i ed T cells o exp ess
syn he ic ecep o s ha a e di ec ed agains a pa icula umo an igen. The majo his ocompa ibili y complex is no
in ol ed in hese; ins ead, he umo an igen is di ec ly iden i ied. The use o his he apy in he las ew yea s has been
success ul, wi h a educ ion in emission a es o up o 80% o hema ologic cance , pa icula ly o acu e lymphoblas ic
leukemia (ALL) and non-Hodgkin lymphomas, such as la ge B cell lymphoma. CAR he apy has he po en ial o o e a
apid and sa e ea men egime o ea non-solid and solid umo s. CAR-T cell he apy's mos signi ican bene i o e
o he cance ea men s is i s quick ime in e en ion a single in usion o CAR T cells. Addi ionally, he pa ien only has
o be p ope ly ca ed o and obse ed o wo o h ee weeks. CAR T cell he apy is conside ed a "d ug o he p esen
day," and because he cells may li e in he hos body o a long ime and ha e he capaci y o con inuously iden i y and
elimina e cance cells a e elapse, i s e ec i eness may las o decades. The p esen e iew insigh in o he s uc u e
and e olu ion o chime ic an igen ecep o s, we hen epo on he ools used o p oduc ion o CAR-T cells. Finally, we
add ess he challenges posed by CAR-T cells.
Keywo ds: Cance ; CAR-T cell; Chime ic an igen ecep o ; Acu e lymphoblas ic leukemia
1. In oduc ion
Chime ic an igen ecep o (CAR T) cell he apy is a e olu iona y new pilla in cance ea men (1). Al hough ea men
wi h CAR T cells p oduced ema kable clinical esponsible wi h ce ain subse s o B cell leukemia o lymphoma many
challenges limi he he apeu ic e icacy o CAR-T cell in solid umou and haema ological malignancy (2). CARs a e
enginee ed syn he ic ecep o ha unc ions o edi ec lymphocy es, mos commonly T cells, o ecognize and
elimina e cells exp essing a speci ic a ge an igen. When we pionee ed he i s CAR design in he la e 1980s and
ea ly1990s, i was widely known ha T cell A e e y powe ul e ec o s in he igh agains cance , bu he applica ion
o hese cells o cance pa ien s su e ed om wo majo limi a ion(3). Fi s T cell ecogni ion depends on he
exp essions o majo his ocompa ibili y complex molecules and an igen p ocessing machine y, and many umou s
silence hese pa hways as pa o hei escape om immune ecogni ion(4). second many umou s ha do no exp ess
cos imula o y molecules equi ed o igge ing he ull po ency o T cell o en ende umou -speci ic T cell
non unc ional esea che s ha e designed CARs o o e an al e na i e o con en ional T cell ecep o s (TCRs) and
ci cum en hese hu dles(1,3). The unp eceden ed success o an i CD19 CAR-T cell he apy agains B cell malignancies
esul ed in i s app o ed by he US ood and d ug adminis a ion (FDA) in 2017.howe e , he e a e majo limi a ions o
CAR-T cell he apy ha s ill mus be add essed including li e- h ea ening CAR-T associa ed oxici ies, limi ed e icacy
agains pe sis ence, poo a icking and umou in il a ion, and he immunosupp essi e ((2,5).
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1.1. Backg ound
Chime ic an igen ecep o s (CARs) a e enginee ed ecep o s ha p o ide immune e ec o cells (T cells) wi h a
cus omized speci ici y (6). CARs consis o h ee componen s: an ex acellula domain o an igen ecogni ion de i ed
om a single-chain a iable agmen (scF ) o an an ibody, a ansmemb ane segmen , and an in acellula T cell
ac i a ion domain known as CD3 (7). The pu pose o CAR-T cell he apy is o guide a pa ien ’s o dono ’s T cells o
p ecisely loca e and elimina e cance cells. This app oach holds signi ican po en ial o ea ing hema ologic cance s as
well as solid umo s, wi hou being es ic ed by majo his ocompa ibili y complex (8).
Immuno he apy has e olu ionized cance ea men , o e ing a lash o hope o pa ien s acing la e-s age me as a ic
umo s. Science magazine acknowledged i s impac , designa ing i as he “B eak h ough o he Yea ” in 2013(9).
1.2. Enginee ing o CAR-T cells
Enginee ed ecep o s named chime ic an igen ecep o s (CARs) ha e he abili y o ans e an a bi a y speci ici y on o
an immune e ec o cell (T cell). CARs a e composed o h ee componen s: a ansmemb ane domain, an in acellula T
cell ac i a ion domain o CD3, and an ex acellula an igen ecogni ion domain o he single-chain agmen a ian
(as x), which is gene a ed om an an ibody(10).
The goal o CAR-T cell he apy is o di ec T cells om a dono o pa ien o speci ically a ge and kill ume cells. Solid
ume s and haema ologic malignancies wi hou signi ican cons ain s on he majo his ocompa ibili y complex can
bene i g ea ly om his ea men (11).
1.3. S uc u e o CAR-T cells:
CARs in ol es mainly ec odomain, ansmemb ane domain and endodomain(12).
Figu e 1 S uc u e o CAR-T cell
1.4. Ec odomain
The segmen o a memb ane p o ein ha is exposed o he ou side en i onmen and no loca ed wi hin he cy oplasm
is e e ed o as he ec odomain. In his ins ance, he signal pep ide, he an igen ecogni ion a ea, and he space
cons i u e he ec odomain (7, 8).
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A signal pep ide's ole is o di ec he de eloping p o ein in o he endoplasmic e iculum (13). The a iable egions o
he hea y and ligh chains o an an ibody a e connec ed h ough a lexible linke o c ea e he single-chain a iable
agmen (scF ), which ac s as he signal pep ide o he ec odomain in a CAR. Any singula an igen capable o binding
o a ge s wi h high a ini y can be de ec ed by an an igen ecogni ion domain, equen ly ound as a single-chain
a iable agmen (scF ) wi h a undamen al ec odomain and addi ional specialized ecogni ion componen s (14, 15).
The space ac s as a b idge be ween he ansmemb ane domain and he an igen binding domain. The hinge egion o
IgG1 ep esen s he simples o m o space and is su icien o mos scF -based cons uc s.
1.5. T ansmemb ane domain
The ansmemb ane domain, consis ing o a hyd ophobic alpha helix ha ex ends ac oss he memb ane, ep esen s he
closes po ion o he endodomain o he memb ane (6, 8, 9). The e is a ela ionship be ween he s abili y o he ecep o
and he ansmemb ane domain. The a i icial TCR could in eg a e wi h he na i e TCR when he na i e CD3-ze a
ansmemb ane domain is a ailable (16, 17). A p esen , he mos s able ecep o is he ansmemb ane domain o
CD28.
1.6. Endodomain
The mos commonly ound componen o he endodomain, which is he ope a ional end o he ecep o , is CD3ζ, which
includes h ee immuno ecep o y osine-based ac i a ion mo i s (ITAMs)(8,9). A signal is ansmi ed o he T cell when
he ecep o s clus e and ac i a e upon ecognizing an an igen. Co-s imula o y signaling is essen ial du ing his phase
(17).
1.7. E olu ion o CAR-T cells
Based on he endodomain's s uc u e, CAR-T cells can be ca ego ized in o ou gene a ions since he i s c ea ion o
CARs in 1989. A g ea illus a ion o how undamen al esea ch can be applied in he clinic is he de elopmen o CAR
ea men .
Figu e 2 E olu ion o CAR-T cell
1.8. Fi s gene a ion
In he i s gene a ion o CARs, signals om he endogenous T cell ecep o (TCR) we e p ima ily ansmi ed by a single
s uc u e de i ed om he CD3 ζ-chain o FcεRIγ om he in acellula domain (17, 18). Howe e , o e ec i ely
elimina e umo cells, exogenous IL-2 needed o be adminis e ed because hese CAR-T cells couldn' p oduce su icien
in e leukin-2 (IL-2) on hei own. The e o e, he combined use o cy okines p o ed ad an ageous o he ini ial
gene a ion o CAR-T cell he apies ha u ilized single-chain ecep o s (19).
Recen s udies indica e ha he apop o ic signal may be mi iga ed by emo ing phospho yla ion om ITAM A and C in
he CD3ζ signaling po ion, which bene i s he ongoing exp ession o he ansgene (20, 21). None heless, mo e
ex ensi e clinical esea ch has been ca ied ou wi h CD3ζ-chained CAR-T cells in compa ison o FcεRIγ-chained CAR-T
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cells. This migh be a ibu ed o he p esence o one ITAM in he FcεRIγ chain e sus h ee in he CD3ζ chain.
Con e sely, al hough CAR-T cells u ilizing he CD3ζ chain demons a ed lowe exp ession le els in i o, hey p o ed
mo e e ec i e a ac i a ing T cells and elimina ing umo cells. The ansmemb ane domain o CAR-T cells comp ises a
homologous o he e ologous dime o CD3, CD8, and CD28. This ecep o is capable o acili a ing op imal cellula
ac i a ion h ough CAR dime iza ion and i s unc ional link wi h he endogenous TCR. Va ious cance s ha e been
ea ed wi h CD10-CAR-T cells, scF (G250)-CAR-T cells, GD2-CAR-T cells, alpha- ola e ecep o (FR)-CAR-T cells, and
ca cinoemb yonic Ag-speci ic CD3ζ (MFEζ)-CAR-T cells(22–27). Howe e , due o cons ained p oli e a ion, a limi ed
li espan in i o, and inadequa e cy okine elease, mos ea ly expe imen s in ol ing i s -gene a ion CAR-T cells did no
p oduce he expec ed ou comes.
1.9. Second gene a ion
T cell ac i a ion is commonly desc ibed as a p ocess equi ing wo signals. This p ocess in ol es h ee p ima y ypes o
ecep o s: co-s imula o y ecep o s, cy okine ecep o s, and T-cell an igen ecep o s. The ini ial signal is gene a ed
when he T-cell ecep o (TCR) ecognizes he an igenic pep ide-MHC complex p esen on an igen-p esen ing cells. The
seconda y signal comes om a co-s imula o y molecule, such as CD28/B7, which p omo es he p oduc ion o IL-2,
c ucial o comple ing T cell ac i a ion and p e en ing cell dea h. Naï e T cells, e en when s imula ed by an an igen,
canno pe o m hei no mal unc ions wi hou he co-s imula o y signal. The e o e, CARs ha consis solely o he CD3ζ
sequence canno e ec i ely ac i a e CAR-T cells in he absence o his co-s imula o y signal. To p o ide addi ional
signals o T cells, second-gene a ion CARs inco po a e in acellula signaling domains om a ious co-s imula o y
p o ein ecep o s, such as CD28 o 4-1BB and OX40, in o he cy oplasmic ail o he CARs. These modi ica ions can
imp o e he p oli e a ion, e ec i eness, and du abili y o CAR-T cells, as well as p olong hei li espan in i o (28–30).
In addi ion o being essen ial o he o ma ion o memo y and e ec o cells, CD28-media ed co-s imula ion in luences
he p oli e a ion and su i al o lymphocy es. CD134 can boos IL-2 p oduc ion and suppo p oli e a ion. The abili y
o CD137 o main ain he signaling o T cell esponses is i al o bo h he su i al o T cells and he memo y o CD8+ T
cells (31–33). When used in he ea men o B cell cance s, he scF CD19-CD137-CD3-CAR-T cells, MO 19-BBζ-CAR-T
cells, and scF CD19-CD28-CD3ζ-CAR-T cells demons a ed be e ou comes compa ed o he i s gene a ion (34, 35).
Al hough di ec compa isons a e lacking, i seems ha 4-1BBζ-CAR-T cells ha e a longe pe sis ence han CD28ζ-CAR-
T cells (36). CD28ζ-CAR-T cells a e capable o s imula ing, de eloping, and expanding in a consis en manne (37).
Con e sely, ea ly exhaus ion associa ed wi h 4-1BBζ-CAR-T cells may limi hei an i umo e ec i eness (38, 39).
1.10. Thi d gene a ion
To enhance po ency h ough inc eased cy okine p oduc ion and imp o ed killing capabili ies, a ious signaling
domains, including CD3ζ-CD28-OX40 o CD3ζ-CD28-41BB, we e combined o de elop hi d-gene a ion CARs (40).
While scF CD20-CD28-CD137-CD3ζ-CAR-T cells and HER2-CAR-T cells we e used o ea lymphoma and colon cance ,
he ou comes did no su pass hose achie ed wi h second-gene a ion he apies (41, 42). I is possible ha he limi ed
numbe o cases s udied con ibu ed o his esul . The e o e, u he esea ch is necessa y o e alua e he e ec i eness
and sa e y o hese ea men s, and he selec ion o co-s imula o y molecules plays a i al ole.
1.11. Fou h gene a ion
T cell edi ec ed o uni e sal cy okine-media ed killing (TRUCKs) e e s o he ou h-gene a ion CARs, de eloped by
in eg a ing IL-12 in o he ounda ional design o second-gene a ion cons uc s. TRUCKs enhance T-cell ac i a ion and
also ec ui inna e immune cells o a ge and elimina e cance cells ha do no exp ess an igens wi hin he ea ed a ea.
Explo ing how TRUCKs in luence he umo mic oen i onmen h ough he con olled elease o ansgenic immune
modi ie s could p o ide aluable insigh s. Addi ionally, hese TRUCK T cells show e icacy in managing au oimmune
diso de s, me abolic issues, and i al in ec ions (43).
When aken as a whole, hese subsequen e sions o CAR-T cell he apy ha e g ea ly inc eased in e es in cance
ea men (44).
1.12. Mechanism o ac ion
Hos cells a e collec ed and a e gene ically modi ied ex i o o exp ess a CAR a ge ing a umou speci ic an igen. The
ca cons uc consis s o an ex a cellula domain wi h a single chain a iable agmen ha a ge s he an igen o
in e es as well as an in a cellula domain ha cons uc in he T cell memb ane an ini ia e an in acellula signalling
cascade (45).
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The pa ien ’s mononuclea cells a e collec ed, which is ypically accompanied by leukaphe esis. Manu ac u e s o CAR
T cells is a complex, expensi e, and ime-in ensi e p ocess, The T-cell subse is en iched, gene ally modi ied o exp ess
he CAR o in e es , sub sequen expanded ex i o, and s o ed un il use (46). Gene ic modi ica ion o cells is
accomplished by one o h ee majo me hods cu en ly used in clinical p ac ice: - e o i al ec o s, len i i al ec o s,
o he ansposon sys em a e immunopheno ypic con i ma ion o success ul gene ic modi ica ion, a g ow h pla o m
is used o apidly expand he CAR- T cells (47).
Figu e 3 Mechanism o CAR-T cell he apy
1.13. Tools o ansduc ion o CAR-T cells
To in oduce a o eign gene in o human cells, a speci ic ool is necessa y. Cu en ly, gene in eg a ion using ec o s can
be ca ied ou h ough wo p ima y me hods: i al sys ems and non- i al sys ems.
Vi al ec o s a e p e e ed in gene he apy o bo h esea ch and clinical applica ions due o hei high e iciency in
ans e ing genes, quick abili y o achie e he necessa y quan i y o cul u ed T cells, and he a ie y o i uses a ailable,
each wi h dis inc exp ession p o iles. Mos i al sys ems can inco po a e genes om in e es ing and aluable cells and
can p o ide he s uc u al p o eins and enzymes essen ial o he gene a ion o in ec ious i al pa icles ca ied by
ec o s. Examples o i al ec o s include e o i uses like len i i uses, adeno i uses, and adeno-associa ed i uses.
Among hese, gene ically modi ied e o i uses a e he mos commonly used o gene deli e y (48). A g oup o
e o i uses is known as Re o i idae. This g oup a ies in aspec s such as hos ange, pa hogenic po en ial, s uc u e
o he genome, sequences o amino acids and nucleo ides, and hos ange. Ne e heless, he i al ec o s can pose heal h
isks. The achie ed i e is inadequa e, he abili y o ca y is limi ed, and he inse ion mu a ions used o s imula e he
immune esponse may lead o cance and oxici y (48, 49). The bene i s o non- i al gene he apy compa ed o o he
cance ea men s include enhanced e ec i eness, p ecise a ge ing, non-in ec ious na u e, unlimi ed ca ie capaci y,
cus omizable chemical composi ion, and high p oduc ion a ailabili y. Examples o non- i al ec o s comp ise molecula
conjuga es, liposomes, polyme ize s, and naked DNA(50,51). Minici cle DNA ec o s ep esen a no el class o non- i al
ec o s c ea ed in bac e ia om a pa en al plasmid, capable o p oducing high le els o ansgene exp ession in i o
while lacking plasmid bac e ial DNA sequences. This me hod is applicable in clinical se ings (52, 53).
1.14. P oduc ion o CAR-T cell
The p ocess o c ea ing CAR-T cells in ol es mul iple s eps. Addi ionally, conduc ing quali y con ol assessmen s a
e e y s age o he p o ocol is essen ial (54).
Leukocy es a e ob ained om he pa ien o dono h ough a p ocess called leukaphe esis (55). Nex , T cells a e isola ed
and cleaned o dis inguish hem om he leukocy es. Then, speci ic an ibody bead conjuga es o ma ke s a e employed
o sepa a e he T cell subse s based on hei CD4/CD8 composi ion. The inal s ep in ol es cul u ing he T cells o
ac i a e hem. This p ocedu e includes pu i ying he au ologous an igen-p esen ing cells (APCs) om he pa ien o
dono , o u ilizing beads ha a e coa ed wi h an i-CD3/an i-CD28 monoclonal an ibodies, o jus an i-CD3 an ibodies,
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wi h o wi hou eede cells and g ow h ac o s such as IL-2, which is commonly used because i enhances apid T cell
p oli e a ion; u he adjus men s o he cul u e condi ions a e made o di ec T cells owa ds a speci ic pheno ype (56).
Vi al ec o s ha a e inco po a ed in CARs guide he e e se ansc ip ion o RNA in o DNA, which hen pe manen ly
in eg a es in o he genome o he pa ien 's cells. In he ac i a ion p ocess, he i al ec o is elimina ed om he cul u e
h ough media exchange and/o dilu ion. Due o hei be e p o ile o in eg a ion si es, len i i al ec o s a e mo e
commonly u ilized in clinical ials compa ed o gamma e o i al ec o s.(57)
mRNA ans ec ion and he Sleeping Beau y ansposon sys em ep esen addi ional me hods. Howe e , many
ques ions emain un esol ed, such as he equi emen o mul iple in usion cycles wi h empo a y mRNA ans ec ion
and he isks o inse ional mu agenesis and ansposon emobiliza ion when using he Sleeping Beau y ansposon
sys em (58).
CAR-T cells a e cul i a ed using h ee dis inc bio eac o cul u e sys ems: CliniMACS P odigy, G-Rex, and WAVE
Bio eac o (59). A signi ican d awback o he i s wo me hods is ha he lask mus be opened du ing cell inocula ion.
Con e sely, he CliniMACS P odigy sys em se es as a comp ehensi e ool ha e ec i ely en iches, ac i a es,
ansduces, and cul i a es he cells (60). Once he cells each he necessa y quan i y o he apeu ic use, hey a e
ex ac ed and adminis e ed o he pa ien s.
1.15. The clinical success o CAR-T cell he apy
A pa ien a NCI su e ing om ad anced ollicula lymphoma and pa ien s a MSKCC wi h e ac o y CLL and elapsed
B-cell ALL bo h demons a ed p og ess ollowing second-gene a ion CAR T cell he apy (61, 62). A e o i al ec o
named MSGV was u ilized o deli e a CD19-speci ic CAR as pa o he ea men a NCI. This CAR was designed o
a ge he CD19 p o ein ound on he su ace o B-lineage cells, using an an i-CD19 scF de i ed om he mu ine
monoclonal an ibody FMC63. I inco po a ed bo h a CD3z endodomain and a CD28 cos imula o y endodomain.
Following lymphodeple ion, he pa ien ecei ed wo in usions o CAR T cells and eigh doses o IL-2. As a esul o his
he apy, he pa ien unde wen selec i e elimina ion o B-lineage cells and achie ed a pa ial emission o he lymphoma
(61). Au ologous CD19- a ge ed CAR T cells ea u ing he second-gene a ion CAR (19-28z) we e e alua ed o sa e y
and long-las ing e ec s in pa ien s wi h B-ALL and CLL ha had ei he elapsed o we e esis an o chemo he apy in
he MSKCC Phase 1 ial. Pa ien s who had p e iously ecei ed cyclophosphamide aining exhibi ed a pa ial esponse,
whe eas hose who had no been ained showed no measu able eac ions o hei disease (62).
When D . Ca l June and his eam a he Uni e si y o Pennsyl ania sha ed hei indings ha h ee adul pa ien s
su e ing om ad anced ch onic lymphocy ic leukemia (CLL) achie ed ei he comple e o pa ial emission a e
unde going CD19-speci ic CAR T cell he apy, i ep esen ed a majo b eak h ough in he applica ion o CAR T cell
he apy(63,64).
The cons uc u ilized in his ial included he 4-1BB cos imula o y endodomain, he CD3z signaling endodomain, and
an an i-CD19 scF de i ed om FMC63. An EF1-a p omo e -d i en len i i al ec o was employed o exp ess his
cons uc . Following injec ion, he CAR T cell coun s in pa ien s inc eased signi ican ly, o en by a ac o o 1,000. These
esul s enabled he ea men o ad anced cases o CLL and o he B-cell malignancies using second-gene a ion CAR T
cell he apy.
The ou comes o hese clinical s udies e ealed ha lymphodeple ion p io o ea men —speci ically a ype o
chemo he apy ha educes he immune cell coun —is essen ial o he success o CAR T cell he apy. Con e sely, i
appea s ha IL-2 is no necessa y. D . S e en Rosenbe g's eam was he i s o demons a e ha lymphodeple ing
chemo he apy is e ec i e. They ound ha he combina ion o cyclophosphamide and luda abine led o he in i o
g ow h and mo emen o injec ed umo - eac i e T cells owa d umo loca ions (65–67). The p ocess o
lymphodeple ion migh include educing he coun o na i e lymphocy es ha compe e wi h he in used T cells and
inc easing he ci cula ing le els o T cell g ow h ac o s such as IL-15. This would p omo e mo e e ec i e g ow h o he
adminis e ed T cells wi hin he hos 's body (68).
1.16. Challenges o CAR-T cell he apy
Al hough CAR-T cells ha e been u ilized in clinical se ings, se e al ques ions pe sis , including he mos sui able ec o
and he sa e y p o ile o e he long e m. A c ucial elemen o he CAR-T cell p oduc is he i al ec o esponsible o
in oducing he CARs in o he T cells. La ge quan i ies o he CAR-encoding i al ec o can be p oduced and s o ed a -
80 °C o as long as nine yea s (69). Since hese cells will be in used in o he pa ien s, i is c ucial o he ec o o be
s e ile. The hi d gene a ion appea s o be he sa es minimum len i i al ec o ound so a (70). I is essen ial o
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conduc quali y con ol es ing on sa e y, s e ili y, i e , pu i y, and po ency. Gi en ha he e a e mul iple sou ces o
ec o s, i is i al o e alua e hei unc ion, s abili y, and pu i y. Ideally, CAR-T cells should be p oduced using a single
ec o while moni o ing hese a iables. A conce n ha a ises is he po en ial o inse ional mu agenesis esul ing om
he in eg a ion o ec o DNA in o hos cells. In compa ison o o he ec o s, len i i al ec o s migh pose a lowe isk
o mu agenesis (71). On ano he no e, e o i al and len i i al ec o s possess he po en ial o induce cance . The long-
e m sa e y o using i al ec o s in CAR-T cell he apy emains unce ain. Thus, i is i al o moni o any possible long-
e m nega i e e ec s linked o hese ec o s. The impac o du able CAR-T cells on u u e p egnancies is s ill no ully
unde s ood.
To ensu e e ec i e handling o ma e ials and pa ien scheduling h oughou he he apy, he e needs o be seamless
coo dina ion among he collec ion, manu ac u ing, and ea men loca ions. Consequen ly, es ablishing a s anda dized
manu ac u ing p ocess o CAR-T cells is essen ial o iden i y he c i ical quali y a ibu es and desi ed p oduc
cha ac e is ics. Fu he mo e, since a ious p oduc s exhibi di e ing CAR iabili y, pheno ype, and posi i i y, gaining
deepe insigh s in o hese p ocesses is impo an (72). Leukaphe esis p oduces a di e se a ay o s a ing ma e ials,
making i di icul o compa e he esul ing p oduc s.
While CAR-T cell he apy has demons a ed encou aging esul s in ea ing hema ological cance s, solid umo s p esen
ongoing challenges due o immune-supp essi e umo en i onmen s, he loss o an igens in umo cells, and a sho age
o speci ic an igens (73, 74). Consequen ly, no able p og ess has been achie ed by in eg a ing CARs wi h a ious
e ec o molecules; including PD1 swi ch ecep o s, an i-oxidan enzymes, ma ix deg ada ion enzymes, and o he s, o
enhance he e icacy o CAR-T cell he apy (75–79). Se e e cy okine elease synd ome is he p ima y side e ec
associa ed wi h CAR-T cell he apy (CRS). I is i al o minimize side e ec s wi hou comp omising he apeu ic
e ec i eness, e en hough mos ad e se eac ions ollowing CAR-T cell he apy can be managed wi h exis ing
ea men s. Simila ly, imp o ing he sa e y o CAR-T cell he apy is essen ial o inc easing he speci ici y o he modi ied
T cells (80).
2. The majo challenges include
2.1. Se e e ad e se e en s
CAR T cell he apy has shown g ea p omise in he ea men o hema ological cance s, bu one majo conce n wi h his
app oach is he po en ial o li e- h ea ening ad e se e en s. Two o he mos common ad e se e en s a e cy okine
elease synd ome (CRS) and immune e ec o cell-associa ed neu o oxici y synd ome (ICANS) (81).CRS is media ed by
he cy okines IL-1 and IL-6, which can cause e e , hypo ension, and o he sys emic symp oms. To mi iga e he isk o
CRS and neu o oxici y, he FDA app o ed he use o he humanized an i-IL6 ecep o an ibody ocilizumab in 2017 o
CAR T cell he apy. O he po en ial ea men s o hese ad e se e en s include he IL-1 ecep o an agonis anakin a
and he an i-IL6 chime ic an ibody sil uximab(82). The se e i y o ad e se e en s associa ed wi h CAR T cell he apy is
in luenced by se e al ac o s, including he p e ea men umo bu den, lymphodeple ion egimen in ensi y, and CAR
T cell dose (83). Du ing CD19-CAR T cell he apy, ele a ed cy okine le els ha e been associa ed wi h p io umo bu den
(84, 85). Tumo load can impede he a es o ull emission and po en ially o e all su i al, bu i does no seem o
in luence CD19-CAR T cell g ow h peaks (86).
2.2. High cos o manu ac u ing au ologous CAR T cells
One d awback o he p esen CAR T cell he apy is he expensi e cha ges o p oducing au ologous CAR T cells, which
can cause pa ien s wi h se e e CRS o spend up o $500,000 on ea men o e all (87). I also akes be ween 21 and 35
days on a e age o manu ac u e au ologous CAR T cells. Du ing his wai ing pe iod, pa ien s migh need b idging
he apy and occasionally pass away om quickly p og essing illness wi hou ecei ing he bene i s o CAR T cell
he apy. Addi ionally, T cells om heal hy dono s may be mo e ac i e han T cells om sick pa ien s due o i edness.
The e o e, a numbe o app oaches a e being in es iga ed o make his ea men mo e accessible and economical, such
as he use o comme cially a ailable allogenic CAR (allo-CAR) T cells and in i o CAR T cell p oduc ion. Gene a ing CAR
T cells in i o is an in iguing cos -e ec i e ac ic. By deli e ing mRNA encoding he FAP- a ge ing CAR in lipid
nanopa icles (LNPs), ansien CAR T cells can be p oduced in i o (88).
3. Conclusion
CAR-T ea men o pa ien s wi h umo s has shown p omising ou comes; howe e , many emaining challenges need
o be conside ed. The high quali y o CAR-T p oduc s needs o be ensu ed h ough op imiza ion o p o ocols, and he
long- e m sa e y equi es u he s udy.
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Compliance wi h e hical s anda ds
Disclosu e o con lic o in e es
No con lic o in e es o be disclsoed.
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