Compositions and methods targeting complement component 3 for inhibiting tumor growth

1. A method of activating cancer associated fibroblasts (CAFs) ex vivo comprising eliminating expression of complement component 3 (C3) in CAFs obtained from a subject having cancer,

wherein the expression of C3 is eliminated by expression of a CRISPR system directed to a C3 gene in the CAFs,

wherein the activated CAFs comprise increased expression of Ccl5 as compared to CAFs where C3 is not eliminated, and

wherein the activated CAFs increase tumor-infiltrating lymphocytes (TILS) in a tumor of the subject.

2. The method of claim 1 , wherein the activated CAFs further comprise expression of:

Car4, Mtrr, Fosl1, Cdca2, Mex3d, Gjb5, Tcfl5 and Rbml2; or

Gli1, Gli2, Ccl19, Cd52, Wnt4, Wntl1, Ctu1, Ccr5, Cd68, Wnk4, H2-Q1, H2-Q2, H2-Ob, Slfn1, Slfn4, Slfn9, Slfn8, Fgfl2, Kcnkl2, Fcgr4, Clcn1, Clcn2, Kcnkl2 and 117; or

Cfp, C1qa, C1rl and C5ar1.

3. The method of claim 1 , further comprising adoptively transferring the activated CAFs to the subject.

4. The method of claim 3 , wherein adoptively transferring the activated CAFs decreases lymphangiogenesis in a tumor of the subject.

5. The method of claim 3 , wherein adoptively transferring the activated CAFs is administered in combination with an immunotherapy.

6. The method of claim 5 , wherein the immunotherapy is an immune checkpoint blockade therapy or an adoptive T cell therapy.

7. The method of claim 6 , wherein the immune checkpoint blockade therapy comprises anti-TIM3, anti-CTLA4, anti-PD-L1, anti-PD1, anti-TIGIT, anti-LAG3, or combinations thereof.

8. The method of claim 6 , wherein the adoptive T cell therapy comprises CAR T cell therapy.

9. The method of claim 1 , wherein the cancer comprises a cancer of blood, kidney, skin, bone, bladder, colon, brain, breast, head, neck, endometrium, lung, testes, ovary, pancreas, or prostate.

10. The method of claim 1 , further comprising detecting podoplanin (PDPN) expression in CAFs.

11. The method of claim 1 , wherein the activated CAFs further comprise expression of one or more genes selected from the group consisting of Tcf24, Slco5a1, 6720483E21Rik, Gm29107, Npas2, 4930558J18Rik, Ino80dos, Tmem169, Catip, Plcd4, Sphkap, Fbxo36, Glrp1, Gm19589, Agxt, Rnfl52, Serpinb5, Gli2, 2900009J06Rik, Mfsd4, Klhdc8a, Lgr6, Kcnt2, Pdc, Serpinc1, Dars2, XcII, Cd247, Gm16701, Lmx1a, Fcgr4, Gm20045, B930036N10Rik, Igsf9, 01fr433, Opn3, Chm1, Kif26b, 1700056E22Rik, Gm34342, 4930532G15Rik, Kcnk2, Proser2, Gata3, 4930426L09Rik, Thnsl1, 1700084E18Rik, 1700001022Rik, Cfap157, Zbtb43, Lmxlb, Phfl9, Crb2, Wdr38, Lypd6, Scn2a1, Mettl5os, 2600014E21Rik, Rbm45, Zfp385b, Gm13715, Clgtnf4, Spi1, Ambra1, Abtb2, Tcpl111, Mettl15, Gm26899, Catsper2, Blocls6os, 1810024B03Rik, Gm14029, AV099323, Mcm8, Ism1, Slc52a3, Defb25, Mylk2, Rbml2, Gm14286, Fam209, Gm20721, Gm14325, Gm14327, Phactr3, Tcfl5, Nkain4, Col20a1, Nudt10, Rpgr, Gm26652, Jade3, Uspl1, Cfp, 2310010G23Rik, Smim1012a, Xlr3a, Xlr3c, Pdzd4, Tab3, Gm14798, Spin4, Eda2r, Dlg3, Ercc61, C77370, Uprt, MumlI1, Pfkfb1, Nhs, Ofd1, Prps2, 117, Zbtb10, C030034L19Rik, Naaladl2, Lrrc34, Usp13, Gm15952, D3Ertd254e, 1810062G17Rik, Fgf2os, D3Ertd751e, Gm30074, Ccdc169, P2ry1, Gm6634, Map9, Rbm46, Crabp2, Gm3764, Gm16069, Muc1, S100a14, S100a3, S100a7a, Tchh, Ctss, Ciart, Hist2h3c2, Hist2h2aa1, Zfp697, Dennd2c, Ptpn22, Atxn712, Dpyd, A530020G20Rik, Prss12, Gm43254, Lpar3, Spata1, Gdf6, Rad54b, Rragd, Tmem215, Kif24, AI464131, Ccl19, Gm26881, Zbtb5, Palm2, Akap2, Tnfsfl5, Ptprd, Cdkn2a, E130102H24Rik, Pars2, Echdc2, Dmrta2, Bend5, Sti1, Faah, Tmem69, Hpd1, Col8a2, Gm12942, Gjb5, Tmem54, Gm12976, Serinc2, Cd52, Ubxnl1, Runx3, 6030445D17Rik, C1qa, Wnt4, Tmco4, 4921514A10Rik, Gm29367, Gm20707, Casz1, Gpr157, Nol9, Kcnab2, Megf6, B930041F14Rik, Tmem240, Gm16008, Tnfrsfl8, 9430015G10Rik, Sema3a, Fbx113, Napepld, Crygn, Gm7361, Drc1, Fam184b, Selll3, Wdr19, Limch1, Ras1l1b, C530008M17Rik, Spink2, Gm9958, Cdkl2, Nup54, 2010109A12Rik, Fgf5, 1700010H22Rik, Agpat9, Gm29707, Klhl8, Gm42749, Gm28050, A830010M20Rik, Ccdc18, E130006D01Rik, Myolh, Trpv4, Gm10399, Rnft2, Tbx3, Iqcd, Gm42918, 4932422M17Rik, Kntc1, Gm10369, Pilrb1, 6330418K02Rik, Rbak, Zfp316, Gm20635, Alox5ap, Gm19719, D730045B01Rik, D830026I12Rik, Kcp, Tspan33, Klfl4, D630045J12Rik, 4930599N23Rik, Clen1, 1600015I10Rik, Gm16499, Ndnf, Reep1, Atoh8, Mlap, Wdr54, Cm13, Add2, Gm44089, Rab43, Hdacl1, Srgap3, Fancd2, Fxyd4, 4930540M05Rik, Gm26826, C1r1, Ptpn6, Kcna1, Gm10010, Klrbla, Klrk1, Pbp2, Rerg, Gm15704, Pthlh, Far2os1, Mettl20, Tarm1, Gm15510, Zfp583, Zim1, Zfp772, Dhx34, C5ar1, Pnmal2, Zfp94, Tmem91, Rab4b, Itpkc, Zfp607, Zfp568, Zfp382, Gm26810, Krtdap, Lsr, Zfp536, Vsig101, Ctu1, Lrrc4b, Napsa, Lmtk3, Uevld, Fancf, Nipa1, Mkrn3, Lysmd4, Ticrr, Homer2, Sh3gl3, Arnt2, RP24-118K20.1, Rab30, Tenm4, Kctd21, Wnt11, Dgat2, C2cd3, RP23-299D2.2, Folr2, Hbb-bt, Gm15133, Ppfibp2, Pdzd9, 4933440M02Rik, Corola, Gdpd3, Pagrla, Mylpf, Zfp668, 9130023H24Rik, Ifitm5, Sct, Eps812, Syt8, Tnni2, Gm21781, Rgs17, Adat2, Heca, Map7, Eya4, Slcl8b1, Fam26f, Tube1, Slcl6a10, 9030612E09Rik, Bend3, Fam184a, Sh3rf3, Gm5424, 1700120B22Rik, Dnajcl2, Lrrc3, Gm10146, Slcla6, Hcn2, Plppr3, Hmha1, Mex3d, Celf5, Tle6, Zfp873, Gm4924, Glt8d2, Gm15990, Ankslb, Nts, BbsO, Glipr1, Lgr5, Faml9a2, Lrig3, F420014N23Rik, Gi1, Stac3, Gls2, A430046D13Rik, Camsap3, Lrrc8e, RP23-216D14.4, RP23-156J8.1, RP23-366014.5, 3930402G23Rik, Myom2, Xkr5, Nek3, Gm26909, Thap1, Tex15, 6430573F11Rik, AI429214, Msr1, 1700029J07Rik, AcslI, Asb5, Sh2d4a, Zfp963, 1700030K09Rik, Tmem38a, Cedc130, Ckslbrt, Neto2, 9330175E14Rik, Gm3830, Nol3, Rltpr, Ddx28, Pdpr, Spata21, 2810455005Rik, Kcnk1, 6230400D17Rik, Plau, Dnah12, Tnne1, Ncoa4, Lrrcl8, Gdfl0, A630023A22Rik, 2610528A11Rik, Pnp2, S1c39a2, Tgm1, Phflla, Phfl1c, Setdb2, Gm27010, Wdfy2, Neil2, Blk, Fam167a, Ptk2b, Cdca2, Nefm, Nef1, Gm27222, Gm16867, Hr, Dmtn, Cysltr2, Tnfsfl1, Pcdh9, Tpm3-rs7, Ggact, Trpc6, Vstm5, Gm7808, Swsap1, Rgl3, Zfp653, Gm16845, Cnn1, Tbx20, Tmem136, Nlrx1, Hinfp, Ube4a, Plet1, 2310030G06Rik, Gm7293, Elmod1, Lingo1, Rec114, Zwilch, Snx22, Car12, Fam8la, 4933433G15Rik, A130057D12Rik, Lea5, Elovl4, 9430037G07Rik, Adamts7, 4933400C23Rik, Slc9a9, Nphp3, Col6a5, Glyctk, Mstlr, Traip, Col7a1, Als2c1, Gm2415, Acaalb, Acvr2b, Ulk4, Cck, Lyzl4, Cer5, Lif, RasI10a, Nacad, Gm12056, Zrsr1, Papolg, Adralb, Trim7, Col23a1, Jade2, Cdkl3, Fam183b, 2810021J22Rik, Gjc2, Jmjd4, Zkscan6, Usp43, 9130213A22Rik, Kcnab3, Efnb3, Cd68, Zmynd15, 4933427D14Rik, Raplgap2, Rph3a1, Gemin4, Dbil5, Atad5, Adap2, Rhbdl3, Tmem132e, Gm11423, Slfn9, Slfn8, Slfn1, Slfn4, RasI10b, Dusp14, Myol9, Car4, Rad51c, Pctp, Cdc34b, Dlx3, Dlx4, Gm11520, Famll7a, Skap1, Cacnb1, Rapgefl1, Gm11940, Krt36, Hap1, Wnk4, Aarsd1, Gm11551, Nags, Gm11627, 2810433D01Rik, 2410004I01Rik, Arhgap27os2, Milr1, Polg2, Rgs9, D11Wsu47e, Galr2, Mgat5b, Tbcldl6, Aatk, Sectmla, B3gntl1, Heatr1, Ero1lb, Vdac3-ps1, Gpr141, Hist1h2 bp, Hist1h2bk, Hist1h4h, Hist1h3d, Hist1h4c, Hist1h2ab, Hist1h3a, Lrrcl6a, Gm26735, Serpinb1b, Bmp6, Sirt5, Nhlrc1, A830005F24Rik, Ptpde1, Ippk, Omd, Diras2, Syk, Gm5449, Unc5a, Fancc, Cdcl4b, 1810034E14Rik, Gm37276, Mtrr, Adamtsl6, Cep72, Gm17259, Rasgrf2, Msh3, Mtx3, Cmya5, S100z, Gm6169, Gm26619, Gm9828, Naip2, Naip6, Ppwd1, Gm10735, Hen1, Gm7120, Gm26520, Fam228b, Kcns3, Greb1, 5730507CO1Rik, Gm3944, Gm10479, 2410018L13Rik, 9030624G23Rik, Taf1b, Dus41, Ispd, Prpsll3, Gpr135, 4930447C04Rik, Tmem30b, Hspa2, Exd2, Zfyve1, 2410016006Rik, Acot4, Dnal1, Pnma1, Lin52, Gm16381, Eml5, Serpina3f, Wdr25, Tecpr2, Amn, Gm266, 2810029C07Rik, Ptger4, Fyb, Slc1a3, 4930556M19Rik, Fbxl7, Nipal2, Spag1, Baalc, Lrpl2, Zfpm2, Angpt1, Has2, Gm16006, Gsdmc, Zfat, Ly6k, Gm1, Mapkl5, Nrbp2, Smpd5, Kifc2, Gpt, Zfp251, Apol7a, Phf21b, Ppp6r2, Alg10b, Prickle1, A130051J06Rik, Slc4a8, Krt7, Soat2, Gm26518, Hoxc5, D930007P13Rik, Slx4, Sr1, 2610020C07Rik, Snx29, 3110001I22Rik, Pkp2, Gm15648, Rtn4r, Tbx1, Clcn2, Chrd, BC106179, Cldn1, Fgfl2, Bdh1, Ccdcl4, Zbtbllos1, Evalc, Dopey2, Zfp97, Gm7535, Zfp944, Kifc5b, Syngap1, Ip6k3, Gm26549, Zfp811, Zfp799, Zfp870, Zfp563, Myolf, H2-Ob, Aif1, H2-Q1, H2-Q2, Pou5f1, Gm20442, Ppplrl8os, BC023719, Gm8909, Ankrd66, Enpp5, 1600014C23Rik, Rsph9, Cul9, Plcl2, Prr22, Vav1, Cdkl4, Thumpd2, Kcnkl2, Gm6225, Gm17430, Dsc2, Rprdla, Gm3550, Prob1, Pcdhbl7, Gprl51, Eif3j2, Cdo1, Enolb, Dtwd2, Gm4841, Spink10, Pmaip1, Lipg, Katnal2, 8030462N17Rik, Epg5, Hsbpll1, Sytl2, Fosl1, Ctsw, Ccdc88b, Macrod1, Pcna-ps2, Best1, Ms4a6b, Mpeg1, Vps13a, 2410080I02Rik, 4430402I18Rik, Cd274, Ranbp6, Rnls, Exoc6, Sorbs1, Tctn3, Slit1, Lox14, Neurlla, Gpam, B230217012Rik, and Sfxn4.

12. A method of increasing tumor-infiltrating lymphocytes (TILS) in a tumor of a subject having cancer comprising:

obtaining cancer associated fibroblasts (CAFs) from a tumor of the subject; activating the CAFs by ex vivo eliminating expression of complement component 3 (C3) in the CAFs by expressing a CRISPR system directed to a C3 gene in the CAFs,

wherein the activated CAFs comprise increased expression of Ccl5 as compared to CAFs where C3 is not eliminated; and

adoptively transferring the activated CAFs to the subject.

13. The method of claim 12 , further comprising isolating CAFs that do not express PDPN from the activated CAFs before adoptively transferring the activated CAFs to the subject.

14. The method of claim 12 , wherein the activated CAFs further comprise expression of:

Car4, Mtrr, Fosl1, Cdca2, Mex3d, Gjb5, Tcfl5 and Rbm12; or

Gli1, Gli2, Ccl19, Cd52, Wnt4, Wntl1, Ctu1, Ccr5, Cd68, Wnk4, H2-Q1, H2-Q2, H2-Ob, Slfn1, Slfn4, Slfn9, Slfn8, Fgfl2, Kcnkl2, Fcgr4, Clcn1, Clcn2, Kcnkl2 and 117; or

Cfp, C1qa, C1rl and C5ar1.

15. The method of claim 12 , wherein adoptively transferring the activated CAFs decreases lymphangiogenesis in a tumor of the subject.

16. The method of claim 12 , wherein adoptively transferring the activated CAFs is administered in combination with an immunotherapy.

17. The method of claim 16 , wherein the immunotherapy is an immune checkpoint blockade therapy or an adoptive T cell therapy.

18. The method of claim 17 , wherein the immune checkpoint blockade therapy comprises anti-TIM3, anti-CTLA4, anti-PD-L1, anti-PD1, anti-TIGIT, anti-LAG3, or combinations thereof.

19. The method of claim 17 , wherein the adoptive T cell therapy comprises CAR T cell therapy.

20. The method of claim 12 , wherein the cancer comprises a cancer of blood, kidney, skin, bone, bladder, colon, brain, breast, head, neck, endometrium, lung, testes, ovary, pancreas, or prostate.