Tumor targeting antibodies bridge innate to adaptive immunity. Yang-Xin Fu MD PhD Professor of Pathology and Immunology UT Southwestern

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1 Tumor targeting antibodies bridge innate to adaptive immunity Yang-Xin Fu MD PhD Professor of Pathology and Immunology UT Southwestern

2 Tumor targeting antibodies are screened and selected to kill tumor cells Tumor targeting antibodies oncogenic receptors: anti-her2/neu CD2 (cell lineage) CD47 (do not eat me signal) Bi-specific antibody: increase efficacy and reduce toxicity 2

3 How can anti-her2/neu antibody reduce tumor burden: complex mechanisms? Oncogenic blockade vs FcR mediated kill? NK TUMOR We select WT vs Rag-1 KO mice for stronger ADCC but lack T /B cells to test its impact TUMOR 3

4 Tumor volume (mm 3 ) Tumor targeting Ab controls in T cell dependent fashion WT migg Wt -neu Rag -/- migg TUBO derived from MMTV-Rat neu Tg: oncogenic signaling dependent 75 5 Rag -/- -neu Rag-1+Ab Days after tumor inoculation * Wt+Ab Similar T cell dependency is observed using TKI: 1% relapse in Rag-1 KO mice But % in WT mice Are T cells essential for Ab-mediated tumor regression? Nature Med, 27, Cancer Cell, 21 and 214; CCR 213, Mol. Ther

5 How do antibodies trigger immune response? Anti-EGFR or anti-her2/neu CD8 ADCC cytokines IFN Increased Cross-priming Stress protein or DNA TLRs? MyD88 MyD88/HMGB-1, Type I IFN, type II IFN, DC, and T cell dependent 5

6 Major Mechanism for Anti-CD2 Antibodies: Is ADCC vs CDC? ADCC vs CDC: which is more important N Engl J Med 212;366:

7 V o lu m e o f th e tu m o r(m m 3 ) V o lu m e o f th e tu m o r(m m 3 ) V o lu m e o f th e tu m o r(m m 3 ) The therapeutic effect of anti-cd2 treatment is CD8 T cell dependent A C trl A n ti-m C D 2 B C trl A n ti-m C D 2 C C trl A n ti-c D 8 A n ti-m C D 2 A n ti-m C D 2 + a n ti-c D nude T im e a fte r tu m o r in o c u la tio n (d ) T im e a fte r tu m o r in o c u la tio n (d ) T im e a fte r tu m o r in o c u la tio n (d )

8 T u m o r v o lu m e (m m 3 ) S F C s p e r 3 * 1 5 c e lls Anti-CD2 DC?? CD8 + T cell Tumor control 1.8*1 6 A2 S.C. 1μg of anti-mcd2 (Anti-mCD2) or higg (Ctrl) i.p. CD11c-DTR D12 D15 D11 DT i.p. every other day 8 h Ig G 1 h Ig G 1 + D T A n ti-m C D 2 6 A n ti-m C D 2 + D T h Ig G 1 A n ti-m C D 2 h Ig G 1 + D T 1 5 A n ti-m C D 2 + D T T im e a fte r tu m o r in o c u la tio n (d ) M e d iu m A 2 DC is necessary for anti-cd2 treatment 8

9 T u m o r v o lu m e (m m 3 ) S F C s p e r 2 *1 5 c e lls The therapeutic function of anti-cd2 is macrophage-dependent 1.8*1 6 A2 S.C. 1μg of anti-mcd2 BALB/c D12 D15 D11 Clophosome i.p. twice a week D26 IFN-γ ELISPOT 1 C trl C lo p h o s o m e A n ti-m C D 2 8 A n ti-m C D 2 + C lo p h o s o m e 5 4 P <. 1 C trl A n ti-m C D 2 C trl + C lo p h o s o m e 6 3 A n ti-m C D 2 + C lo p h o s o m e T im e a fte r tu m o r in o c u la tio n (d ) M e d iu m A 2 9 How can both DC and macrophages are essential?

10 IF N - (p g /m l) Anti-CD2 induced increase of DC cross-presentation is macrophage-dependent Isolated DCs from A2-HA-bearing mice treated with anti-cd2 +/- MO CL4 T cells P <. 5 Cross priming IFNg MO dep. IF N (p g /m l) Isolated cell For type I IFN C trl P <. 1 A n ti-m C D 2 S F C s p e r 3 *1 5 c e lls 6 C trl 4 2 T cell responses are IFNRA dependent A n ti-m C D 2 P =. 1 A n ti-m C D 2 + a n ti-ifn a r1 C trl A n ti-m C D 2 A n ti-m C D 2 + C lo p h o s o m e D C M a c ro p h a g e D C M a c ro p h a g e M e d iu m A 2 Anti- CD2 Mɸ IFN-I DC CD8 + T cell Tumor control 1

11 CD47 blockade permits rapid phagocytosis of tumor cells: Innate checkpoint blockade? Do not eat me CD47 is innate checkpoint that prevent phagocytosis Eat me signals NSG mice Cell, 29 ALL model Blood; 71(4);

12 Tumor volume(mm 3 ) Tumor volume (mm 3 ) T u m o r v o lu m e (m m 3 ) T u m o r v o lu m e (m m 3 ) Tumor volume (mm 3 ) T cells are essential for anti-cd47-mediated tumor control in syngenic tumor models A2 (lymphoma) systemic local MC38(colon cancer) a A 2 (i.p. a n ti-c D 4 7 ) R a tig a n ti-c D ra tig G a n ti-m C D 4 7 c c RatIg anti-cd D a y s a fte r tu m o r in o c u la tio n * 4 2 * D a y s a fte r tu m o r in o c u la tio n 2 ** Days after tumor inoculation d Nude ratigg Nude anti-mcd47 ns Days after tumor inoculation e RatIg anti-mcd47 Nude mice Days after tumor inoculation *

13 Tumor volume (mm 3 ) But the protection of anti-cd47 Ab is dependent on CD8 T cells A: CD8 dependent B: memory response by rechallenge Naive anti-cd Days after tumor rechallenge 13

14 Therapeutic effect of anti-cd47 depends on type I IFNs and DC responding to IFN for cross priming. re la tiv e c o p y n u m b e (x 1-3 ) Tumor volume (mm 3 ) Tumor volume (mm 3 ) A: IFN production by DC M o n o c y te IF N R a tig a n ti-c D 4 7 * ns M a c ro p h a g e * * D C B: IFNAR signaling RatIg anti-ifnar anti-cd47 anti-cd47+anti-ifnar Days after tumor inoculation C: IFNAR on DC ** CD11c-cre x IFNAR fl/fl = no IFNAR on DC Ifnar1 fl/fl +RatIg Ifnar1 fl/fl + anti-cd47 Cd11c cre Ifnar1 fl/fl + RatIg Cd11c cre Ifnar1 fl/fl + anti-cd47 ** Days after tumor inoculation Which pathway to produce IFN: MyD88, like anti-neu? 14

15 Tumor size (mm 3 ) Tumor volume (mm 3 ) Neither MyD88 nor Trif, two major pathways for IFN production, is essential for tumor control by anti-cd47 1 RatIg 8 anti-cd47 MyD88 -/- +RatIg 6 MyD88 -/- +anti-cd RatIg anti-cd47 Trif-/-+RatIg Trif-/-+anti-CD47 2 ns 2 ns Days after tumor inoculation Days after tumor inoculation Xu et al Nature Med

16 Tumor volume (mm 3 ) Fold induction IFN- spots/1 6 cells STING signaling is essential for type I IFNs Sting dependent: tumor control DC IFN production Cross-Priming c d e 8 RatIg anti-cd47 6 Sting mut +RatIg Sting mut +anti-cd * IFN * RatIg anti-cd47 CD8 T cells response 2 RatIg 15 *** *** anti-cd * 2 ns Days after tumor inoculation WT Sting mut WT Sting mut 16

17 Tumor size (mm 3 ) Tumor size (mm 3 ) STING from DC but not macrophages is required for anti-cd47 therapy A B 15 Sting f/f +rat Ig Sting f/f +anti-mcd47 1 Lyz-cre;Sting f/f +rat Ig Lyz-cre;Sting f/f 5 +anti-mcd47 ** ns 2 Sting f/f +rat Ig Sting f/f +anti-mcd47 15 Cd11c-cre;Sting f/f +rat Ig 1 Cd11c-cre;Sting f/f +anti-mcd47 5 ns *** Days after tumor inoculation Days after tumor inoculation Why can DC but not macrophages be essential for innate sensing? Which type of DNA or DNA complex senses cgas/sting pathway? 17

18 % of EdU + cells Anti-CD47 increases DNA and tumor uptake by both DC and macrophages CFSE-labeled MC38 tumor cells + BMDC or BMM Ab 1 h. A Edu labeled tumor cells were inoculated Ab 4 h cells harvested Gate: CD45+CD11b+: Increased DNA uptake by anti-cd47 B 15 1 RatIg anti-cd47mab ** * 5 BMDC BMM

19 Cytosolic DNA (Relative copy number) Fold Change (relative to RatIg) DCs accumulate more cytosolic DNA after CD47 blockade ex vivo 3 *** *** 2 RatIg Anti-mCD47(24h) Anti-mCD47(72h) Cytosolic mtdna Rat Ig Anti-mCD47 ** 1 2 gdna mtdna Mono M DC

20 Anti-CD47 promotes tumor-derived mtdna Cytosolic DNA (Relative copy number) Cytosolic DNA (Relative copy number) Host VS Tumor? Genomic DNA VS Mitochondrial DNA? gdna mtdna 8 RatIg Anti-mCD mig ** Anti-hCD ns mouse ns human 2 ns mouse human

21 IFN- spots/1 6 cells DNA abundance (%) Fold Change (relative to RatIg) Tumor mtdna is required for type I IFN production and cross-priming in DC A DDC reduces mtdna B: 1 ns *** Non-ddC ddc 6 4 IFN- * Rat Ig Anti-mCD ns gdna mtdna mtdna depleted Macrophage DC C 6 4 *** *** Rat Ig Anti-mCD47 2 mtdna depleted ns

22 Phagosomal PH Phagosomal PH Phagosomal mtdna (relative to RatIg) CD47-SIRPα signaling in DCs controls the phagosomal PH and DNA degradation A: mtdna reduced in macrophages but not DC 3 ** ns RatIg ** ** anti-cd47(') anti-cd47(3') 2 anti-cd47(9') 1 Macrophage DC B: ph in macrophage is reduced C: ph in DC increase Macrophage 5 M alone M +Tumor(ratIg) 4 M +Tumor(anti-CD47) Time of chase (min) 8 7 DC 6 DC alone 5 DC+Tumor(ratIg) DC+Tumor(anti-CD47) Time of chase (min) *

23 Tumor Volume (mm 3 ) Tumor Volume (mm3) T u m o r V o lu m e (m m 3 ) T u m o r V o lu m e (m m 3 ) T u m o r V o lu m e (m m 3 ) % C D 3 + C D 8 + in tu m o r Cell counts CD47 on tumor cells plays dominate role in evasion and PD-L1 is an adaptive resistance A B C D M C 3 8 M C 3 8 -C D 4 7 K O * M C 3 8 M C 3 8 -C D 4 7 K O M C 3 8 -C D 4 7 K O + a n ti-c D 8 * * * 4 3 * * * ISO MC38 MC38-CD47 KO 5 * * * * T im e a fte r tu m o r in o c u la tio n (d a y ) T im e a fte r tu m o r in o c u la tio n (d a y ) M C 3 8 M C 3 8 -C D 4 7 K O PDL1 E MC38 MC38-CD47 KO MC38-PDL1 KO MC38-CD47/PDL1 DKO *** F WT:MC38 WT:MC38-CD47/PDL1 DKO Rag-/-:MC38 Rag-/-:MC38-CD47/PDL1 DKO *** G 1 5 M C 3 8 M C 3 8 -C D 4 7 /P D L 1 D K O M C 3 8 -C D 4 7 /P D L 1 D K O + a n ti-c D 8 * * Time after tumor inoculation (day) Time after tumor inoculation (day) T im e a fte r tu m o r in o c u la tio n (d a y )

24 T u m o r V o lu m e (m m 3 ) IFNAR and STING/IFN on host cells are required for CD47 /PDL1-mediated evasion T u m o r V o lu m e (m m 3 ) T u m o r V o lu m e (m m 3 ) A W T :M C 3 8 W T :M C 3 8 -C D 4 7 /P D L 1 D K O IF N A R -/-:M C 3 8 IF N A R -/-:M C 3 8 -C D 4 7 /P D L 1 D K O B W T :M C 3 8 W T :M C 3 8 -C D 4 7 /P D L 1 D K O S T IN G m u t :M C 3 8 S T IN G m u t :M C 3 8 -C D 4 7 /P D L 1 D K O 1 * * * * * * * T im e a fte r tu m o r in o c u la tio n (d a y ) C T im e a fte r tu m o r in o c u la tio n (d a y ) W T :M C 3 8 W T : M C 3 8 -C D 4 7 /P D L 1 D K O C D 1 1 C -S T IN G -/-:M C 3 8 C D 1 1 C -S T IN G -/-:M C 3 8 -C D 4 7 /P D L 1 D K O 1 5 * * * * T im e a fte r tu m o r in o c u la tio n (d a y )

25 Binding RBC is the major barrier for systemic use of anti-cd47 How can we target tumor better? A B RBC MC38 SIRPa anti-pdl1-sirpa Tumor cells have both PD-L1 and CD47 while RBC have only CD47. Bi-specific anti-pdl1-sirpα maintaining binding to tumor cells while greatly reducing binding to RBC

26 T u m o r V o lu m e (m m 3 ) T u m o r V o lu m e (m m 3 ) T u m o r V o lu m e (m m 3 ) T u m o r V o lu m e (m m 3 ) Co-targeting of CD47 and PDL1 on tumor cells is required for tumor growth inhibition A In tra tu m o ra l tre a tm e n t B S y s te m ic tre a tm e n t h Ig G a n ti-p D L 1 S IR P a a n ti-p D L 1 -S IR P a h Ig G a n ti-p D L 1 -S IR P a a n ti-p D L 1 + S IR P a 5 * * * 1 5 * * D 1 5 T im e a fte r tu m o r in o c u la tio n (d a y ) M C 3 8 -C D 4 7 K O tu m o r E 1 5 T im e a fte r tu m o r in o c u la tio n (d a y ) M C 3 8 -P D L 1 K O tu m o r 1 h Ig G a n ti-p D L 1 -S IR P a 1 h Ig G a n ti-p D L 1 -S IR P a T im e a fte r tu m o r in o c u la tio n (d a y ) T im e a fte r tu m o r in o c u la tio n (d a y )

27 T u m o r V o lu m e (m m 3 ) T u m o r V o lu m e (m m 3 ) Anti-PDL1-SIRPα enhances intratumoral DC innate sensing 1 8 W T : h Ig G W T : a n ti-p D L 1 -S IR P a S T IN G -/-: h Ig G S T IN G -/-: a n ti-p D L 1 -S IR P a 1 5 S T IN G -/-: h Ig G S T IN G -/-: a n ti-p D L 1 -S IR P a C D 1 1 c -S T IN G -/-: h Ig G C D 1 1 c -S T IN G -/-: a n ti-p D L 1 -S IR P a * 5 * T im e a fte r tu m o r in o c u la tio n (d a y ) T im e a fte r tu m o r in o c u la tio n (d a y )

28 Summary Therapeutic effect of anti-cd47 depends on CTL. DCs but not macrophage are required for anti-cd47 Abmediated cross priming While macrophages degrade DNA faster than DC Anti-CD47 Ab-mediated type I IFN induction or cross priming by host DC depends on cgas-sting pathway DC can better sense cytosol mtdna through cgas-sting pathway 28

29 Acknowledgements: CAS collaborative group Radiation Liufu Deng Sog Auh/Youjin Lee Byron Burnette Anti-CD47 Ab Michelle Xu Xiaojuan Liu Christine Pu Hua Peng Anti-Neu SeaGwang Park Yang Wang Eric Morterson Shengdien Wang Ab-IFN Xuanming Yang Yang Wang Ab-LIGHT Haidong Tang Yang Wang CD2 Zhenhua Ren Hua Peng UC Ralph Weichselbaum Tom Gajewski Hans Schreiber UTSW Zhijian James Chen Alphamab Luan Yan Ting Xu 29

30 T u m o r v o lu m e (m m 3 ) Large tumors have more CTLA+ Treg and resist to anti- CD2 therapy: new strategy for combination 2 5 h Ig G + h a m s te r Ig G A n ti-m C D 2 + h a m s te r Ig G 2 A n ti-c T L A -4 + h Ig G A n ti-m C D 2 + a n ti-c T L A T im e a fte r tu m o r in o c u la tio n (d ) Anti-CTLA4 therapy overcomes anti-cd2 resistance 3

31 P e rc e n t s u rv iv a l Cell counts P e rc e n t s u rv iv a l Pretreatment with chemotherapy induces more eat me signal and synergize with anti-pdl1-sirpα for tumor eradication A B 1 h Ig G * D O X a n ti-p D L 1 -S IR P a D O X + a n ti-p D L 1 -S IR P a 5 Calreticulin T im e a fte r tu m o r in o c u la tio n (d a y ) C 1 h Ig G D O X + a n ti-p D L 1 -S IR P a * * Z -V A D -F M K Z -V A D -F M K + D O X + a n ti-p D L 1 -S IR P a T im e a fte r tu m o r in o c u la tio n (d a y )

32 IF N -r s p o ts /1 6 c e lls Tumor Volume (mm3) T u m o r V o lu m e (m m 3 ) T u m o r V o lu m e (m m 3 ) CD8 + T cells are essential for the therapeutic effect of anti-pdl1-sirpα A h Ig G R a g -/- a n ti-p D L 1 -S IR P a B h Ig G a n ti-p D L 1 -S IR P a a n ti-c D 8 a n ti-p D L 1 -S IR P a + a n ti-c D * * * T im e a fte r tu m o r in o c u la tio n (d a y ) T im e a fte r tu m o r in o c u la tio n (d a y ) C 3 * 2 * D 15 1 higg anti-pdl1-sirpa FTY72 anti-pdl1-sirpa+fty ns h Ig G a n ti-p D L 1 S IR P a a n ti-p D L 1 -S IR P a Time after tumor inoculation (day)

33 Is Tumor expressed-pdl1 essential for tumor growth or PD-L1 blockade therapy? No and no Generate PD-L1 KO tumor PD-L1 from host cells but not tumor is essential for suppression 33

34 Type I IFN is essential for various anti-cancer therapy induced tumor regression: such as RT, anti-her2/neu, anti-cd47 and some chemo drugs IFN has been used in some cancer therapy to suppress tumor cell proliferation Can tumor targeting antibodies bring sufficient IFN to tumor tissues for immune responses:? 34