Reducing CD33 expression to selectively protect therapeutic cells
Systems and methods to selectively protect therapeutic cells by reducing CD33 expression in the therapeutic cells and targeting non-therapeutic cells with an anti-CD33 therapy. The selective protection results in the enrichment of the therapeutic cells while simultaneously targeting any diseased, malignant and/or non-therapeutic CD33 expressing cells within a subject.
1. A genetic construct comprising a CD33 blocking molecule selected from SEQ ID NO: 8 or SEQ ID NO: 9.
2. The genetic construct of claim 1 , wherein the genetic construct is within a viral vector.
3. The genetic construct of claim 2 , wherein the viral vector is a lentiviral vector, a foamy viral vector, or an adenoviral vector that optionally comprises a PGK promoter.
4. The genetic construct of claim 1 , further comprising a therapeutic gene.
5. The genetic construct of claim 4 , wherein the therapeutic gene:
(i) comprises FancA, FancB, FancC, FancD1, FancD2, FancE, FancF, FancG, FancI, FancJ, FancL, FancM, FancN, FancO, FancP, FancQ, FancR, FancS, FancT, FancU, FancV, or FancW; or
(ii) encodes a checkpoint inhibitor, a gene editing molecule, a chimeric antigen receptor that specifically binds a cellular antigen or a T-cell receptor that specifically binds a cellular antigen; or
(iii) comprises γC, JAK3, IL7RA, RAG1, RAG2, DCLRE1C, PRKDC, LIG4, NHEJ1, CD3D, CD3E, CD3Z, CD3G, PTPRC, ZAP70, LCK, AK2, ADA, PNP, WHN, CHD7, ORAI1, STIM1, CORO1A, CIITA, RFXANK, RFX5, RFXAP, RMRP, DKC1, TERT, TINF2, DCLRE1B, or SLC46A1; or
(iv) comprises factor VIII (FVIII), FVII, von Willebrand factor (VWF), FI, FII, FV, FX, FXI, or FXIII); or
(v) comprises F8 or F9; or
(vi) comprises γ-globin; soluble CD40; CTLA; Fas L; an antibody to CD4, CD5, CD7, CD52; an antibody to IL1, IL2, IL6; an antibody to TCR specifically present on autoreactive T cells; IL4; IL10; IL12; IL13; IL1Ra, sIL1RI, sIL1RII; sTNFRI; sTNFRII; an antibody to TNF; P53, PTPN22, and DRB1*1501/DQB1*0602; globin family genes; WAS; phox; dystrophin; pyruvate kinase (PK); CLN3; ABCD1; arylsulfatase A (ARSA); SFTPB; SFTPC; NLX2.1; ABCA3; GATA1; ribosomal protein genes; TERC; CFTR; LRRK2; PARK2; PARK7; PINK1; SNCA; PSEN1; PSEN2; APP; SOD1; TDP43; FUS; ubiquilin 2; or C9ORF72; or
(vii) comprises ABLI, AKT1, APC, ARSB, BCL11A, BLC1, BLC6, BRCA1, BRCA2, BRIP1, C46, CAS9, C-CAM, CBFAI, CBL, CCR5, CD19, CDA, C-MYC, CRE, CSCR4, CSFIR, CTS-I, CYB5R3, DCC, DHFR, DLL1, DMD, EGFR, ERBA, ERBB, EBRB2, ETSI, ETS2, ETV6, FCC, FGR, FOX, FUSI, FYN, GALNS, GLB1, GNS, GUSB, HBB, HBD, HBE1, HBG1, HBG2, HCR, HGSNAT, HOXB4, HRAS, HYAL1, ICAM-1, iCaspase, IDUA, IDS, JUN, KLF4, KRAS, LYN, MCC, MDM2, MGMT, MLL, MMACI, MYB, MEN-I, MEN-II, MYC, NAGLU, NANOG, NF-1, NF-2, NKX2.1, NOTCH, OCT4, p16, p21, p27, p57, p73, PALB2, RAD51C, ras, at least one of RPL3 through RPL40, RPLPO, RPLP1, RPLP2, at least one of RPS2 through RPS30, RPSA, SGSH, SLX4, SOX2, VHL, or WT-I; or
(viii) any two or more of (i)-(vii).
6. The genetic construct of claim 1 , cloned between SEQ ID NO: 18 and SEQ ID NO: 19.
7. The genetic construct of claim 1 , wherein the CD33 blocking molecule comprises a wobble base pair.
8. A kit comprising a genetic construct of claim 1 and a CD33-targeting agent.
9. The kit of claim 8 , wherein the CD33-targeting agent comprises:
(i) an anti-CD33 antibody, an anti-CD33 immunotoxin, an anti-CD33 antibody-drug conjugate, an anti-CD33 antibody-radioisotope conjugate, an anti-CD33 bispecific antibody, an anti-CD33 bispecific immune cell engaging antibody, an anti-CD33 trispecific antibody, and/or an anti-CD33 chimeric antigen receptor (CAR) with one or more binding domains; or
(ii) Hp67.6, lintuzumab, SGN-CD33A, and/or AMG 330; or
(iii) a binding domain derived from Hp67.6, lintuzumab, SGN-CD33A, and/or AMG 330; or
(iv) the CDRs of Hp67.6, lintuzumab, SGN-CD33A, and/or AMG 330 and/or a sequence combination of
a variable light chain comprising SEQ ID NO: 39 and a variable heavy chain comprising SEQ ID NO: 40;
a variable light chain comprising SEQ ID NO: 47 and a variable heavy chain comprising SEQ ID NO: 48;
a variable light chain comprising a CDRL1 of SEQ ID NO: 41, a CDRL2 of SEQ ID NO: 42, and a CDRL3 of SEQ ID NO: 43 and a variable heavy chain comprising a CDRH1 of SEQ ID NO: 44, a CDRH2 of SEQ ID NO: 45, and a CDRH3 of SEQ ID NO: 46;
a variable light chain comprising a CDRL1 of SEQ ID NO: 49, a CDRL2 of SEQ ID NO: 50, and a CDRL3 of SEQ ID NO: 51 and a variable heavy chain comprising a CDRH1 of SEQ ID NO: 52, a CDRH2 of SEQ ID NO: 53, and a CDRH3 of SEQ ID NO: 54; and/or
a variable light chain comprising a CDRL1 of SEQ ID NO: 98, a CDRL2 of SEQ ID NO: 99, and a CDRL3 of SEQ ID NO: 100 and a variable heavy chain comprising a CDRH1 of SEQ ID NO: 101, a CDRH2 of SEQ ID NO: 102, and a CDRH3 of SEQ ID NO: 103; or
(v) an antibody-drug conjugate or an antibody-radioisotope conjugate wherein the drug or radioisotope are selected from taxol, taxane, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracinedione, mitoxantrone, mithramycin, maytansinoid, dolastatin, auristatin, calicheamicin, pyrrolobenzodiazepine, nemorubicin PNU-159682, anthracycline, vinca alkaloid, trichothecene, CC1065, camptothecin, elinafide, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, ricin, CC-1065, duocarmycin, diphtheria toxin, snake venom, cobra venom, mistletoe lectin, modeccin, pokeweed antiviral protein, saporin, Bryodin 1, bouganin, gelonin, Pseudomonas exotoxin, iodine-131, indium-111, yttrium-90, lutetium-177, astatine-211, bismuth-212, and/or bismuth-213, and/or wherein the antibody-drug conjugate comprises gemtuzumab ozogamicin (GO); or
(vi) a linker; or
(vii) a bispecific antibody comprising a combination of binding variable chains or a binding CDR combination of Hp67.6, lintuzumab, SGN-CD33A, and/or AMG 330 and/or a sequence combination of
a variable light chain comprising SEQ ID NO: 39 and a variable heavy chain comprising SEQ ID NO: 40;
a variable light chain comprising SEQ ID NO: 47 and a variable heavy chain comprising SEQ ID NO: 48;
a variable light chain comprising a CDRL1 of SEQ ID NO: 41, a CDRL2 of SEQ ID NO: 42, and a CDRL3 of SEQ ID NO: 43 and a variable heavy chain comprising a CDRH1 of SEQ ID NO: 44, a CDRH2 of SEQ ID NO: 45, and a CDRH3 of SEQ ID NO: 46;
a variable light chain comprising a CDRL1 of SEQ ID NO: 49, a CDRL2 of SEQ ID NO: 50, and a CDRL3 of SEQ ID NO: 51 and a variable heavy chain comprising a CDRH1 of SEQ ID NO: 52, a CDRH2 of SEQ ID NO: 53, and a CDRH3 of SEQ ID NO: 54; and/or
a variable light chain comprising a CDRL1 of SEQ ID NO: 98, a CDRL2 of SEQ ID NO: 99, and a CDRL3 of SEQ ID NO: 100 and a variable heavy chain comprising a CDRH1 of SEQ ID NO: 101, a CDRH2 of SEQ ID NO: 102, and a CDRH3 of SEQ ID NO: 103; or
(viii) any two or more of (i)-(vii).
10. The kit of claim 8 , wherein the CD33-targeting agent comprises a bispecific antibody comprising at least one binding domain that activates an immune cell.
11. The kit of claim 10 , wherein the binding domain that activates an immune cell:
(i) binds CD3, CD28, CD8, NKG2D, CD8, CD16, KIR2DL4, KIR2DS1, KIR2DS2, KIR3DS1, NKG2C, NKG2E, NKG2D, NKp30, NKp44, NKp46, NKp80, DNAM-1, CD11b, CD11c, CD64, CD68, CD119, CD163, CD206, CD209, F4/80, IFGR2, Toll-like receptors 1-9, IL-4Rα, or MARCO; or
(ii) comprises a variable light chain comprising a CDRL1 of SEQ ID NO: 55, a CDRL2 of SEQ ID NO: 56, and a CDRL3 sequence of SEQ ID NO: 57 and a variable heavy chain comprising a CDRH1 of SEQ ID NO: 58, a CDRH2 of SEQ ID NO: 59, and a CDRH3 of SEQ ID NO: 60; or
(iii) comprises SEQ ID NO: 61; or
(iv) comprises a variable light chain comprising a CDRL1 of SEQ ID NO: 62, a CDRL2 of KVS, and a CDRL3 sequence of SEQ ID NO: 63 and a variable heavy chain comprising a CDRH1 of SEQ ID NO: 64, a CDRH2 of SEQ ID NO: 65, and a CDRH3 of SEQ ID NO: 66; or
(v) comprises a variable light chain comprising a CDRL1 of SEQ ID NO: 67, a CDRL2 of KVS, and a CDRL3 sequence of SEQ ID NO: 63 and a variable heavy chain comprising a CDRH1 of SEQ ID NO: 69, a CDRH2 of SEQ ID NO: 70, and a CDRH3 of SEQ ID NO: 71; or
(vi) comprises a variable light chain comprising a CDRL1 of SEQ ID NO: 72, a CDRL2 of KVS, and a CDRL3 sequence of SEQ ID NO: 63 and a variable heavy chain comprising a CDRH1 of SEQ ID NO: 69, a CDRH2 of SEQ ID NO: 75, and a CDRH3 of SEQ ID NO: 76; or
(vii) comprises a variable light chain comprising a CDRL1 of SEQ ID NO: 77, a CDRL2 of KVS, and a CDRL3 sequence of SEQ ID NO: 78 and a variable heavy chain comprising a CDRH1 of SEQ ID NO: 79, a CDRH2 of SEQ ID NO: 80, and a CDRH3 of SEQ ID NO: 81; or
(viii) comprises a variable light chain comprising a CDRL1 of SEQ ID NO: 82, a CDRL2 of SEQ ID NO: 83, and a CDRL3 sequence of SEQ ID NO: 84 and a variable heavy chain comprising a CDRH1 of SEQ ID NO: 85, a CDRH2 of SEQ ID NO: 86, and a CDRH3 of SEQ ID NO: 87;
(ix) comprises a TCR; or
(x) comprises a variable light chain comprising SEQ ID NO: 88 and a variable heavy chain comprising SEQ ID NO: 89; or
(xi) any two or more of (i)-(x).
12. The kit of claim 8 , wherein the CD33-targeting agent comprises a chimeric antigen receptor (CAR) comprising one or more binding domains.
13. The kit of claim 12 , wherein:
(i) the CAR comprises an anti-CD33 binding domain and a binding domain that activates an immune cell; or
(ii) the CAR comprises an effector domain selected from 4-1BB, CD3ε, CD3δ, CD3ζ, CD27, CD28, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt, NKG2D, OX40, ROR2, Ryk, SLAMF1, Slp76, pTa, TCRa, TCRB, TRIM, Zap70, PTCH2, or any combination thereof; or
(iii) the CAR comprises a cytoplasmic signaling sequence derived from CD3 zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d;
(iv) the CAR comprises an intracellular signaling domain and a costimulatory signaling region; or
(v) the CAR comprises a spacer region; or
(vi) the CAR comprises a transmembrane domain;
(vii) the CAR comprises a costimulatory signaling region comprises the intracellular domain of CD27, CD28, 4-1BB, OX40, CD30, CD40, lymphocyte function-associated antigen-1, CD2, CD7, LIGHT, NKG2C, or B7-H3; or
(viii) any two or more of (i)-(vii).
14. A method of genetically-modifying a cell to provide a therapeutic gene and to have reduced CD33 expression, the method comprising exposing the cell to an effective amount of a genetic construct of claim 4 .
15. A method of protecting a cell from an anti-CD33 treatment comprising genetically-modifying the cell with a genetic construct of claim 1 .
16. The method of claim 15 , wherein:
the cell is a therapeutic cell; and/or
the protecting is in vivo.