Hair­pins and Scis­sors - De­liv­er­ing a Non-Gene Edit­ed Al­lo­gene­ic CAR T Cell Ther­a­py for the Mass­es

Overview

The li­cens­ing of two Chimeric Anti­gen Re­cep­tor (CAR) T cell ther­a­pies for the treat­ment of B cell ma­lig­nan­cies un­der­scores the po­ten­tial of cell based im­mune ther­a­py to de­liv­er im­pres­sive durable clin­i­cal re­spons­es¹. These prod­ucts are au­tol­o­gous in na­ture which in­volves col­lect­ing im­mune cells from the pa­tient that are used to man­u­fac­ture the CAR T cells. Once pro­duced, these CAR T cells are then re­in­fused as the clin­i­cal prod­uct to the pa­tient. How­ev­er, there are sig­nif­i­cant chal­lenges to au­tol­o­gous ther­a­py, in­clud­ing prod­uct pro­duc­tion time (which cur­rent­ly takes weeks) dur­ing which the pa­tient’s dis­ease may progress, and the high­ly vari­able qual­i­ty of the start­ing ma­te­r­i­al, which can re­sult in man­u­fac­tur­ing fail­ures.

Al­lo­gene­ic CAR T cell ther­a­py, an off the shelf ap­proach that can be ad­min­is­tered when re­quired, is the ide­al so­lu­tion. This ap­proach gen­er­ates cells from a healthy donor to form a bank of CAR T cells that can be used as need­ed. The key chal­lenge of al­lo­gene­ic CAR T is over­com­ing a tox­i­c­i­ty as­so­ci­at­ed with the recog­ni­tion of healthy pa­tient tis­sues by the al­lo­gene­ic CAR T cells. This is me­di­at­ed by the T cell Re­cep­tor (TCR). Dis­rupt­ing the TCR un­der­pins all cur­rent al­lo­gene­ic CAR T strate­gies².

Hair­pins and Scis­sors

Cur­rent­ly, gene edit­ing tech­nolo­gies used to gen­er­ate al­lo­gene­ic CAR T are at an ear­ly stage of clin­i­cal de­vel­op­ment. The dif­fer­ent gene edit­ing ap­proach­es are all based on cut­ting the genome with­in one of the genes that en­code the TCR, which per­ma­nent­ly re­duces ex­pres­sion of the en­tire TCR com­plex. Whilst an el­e­gant ap­proach, this scis­sor strat­e­gy has been chal­leng­ing to move in­to clin­i­cal test­ing due to po­ten­tial prod­uct safe­ty con­cerns – main­ly en­sur­ing the ab­sence of ‘off-tar­get’ genome cut­ting dur­ing gene edit­ing³.

Al­ter­na­tive­ly, tar­get­ing gene ex­pres­sion at the mR­NA lev­el does not in­volve cut­ting the genome and avoids jeop­ar­diz­ing genome in­tegri­ty. To de­liv­er this mR­NA ‘edit­ing’, Celyad On­col­o­gy em­ploys short hair­pin RNA (shRNA), a method used over sev­er­al decades to knock­down gene ex­pres­sion⁴. The ap­proach in­volves us­ing an shRNA that has a com­ple­men­tary se­quence to that of the tar­get gene. In oth­er words, a tar­get­ed shRNA can specif­i­cal­ly re­duce the lev­el of a de­sired pro­tein such as the TCR com­plex by in­ter­fer­ing with mR­NA and not by cut­ting the genome⁵.

Cen­tral to this is the All-in-One vec­tor ap­proach. In one step, a sin­gle reagent (vec­tor) in­tro­duced in­to healthy donor T cells re­sults in the si­mul­ta­ne­ous pro­duc­tion of all el­e­ments in the T cell that can re-di­rect the T cell against tu­mor (the CAR), elim­i­nate the TCR (shRNA) and pro­vide a han­dle where the mod­i­fied cells can be en­riched in man­u­fac­tur­ing (the mark­er).

shRNA in an Al­lo­gene­ic CAR T Cell Plat­form

The CD3 ze­ta sub­unit pro­vides the main sig­nal­ing pow­er to the TCR that en­ables ac­ti­va­tion and en­gage­ment of the T cells killing abil­i­ty. Through the se­lec­tion of an op­ti­mal shRNA and process de­vel­op­ment, tar­get­ing CD3 ze­ta re­sults in durable high-lev­el knock­down of the TCR on pri­ma­ry T cells to a lev­el equiv­a­lent to that seen if the CD3 ze­ta gene was gene edit­ed (Fig­ure 1A). Func­tion­al­ly, this cor­re­lates with an in­abil­i­ty of these cells to re­spond to a mi­to­genic stim­u­lus (aka TCR dri­ven T cell ac­ti­va­tion; Fig­ure 1B) and a cor­re­spond­ing ab­sence of tox­i­c­i­ty when these cells are in­fused in­to the gold stan­dard in vi­vo test mod­el (Fig­ure 2A, B). In­ter­est­ing­ly, the per­sis­tence of the shRNA tar­get­ed T cells was much longer than that of the CRISPR-Cas9 gene edit­ed equiv­a­lent cells (Fig­ure 2C). The mech­a­nism is still be­ing in­ves­ti­gat­ed but clear­ly shows that T cells ex­press­ing a sin­gle shRNA against CD3 ze­ta can form a plat­form suit­able for al­lo­gene­ic CAR T cell ther­a­py.

Fig­ure 1: (A) Sur­face ex­pres­sion of TCR as as­sessed by de­tec­tion of the TCRαβ sub­unit by flow cy­tom­e­try, and (B) IFN-γ se­cre­tion in the su­per­natant up­on 24-hour cul­ture with in­creas­ing con­cen­tra­tion of an­ti-CD3 an­ti­body as TCR mi­to­genic stim­u­lus, of T cells trans­duced with con­trol vec­tor (Con­trol), trans­duced with a vec­tor con­tain­ing shRNA tar­get­ing CD3 ze­ta (shRNA-CD3 ze­ta) or trans­fect­ed with CRISPR tar­get­ing the CD247 gene that en­codes CD3 ze­ta (CRISPR-CD3 ze­ta).

Click on the im­age to see the full-sized ver­sion

Fig­ure 2: (A) Weight ki­net­ics, (B) Ka­plan-Meier sur­vival curves, and (C) T cell en­graft­ment in NSG mice (n=5 per group) in­ject­ed with 20×10⁶ T cells trans­duced with con­trol vec­tor (Con­trol), trans­duced with a vec­tor con­tain­ing shRNA tar­get­ing (shRNA-CD3 ze­ta) or trans­fect­ed with CRISPR tar­get­ing the CD247 gene that en­codes CD3 ze­ta (CRISPR-CD3 ze­ta), 1 day af­ter re­ceiv­ing 1.44 Gy to­tal body ir­ra­di­a­tion.

Click on the im­age to see the full-sized ver­sion

CYAD-211; The First shRNA-Based Non-Gene Edit­ed Al­lo­gene­ic CAR T cell ther­a­py

Celyad On­col­o­gy’s first clin­i­cal can­di­date em­ploy­ing shRNA tech­nol­o­gy in an al­lo­gene­ic con­text is CYAD-211, an an­ti-BC­MA ther­a­py. The three key el­e­ments of CYAD-211 are:

  1. A sec­ond gen­er­a­tion CAR that em­ploys a BC­MA-spe­cif­ic en­gager
  2. A sin­gle shRNA that tar­gets CD3 ze­ta
  3. A mark­er gene that al­lows di­rect en­rich­ment of the en­gi­neered al­lo­gene­ic CAR T cells, as well as non-mod­i­fied cells to be re­moved in a sin­gle man­u­fac­tur­ing step

Pre-clin­i­cal stud­ies con­firmed that T cells en­graft­ed with a BC­MA CAR co-ex­press­ing the CD3 ze­ta tar­get­ing shRNA ex­hib­it­ed ro­bust an­ti-tu­mor ac­tiv­i­ty in vit­ro and in vi­vo with no ev­i­dence of tox­i­c­i­ty (Fig­ure 3). Im­por­tant­ly, the speed of tak­ing this non-gene edit­ed ap­proach from ini­tial con­cept to clin­i­cal tri­al test­ing in around two years is a tes­ta­ment to the po­ten­tial of the tech­nol­o­gy from a reg­u­la­to­ry con­text

Fig­ure 3: Ka­plan-Meier sur­vival curves of NSG mice (n=5 per group) in­tra­venous­ly in­ject­ed with ve­hi­cle or 10⁷ T cells trans­duced with a con­trol vec­tor (mock) or with a vec­tor con­tain­ing a BC­MA-tar­get­ing CAR and a shRNA tar­get­ing CD3 ze­ta, 6 days fol­low­ing in­tra­venous in­jec­tion of 5×10⁶ KMS-11 can­cer cells.

Click on the im­age to see the full-sized ver­sion

The Fu­ture of Non-Gene Edit­ed Al­lo­gene­ic Ap­proach­es

CYAD-211’s fu­ture in-hu­man tri­als aim to prove the po­ten­tial for a sin­gle shRNA al­lo­gene­ic ap­proach. How­ev­er, the small size of the trans­gene and po­ten­tial to ex­press mul­ti­ple shRNAs (mul­ti­plex­ing) will en­able fu­ture clin­i­cal can­di­dates to mod­u­late the ex­pres­sion lev­el of mul­ti­ple gene prod­ucts, there­by gen­er­at­ing T cells with de­sired phe­no­typ­ic and func­tion­al prop­er­ties. Im­por­tant­ly, as com­pared to gene edit­ing, the lev­el of gene knock­down can al­so be con­trolled through the choice of the spe­cif­ic shRNA.

This is par­tic­u­lar­ly im­por­tant where the knock­out of a pro­tein may cause is­sues of vi­a­bil­i­ty, such as with es­sen­tial ki­nas­es, or through the knock­out of a pro­tein like HLA class 1 which would sen­si­tize the CAR T cell to nat­ur­al killer cell elim­i­na­tion.

In ad­di­tion, shRNA mul­ti­plex­ing pro­vides a means to gen­er­ate an op­ti­mal ther­a­peu­tic T cell phe­no­type with a strong con­trol on one of the ma­jor raw ma­te­r­i­al costs since all these el­e­ments are main­tained with­in a sin­gle vec­tor. En­gi­neer­ing mul­ti­ple knock­downs us­ing cur­rent gene edit­ing ap­proach­es will re­quire an in­creas­ing num­ber of clin­i­cal grade reagents, which the All-in-One vec­tor ap­proach avoids.

Over­all, shRNA pro­vides a po­tent plat­form with the pow­er to con­trol the lev­el of sin­gle or mul­ti­ple genes to op­ti­mize al­lo­gene­ic CAR T cell prod­ucts.


Ref­er­ences

  1. June CH, O’Con­nor R S, Kawalekar OU, Ghas­se­mi S & Milone MC. CAR T cell im­munother­a­py for hu­man can­cer. Sci­ence 359, 1361–1365 (2018)
  2. De­pil S, Duchateau P, Grupp SA, Mufti G & Poirot L. ‘Off-the-shelf’ al­lo­gene­ic CAR T cells: de­vel­op­ment and chal­lenges. Nat. Rev. Drug Dis­cov. 19, 185–199 (2020)
  3. Kosic­ki M, Tomberg K & Bradley A. Re­pair of dou­ble-strand breaks in­duced by CRISPR–Cas9 leads to large dele­tions and com­plex re­arrange­ments. Nat. Biotech­nol. 36, 765–771 (2018)
  4. Bara­ta P, Sood AK & Hong DS. RNA-tar­get­ed ther­a­peu­tics in can­cer clin­i­cal tri­als: Cur­rent sta­tus and fu­ture di­rec­tions. Can­cer Treat. Rev. 50, 35-47 (2016)
  5. Gier­ing JC, Grimm D, Storm TA & Kay MA. Ex­pres­sion of shRNA from a tis­sue-spe­cif­ic pol II pro­mot­er is an ef­fec­tive and safe RNAi ther­a­peu­tic. Mol. Ther. 16, 1630-1636 (2008)

AUTHOR

David Gilham

CSO, Celyad Oncology