k e y t r u d a + a v e l u m a b c t x - 8 3 7 1 a v e l ... · c t x - 8 3 7 1 k e y t r u d a c...

1
Stitchmabs allow for rapid testing of bispecific concepts We initially theorized that a bispecific containing anti-PD-1 and PD-L1 arms would function to bring T cells into contact with PD-L1+ tumor cells while also blocking a critical T cell checkpoint In a MLR assay, Stitchmab™ bispecifics of commercial anti -PD-1 and anti-PD-L1 antibodies caused increased IFN- production at picomolar antibody levels This prompted us to generate common light chain bispecific variants CTX-8371 was selected as a PD-1×PD-L1 bispecific from in-house generated anti-PD-1 and anti-PD-L1 antibodies with excellent drug-like properties Compass anti-PD-1 and anti-PD-L1 antibody Fabs have similar affinities as clinical comparator Fabs to Human and Cynomolgus monkey PD-1 and PD-L1 CTX-8371 maintains monoclonal antibody-like binding to PD-1 and PD-L1 from all 3 species as compared to monoclonal parent molecules. CTX-8371 also has strong anti-mouse PD-1 binding but weaker anti-mouse PD-L1 binding 1 Human T cells were expanded in vitro by TCR activation for 7 days and co-cultured with engineered K562 tumor cell targets in the presence of antibodies for 2 days 1 st generation PD-1xPD-L1 bispecifics enhanced T cell activation compared to Keytruda, Nivolumab, and controls Human PBMCs were stimulated with Staphylococcus Enterotoxin A (SEA) and treated with CTX-8371 or other antibodies for 3 days CTX-8371 remained active at sub-nanomolar doses while other antibodies have regressed to the isotype CMV-specific T cells were co-cultured with engineered K562 tumor cell targets and bystander cells in the presence of antibodies for 2 days CTX-8371 treatment increased CMV-specific T cell killing of K562 tumor cell targets at sub-nanomolar doses while the other antibodies regressed to the isotype CTX-8371 increases T cell activation and killing in vitro Background Monoclonal antibody immunotherapies targeting immune checkpoint receptors have shown great promise for a subset of cancer patients. However, novel combination therapies are still needed to increase the benefit of cancer immunotherapy and bring it to broader patient populations. Here we describe the preclinical evaluation of CTX-8371, which combines PD-1 and PD-L1 targeting in one bispecific, tetravalent molecule. Methods Our proprietary Stitchmabs™ bispecific screening platform was used to conduct an unbiased screen of bispecifics comprised of various checkpoint blocking antibodies. This screen yielded the surprising discovery that a bispecific containing both PD-1 and PD-L1 binding arms was more potent than the combination of parent monoclonal antibodies. We then generated common light chain bispecifics containing compatible anti-PD-1 and PD-L1 antibodies and used multiple in vitro assays to identify our lead, CTX-8371. Additional in vitro and in vivo experiments confirmed CTX-8371’s reactivity across species, in vivo anti-tumor effects, and the underlining mechanisms driving its distinctive activity. Results We found that CTX-8371 binds to human and cynomolgus monkey PD-1 and PD-L1 targets with sub-nanomolar affinities and is cross-reactive to mouse PD-1 and PD-L1. Compared to Keytruda®, CTX-8371 increased T cell activation and tumor cell killing in vitro, significantly delayed tumor growth, and prolonged survival in human cell transfer tumor models. Additionally, CTX-8371 demonstrated efficacy in transplantable mouse syngeneic models. Investigation into the mechanisms responsible for the enhanced efficacy of CTX-8371 unexpectedly found that the bispecific causes a massive loss of PD-1 from the T cell surface, which was not observed in response to monoclonal antibodies alone or combined. This robust PD-1 downregulation, potentially mediated through bridging together the T cell and tumor cell, may explain the ability of CTX-8371 to reverse PD-1 suppression more potently than standard blocking antibodies. Conclusions Taken together, the results demonstrate that the bispecific, tetravalent antibody CTX-8371 has increased potency in vitro and in vivo as compared to clinical checkpoint blockade agents. Some of its effects are likely attributable to its unique mechanism of action, driving robust downregulation of cell-surface PD-1. Thus, CTX-8371 has the potential to increase the number of patients that benefit from PD-1/PD-L1 checkpoint blockade. Summary PD-1xPD-L1 Stichmab TM antibodies were superior to monoclonal combination in a MLR assay. This preliminary data led to the creation of CTX-8371, a common light chain bispecific antibody with excellent drug-like properties. CTX-8371 had exceptional in vitro activity and in vivo anti-tumor efficacy compared to approved checkpoint inhibitor immunotherapies and combination thereof. The anti-tumor activity of CTX-8371 might be at least in part explained by its unique MOA leading to massive surface PD-1 loss, mediated through bridging the T cell and tumor cell. Investigation into CTX-8371 MOA is ongoing. CTX-8371, a Novel Bispecific Targeting both PD-1 and PD-L1, is more Potent than Combination anti-PD-1 and PD-L1 Therapy and Provides Enhanced Protection from Tumors in vivo Diana Albu, Pearl Bakhru, Wilson Guzman, Michael Ophir, Rachel McCrory, Beau Carson, Amanda Oliphant, Rachel Rennard, Lucy Liu, Alan Leung, Sara Haserlat, Michael Schmidt, Jose Gonzalo, Bing Gong, Robert Tighe, Ben Wolf Compass Therapeutics, 245 First Street, Cambridge MA + IFN (pg/ml) Stitchmab “A×C” Stitchmab “B×C” Combo “A” + “C” Combo “B” + “C” Single mAb “A” Anti-PD-1 Parent mAb: CTX-5616 Anti-PD-L1 Parent mAb: CTX-6068 CTX-8371 Affinity (nM) Human PD-1 Cyno PD-1 CTX-5616 (a-PD-1) 1.09e-09 1.97e-09 Keytruda 1.81e-09 7.42e-10 Nivolumab 3.52e-09 1.34e-09 Affinity (nM) Human PD-L1 Cyno PD-L1 CTX-6068 (a-PD-L1) 3.08e-09 2.30e-09 Atezolizumab 4.55e-10 1.05e-08 Avelumab 5.05e-10 7.51e-10 -14 -12 -10 -8 -6 0 2 10 6 4 10 6 6 10 6 8 10 6 Cell Binding: CHO/HuPD-1 Log conc (M) MFI CTX-8371 CTX-5616 -13 -12 -11 -10 -9 -8 -7 0 5 10 5 1 10 6 1.5 10 6 Cell Binding: CHO/HuPD-L1 Log conc (M) MFI CTX-8371 CTX-6068 -14 -12 -10 -8 -6 0 5 10 5 1 10 6 1.5 10 6 2 10 6 2.5 10 6 Cell Binding: CHO/CyPD-1 Log conc (M) MFI CTX-8371 CTX-5616 -13 -12 -11 -10 -9 -8 -7 0 2 10 4 4 10 4 6 10 4 8 10 4 Cell Binding: CHO/CyPD-L1 Log conc (M) MFI CTX-8371 CTX-6068 -14 -12 -10 -8 -6 0 5 10 5 1 10 6 1.5 10 6 Cell Binding: CHO/MsPD-1 Log conc (M) MFI CTX-8371 CTX-5616 -14 -12 -10 -8 -6 1 10 4 1.2 10 4 1.4 10 4 1.6 10 4 Cell Binding: A20 (Mouse) Log conc (M) MFI CTX-8371 CTX-6068 0.00001 0.0001 0.001 0.01 0.1 1 10 100 0 2000 4000 6000 8000 10000 SEA assay Concentration (nM) IL-2 (pg/ml) 0.00001 0.0001 0.001 0.01 0.1 1 10 100 1000 35 40 45 50 55 60 65 Tumor cell killing assay Concentration (nM) % Specific Killing Isotype CTX-8371 Keytruda CTX-3834 (Atezolizumab) CTX-5616 + CTX-6068 Human PBMCs were stimulated with anti-CD3 and anti-CD28 antibodies for 3 days before overnight co-culture with the listed antibodies CTX-8371 drove loss of cell-associated PD-1 but only when both arms of the bispecific were engaged Jurkat-PD1, Jurkat-PD-1/PD-L1, or mix of Jurkat-PD-1 + Jurkat-PD-L1 were treated overnight with isotype or CTX-8371 antibodies PD-L1 binding was necessary for CTX-8371-induced loss of cell-associated PD-1 (when both arms of the bispecific were engaged) CTX-8371 drove PD-1 loss predominantly when PD-1 and PD-L1 were in trans Ab Isotype Keytruda CTX-8371 nM: 0.1 1 10 0.1 1 10 0.1 1 10 Atezolizumab Keytruda + Atezolizumab CTX 5616 + 6068 0.1 1 10 0.1 1 10 0.1 1 10 CTX-8371 + 50 nM CTX-6068 0.1 1 10 WB: α-PD-1 50 kD - 37 kD - 75 kD - WB: α-β-Actin 50 kD - 37 kD - 75 kD - Abstract CTX-8371 is effective against mouse syngeneic tumors Unique MOA: CTX-8371 drives loss of PD-1 0 10 20 30 40 0 200 400 600 800 1000 Control Days post tumor inoculation Tumor volume (mm 3 ) 0 10 20 30 40 0 200 400 600 800 1000 CTX-8371 0 5 10 15 20 25 0 200 400 600 800 Days post tumor inoculation Average tumor volume (mm 3 ) Control CTX-8371 Treatment Start *** 0 10 20 30 40 50 0 500 1000 1500 2000 2500 hIgG1 IC Days after tumor cell inoculation Tumor volume (mm 3 ) 0 10 20 30 40 50 0 500 1000 1500 2000 2500 CTX-8371 0 5 10 15 20 25 0 500 1000 1500 Days after tumor cells inoculation Average Tumor Volume (mm 3 ) hIgG1 IC CTX-8371 **** Treatment start 0 10 20 30 40 0 1000 2000 3000 No T cells Days after tumor cell inoculation Tumor Volume (mm 3 ) 0 10 20 30 40 50 0 1000 2000 3000 T cells + IC 0 10 20 30 40 50 0 1000 2000 3000 T cells + Keytruda 0 10 20 30 40 50 0 1000 2000 3000 T cells + Avelumab 0 10 20 30 40 50 0 1000 2000 3000 T cells + PID-8371 0 10 20 30 40 50 0 1000 2000 3000 T cells + PID-5616+PID-6068 0 10 20 30 40 0 500 1000 1500 2000 Tumor Volume Days after tumor cell inoculation Tumor Volume (mm 3 ) No T cells T cells + IC T cells + Keytruda T cells + Avelumab T cells + 8371 T cells + PID-5616+ PID-6068 0 20 40 60 0 20 40 60 80 100 Survival Days after tumor cell inoculation Percent survival No T cells T cells + IC T cells + Keytruda T cells + Avelumab T cells + 8371 T cells + PID-5616+ PID-6068 0 20 40 60 0 1000 2000 3000 hIgG1 Tumor volume (mm 3 ) 0 20 40 60 0 1000 2000 3000 Avelumab Tumor volume (mm 3 ) 0 20 40 60 0 1000 2000 3000 Keytruda Tumor volume (mm 3 ) 0 20 40 60 0 1000 2000 3000 CTX-8371 Tumor volume (mm 3 ) 0 20 40 60 0 1000 2000 3000 Keytruda + Avelumab Tumor volume (mm 3 ) 0 20 40 60 80 0 20 40 60 80 100 Survival Days after tumor cell inoculation Percent survival CTX-8371 Keytruda Avelumab IC hIgG1 Keytruda + Avelumab 0 5 10 15 20 25 0 500 1000 1500 B16F10-HuPD-L1 Tumor Volume (Day 21 cutoff) Days after tumor cell inoculation Average Tumor Volume (mm 3 ) Isotype Avelumab Keytruda CTX-8371 Keytruda + Avelumab 0 5 10 15 20 0 100 200 300 B16F10-HuPD-L1 Tumor Volume (Day 15 cutoff) Days after tumor cell inoculation Average Tumor Volume (mm 3 ) Isotype Avelumab Keytruda CTX-8371 Keytruda + Avelumab * ** **** **** CTX-8371 is effective against mouse syngeneic tumors, although its potency in syngeneic tumors might be underestimated due to the very weak mouse PD-L1 cross-reactivity Upper panel shows individual and average tumor growth curves for EMT-6 mouse breast cancer treated with CTX-8371 or isotype control antibodies Lower panel shows individual and average tumor growth curves for MB49 mouse bladder carcinoma treated with CTX-8371 or isotype control antibodies ****, P<0.0001, ***, P<0.001, Two-way ANOVA and Tukey's multiple comparisons test. CTX-8371 is effective against human xenograft tumors Upper panel shows individual, average tumor growth curves, survival, and body weight change of NSG mice adoptively transferred with CMV-specific human T cells against K562 s.c. tumor challenge. Different groups of mice (n=10) were treated with CTX-8371, Keytruda, anti-PD-1 + anti-PD-L1 monoclonal combination or isotype control antibodies. Lower panel shows individual, average tumor growth curves, survival, and body weight change of NSG mice adoptively transferred with CMV-specific human T cells against Raji s.c. tumor challenge. Different groups of mice (n=8) were treated with CTX-8371, Keytruda, Avelumab, anti-PD-1 + anti-PD-L1 monoclonal combination or isotype control antibodies. ****, P<0.0001, **, P<0.01, Two-way ANOVA and Tukey's multiple comparisons test; ***, P<0.001, Log-rank (Mantel-Cox) test comparing CTX-8371 to Keytruda . CTX-8371 is effective against mouse tumors expressing human PD-L1 Upper graphs show average tumor growth curves, body weight, and survival of huPD-1/PD-L1 transgenic mice bearing MC-38-hPD-L1 tumors. * 2 mice in Keytruda group and 1 mouse in control group were euthanized due to dermatitis Lower graphs depict individual and average tumor growth curves, survival, and body weight change of transgenic C57BL/6-huPD- 1/PD-L1 mice bearing B16-F10-huPD-L1 melanomas. Different groups of mice (n=8) were treated with CTX-8371, Keytruda, Avelumab, Keytruda + Avelumab combination or isotype control antibodies. ****, P<0.0001, **, P<0.01, *, P<0.05, Two-way ANOVA and Tukey's multiple comparisons test. NSG Mice were co- inoculated s.c. with 5e6 K562-A2-CMV-PDL1 and 5e6 CMV-specific T cells (upper panels) or 5e6 Raji-A2-CMV-PDL1 and 5e6 CMV-specific T cells (lower panels) in matrigel Dosing with equimolar amounts of antibody started at day of inoculation and every 3 days thereafter for a total of 5 doses Tumor size and body weight were measured 2- 3 times per week, with mice euthanized when tumors are approaching 2000 mm 3 or mice lost 20% of body weight **** *** 0 10 20 30 40 0 20 40 60 80 100 Survival Days after tumor cell inoculation Percent survival *** 0 5 10 15 20 0 500 1000 1500 2000 K562 Tumor Volume Days after tumor cell inoculation Average Tumor Volume (mm 3 ) No T cells T cells + Isotype T cells + Keytruda T cells + CTX-8371 T cells + CTX-5616 + CTX-6068 ] ** **** 0 10 20 30 40 0 1000 2000 3000 No T cells Days posttreatment start Tumor volume (mm 3 ) 0 10 20 30 40 0 1000 2000 3000 Isotype Control Days posttreatment start Tumor volume (mm 3 ) 0 10 20 30 40 0 1000 2000 3000 Keytruda Days posttreatment start Tumor volume (mm 3 ) 0 10 20 30 40 0 1000 2000 3000 CTX-8371 Days posttreatment start Tumor volume (mm 3 ) 0 10 20 30 40 0 1000 2000 3000 CTX-5616 + CTX-6068 Days posttreatment start Tumor volume (mm 3 ) CTX-8371 is effective against human xenograft tumors CTX-8371 has potent anti-tumor activity against mouse tumors implanted in PD-1/PD-L1 humanized mice with no apparent toxicity EMT6 MB49 0.0001 0.001 0.01 0.1 1 10 0 200 400 600 800 1000 1200 Ab concentration(nM) IFN- (pg/ml) 1 st Gen PD-1xPD-L1 bispecifics No Ab control Keytruda Nivolumab Isotype T cell activation Jurkat-PD-1 + Jurkat-PD-L1 Isotype CTX-8371 0.01 0.1 1 0.01 .01 1 Jurkat-PD-1 Jurkat-PD-1/PD-L1 Ab Isotype CTX-8371 Isotype CTX-8371 nM: 0.01 1 0.01 0.1 1 0.01 0.1 1 0.01 0.1 1 Tumor growth Body weight 1 st Gen PD-1xPD-L1 1 st Gen PD-1xPD-L1 * 1 st Gen PD-1xPD-L1 * Survival 0 5 10 15 20 0 2 4 6 8 10 Body weight change Days after tumor cell inoculation Body weight % change IC hIgG1 Avelumab Keytruda CTX-8371 Keytruda + Avelumab 0 5 10 15 20 25 -5 0 5 10 15 Body weight change Days after tumor cell inoculation % Body Weight Change 0 5 10 15 20 -10 -5 0 5 Body weight change Days after tumor cell inoculation Body weight % change Treatment Tumor-free/Total IC hIgG1 0/8 CTX-8371 3/8 Keytruda 0/8 Avelumab 0/8 Keytruda + Avelumab 1/8 Treatment Tumor-free/Total No T cells 0/8 T cells + IC 0/8 T cells + CTX-8371 2/8 T cells + Keytruda 0/8 T cells + Avelumab 2/8 T cells + a-PD-1 + a-PD-L1 2/8 Discovery of PD-1xPD-L1 synergy via a Stitchmab™ experiment

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Page 1: K e y t r u d a + A v e l u m a b C T X - 8 3 7 1 A v e l ... · C T X - 8 3 7 1 K e y t r u d a C T X - 3 8 3 4 ( A t e z o l i z u m a b ) C T X - 5 6 1 6 + C T X - 6 0 6 8 •

Stitchmabs allow for rapid testing of bispecific concepts

• We initially theorized that a bispecific containing anti-PD-1 and PD-L1 arms would function to bring T

cells into contact with PD-L1+ tumor cells while also blocking a critical T cell checkpoint

• In a MLR assay, Stitchmab™ bispecifics of commercial anti-PD-1 and anti-PD-L1 antibodies caused

increased IFN- production at picomolar antibody levels • This prompted us to generate common light chain bispecific variants

• CTX-8371 was selected as a PD-1×PD-L1 bispecific from in-house generated anti-PD-1 and anti-PD-L1

antibodies with excellent drug-like properties

• Compass anti-PD-1 and anti-PD-L1 antibody Fabs have similar affinities as clinical comparator Fabs to

Human and Cynomolgus monkey PD-1 and PD-L1

• CTX-8371 maintains monoclonal antibody-like binding to PD-1 and PD-L1 from all 3 species as

compared to monoclonal parent molecules.• CTX-8371 also has strong anti-mouse PD-1 binding but weaker anti-mouse PD-L1 binding

1

• Human T cells were expanded in vitro by TCR activation for 7 days and co-cultured with engineered K562 tumor cell targets in the

presence of antibodies for 2 days• 1st generation PD-1xPD-L1 bispecifics enhanced T cell activation compared to Keytruda, Nivolumab, and controls

• Human PBMCs were stimulated with Staphylococcus Enterotoxin A (SEA) and treated with CTX-8371 or other antibodies for 3 days• CTX-8371 remained active at sub-nanomolar doses while other antibodies have regressed to the isotype

• CMV-specific T cells were co-cultured with engineered K562 tumor cell targets and bystander cells in the presence of antibodies for 2

days• CTX-8371 treatment increased CMV-specific T cell killing of K562 tumor cell targets at sub-nanomolar doses while the other antibodies

regressed to the isotype

CTX-8371 increases T cell activation and killing in vitro

Background Monoclonal antibody immunotherapies targeting immune checkpoint receptors have shown great promise for a subset of

cancer patients. However, novel combination therapies are still needed to increase the benefit of cancer immunotherapy and bring it to

broader patient populations. Here we describe the preclinical evaluation of CTX-8371, which combines PD-1 and PD-L1 targeting in one

bispecific, tetravalent molecule.

Methods Our proprietary Stitchmabs™ bispecific screening platform was used to conduct an unbiased screen of bispecifics comprised

of various checkpoint blocking antibodies. This screen yielded the surprising discovery that a bispecific containing both PD-1 and PD-L1

binding arms was more potent than the combination of parent monoclonal antibodies. We then generated common light chain

bispecifics containing compatible anti-PD-1 and PD-L1 antibodies and used multiple in vitro assays to identify our lead, CTX-8371.

Additional in vitro and in vivo experiments confirmed CTX-8371’s reactivity across species, in vivo anti-tumor effects, and the underlining

mechanisms driving its distinctive activity.

Results We found that CTX-8371 binds to human and cynomolgus monkey PD-1 and PD-L1 targets with sub-nanomolar affinities and

is cross-reactive to mouse PD-1 and PD-L1. Compared to Keytruda®, CTX-8371 increased T cell activation and tumor cell killing in

vitro, significantly delayed tumor growth, and prolonged survival in human cell transfer tumor models. Additionally, CTX-8371

demonstrated efficacy in transplantable mouse syngeneic models. Investigation into the mechanisms responsible for the enhanced

efficacy of CTX-8371 unexpectedly found that the bispecific causes a massive loss of PD-1 from the T cell surface, which was not

observed in response to monoclonal antibodies alone or combined. This robust PD-1 downregulation, potentially mediated through

bridging together the T cell and tumor cell, may explain the ability of CTX-8371 to reverse PD-1 suppression more potently than

standard blocking antibodies.

Conclusions Taken together, the results demonstrate that the bispecific, tetravalent antibody CTX-8371 has increased potency in vitro

and in vivo as compared to clinical checkpoint blockade agents. Some of its effects are likely attributable to its unique mechanism of

action, driving robust downregulation of cell-surface PD-1. Thus, CTX-8371 has the potential to increase the number of patients that

benefit from PD-1/PD-L1 checkpoint blockade.

Summary• PD-1xPD-L1 StichmabTM antibodies were superior to monoclonal combination in a MLR assay. This preliminary data led to the

creation of CTX-8371, a common light chain bispecific antibody with excellent drug-like properties.

• CTX-8371 had exceptional in vitro activity and in vivo anti-tumor efficacy compared to approved checkpoint inhibitor

immunotherapies and combination thereof.

• The anti-tumor activity of CTX-8371 might be at least in part explained by its unique MOA leading to massive surface PD-1 loss,

mediated through bridging the T cell and tumor cell.

• Investigation into CTX-8371 MOA is ongoing.

CTX-8371, a Novel Bispecific Targeting both PD-1 and PD-L1, is more Potent than Combination anti-PD-1 and PD-L1 Therapy and Provides Enhanced Protection from Tumors in vivo

Diana Albu, Pearl Bakhru, Wilson Guzman, Michael Ophir, Rachel McCrory, Beau Carson, Amanda Oliphant, Rachel Rennard, Lucy Liu, Alan

Leung, Sara Haserlat, Michael Schmidt, Jose Gonzalo, Bing Gong, Robert Tighe, Ben Wolf

Compass Therapeutics, 245 First Street, Cambridge MA

+

IFN

(pg

/ml)

Stitchmab

“A×C”

Stitchmab

“B×C”

Combo

“A” + “C”

Combo

“B” + “C”

Single mAb

“A”

Anti-PD-1

Parent mAb: CTX-5616

Anti-PD-L1

Parent mAb: CTX-6068

CTX-8371

Affinity (nM) Human PD-1 Cyno PD-1

CTX-5616(a-PD-1)

1.09e-09 1.97e-09

Keytruda 1.81e-09 7.42e-10

Nivolumab 3.52e-09 1.34e-09

Affinity (nM) Human PD-L1 Cyno PD-L1

CTX-6068 (a-PD-L1)

3.08e-09 2.30e-09

Atezolizumab 4.55e-10 1.05e-08

Avelumab 5.05e-10 7.51e-10

-1 4 -1 2 -1 0 -8 -6

0

21 0 6

41 0 6

61 0 6

81 0 6

C e ll B in d in g : C H O /H u P D -1

L o g c o n c (M )

MF

I

C T X -8 3 7 1

C T X -5 6 1 6

-1 3 -1 2 -1 1 -1 0 -9 -8 -7

0

51 0 5

11 0 6

1 .51 0 6

C e ll B in d in g : C H O /H u P D -L 1

L o g c o n c (M )

MF

I

C T X -8 3 7 1

C T X -6 0 6 8

-1 4 -1 2 -1 0 -8 -6

0

51 0 5

11 0 6

1 .51 0 6

21 0 6

2 .51 0 6

C e ll B in d in g : C H O /C y P D -1

L o g c o n c (M )

MF

I

C T X -8 3 7 1

C T X -5 6 1 6

-1 3 -1 2 -1 1 -1 0 -9 -8 -7

0

21 0 4

41 0 4

61 0 4

81 0 4

C e ll B in d in g : C H O /C y P D -L 1

L o g c o n c (M )

MF

I

C T X -8 3 7 1

C T X -6 0 6 8

-1 4 -1 2 -1 0 -8 -6

0

51 0 5

11 0 6

1 .51 0 6

C e ll B in d in g : C H O /M s P D -1

L o g c o n c (M )

MF

I

C T X -8 3 7 1

C T X -5 6 1 6

-1 4 -1 2 -1 0 -8 -6

11 0 4

1 .21 0 4

1 .41 0 4

1 .61 0 4

C e ll B in d in g : A 2 0 (M o u s e )

L o g c o n c (M )

MF

I

C T X -8 3 7 1

C T X -6 0 6 8

0.0

0001

0.0

001

0.0

01

0.0

10.1 1

10

100

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

S E A a s s a y

C o n ce n tra tio n (n M )

IL-2

(p

g/m

l)

0.0

0001

0.0

001

0.0

01

0.0

10.1 1

10

100

1000

3 5

4 0

4 5

5 0

5 5

6 0

6 5

T u m o r c e ll k il l in g a s s a y

C o n ce n tra tio n (n M )

% S

pe

cif

ic K

illi

ng

Is o ty p e

C T X -8 3 7 1

K e y tru d a

C T X -3 8 3 4 (A te z o liz u m a b )

C T X -5 6 1 6 + C T X -6 0 6 8

• Human PBMCs were stimulated with anti-CD3 and anti-CD28 antibodies for 3 days before overnight co-culture with the listed

antibodies• CTX-8371 drove loss of cell-associated PD-1 but only when both arms of the bispecific were engaged

• Jurkat-PD1, Jurkat-PD-1/PD-L1, or mix of Jurkat-PD-1 + Jurkat-PD-L1 were treated overnight with isotype or CTX-8371 antibodies• PD-L1 binding was necessary for CTX-8371-induced loss of cell-associated PD-1 (when both arms of the bispecific were engaged)

• CTX-8371 drove PD-1 loss predominantly when PD-1 and PD-L1 were in trans

Ab Isotype Keytruda CTX-8371

nM: 0.1 1 10 0.1 1 10 0.1 1 10

AtezolizumabKeytruda +

AtezolizumabCTX 5616 + 6068

0.1 1 10 0.1 1 10 0.1 1 10

CTX-8371 + 50 nM

CTX-6068

0.1 1 10

WB

:

α-P

D-1

50 kD -

37 kD -

75 kD -

WB

:

α-β

-Actin

50 kD -

37 kD -

75 kD -

Abstract

CTX-8371 is effective against mouse syngeneic tumors

Unique MOA: CTX-8371 drives loss of PD-1

0 1 0 2 0 3 0 4 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

C o n tro l

D a y s p o s t tu m o r in o c u la tio n

Tu

mo

r v

olu

me

(m

m3)

0 1 0 2 0 3 0 4 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

C T X -8 3 7 1

0 5 1 0 1 5 2 0 2 5

0

2 0 0

4 0 0

6 0 0

8 0 0

D a y s p o s t tu m o r in o c u la t io n

Av

era

ge

tu

mo

r v

olu

me

(m

m3)

C o n tro l

C T X -8 3 7 1

T re a tm e n t

S ta r t

***

0 1 0 2 0 3 0 4 0 5 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

h Ig G 1 IC

D a y s a fte r tu m o r c e ll in o c u la tio n

Tu

mo

r v

olu

me

(m

m3)

0 1 0 2 0 3 0 4 0 5 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

C T X -8 3 7 1

0 5 1 0 1 5 2 0 2 5

0

5 0 0

1 0 0 0

1 5 0 0

D a y s a f te r tu m o r c e lls in o c u la t io n

Av

era

ge

Tu

mo

r V

olu

me

(m

m3)

h Ig G 1 IC

C T X -8 3 7 1

****

T re a tm e n t

s ta r t

0 1 0 2 0 3 0 4 0

0

1 0 0 0

2 0 0 0

3 0 0 0

N o T c e lls

D a y s a fte r tu m o r c e ll in o c u la t io n

Tu

mo

r V

olu

me

(m

m3

)

0 1 0 2 0 3 0 4 0 5 0

0

1 0 0 0

2 0 0 0

3 0 0 0

T c e lls + IC

0 1 0 2 0 3 0 4 0 5 0

0

1 0 0 0

2 0 0 0

3 0 0 0

T c e lls + K e y tru d a

0 1 0 2 0 3 0 4 0 5 0

0

1 0 0 0

2 0 0 0

3 0 0 0

T c e lls + A v e lu m a b

0 1 0 2 0 3 0 4 0 5 0

0

1 0 0 0

2 0 0 0

3 0 0 0

T c e lls + P ID -8 3 7 1

0 1 0 2 0 3 0 4 0 5 0

0

1 0 0 0

2 0 0 0

3 0 0 0

T c e lls + P ID -5 6 1 6 + P ID -6 0 6 8

0 1 0 2 0 3 0 4 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0T u m o r V o lu m e

D a y s a fte r tu m o r c e ll in o c u la t io n

Tu

mo

r V

olu

me

(m

m3

)

N o T ce lls

T c e lls + IC

T c e lls + K e y tru d a

T c e lls + A v e lu m a b

T c e lls + 8 3 7 1

T c e lls +

P ID -5 6 1 6 +

P ID -6 0 6 8

0 2 0 4 0 6 0

0

2 0

4 0

6 0

8 0

1 0 0

S u rv iv a l

D a y s a fte r tu m o r c e ll in o c u la t io n

Pe

rce

nt

su

rviv

al

N o T ce lls

T c e lls + IC

T c e lls + K e y tru d a

T c e lls + A v e lu m a b

T c e lls + 8 3 7 1

T c e lls + P ID -5 6 1 6 +

P ID -6 0 6 8

0 2 0 4 0 6 0

0

1 0 0 0

2 0 0 0

3 0 0 0

h Ig G 1

Tu

mo

r v

olu

me

(m

m3

)

0 2 0 4 0 6 0

0

1 0 0 0

2 0 0 0

3 0 0 0

A v e lu m a b

Tu

mo

r v

olu

me

(m

m3

)

0 2 0 4 0 6 0

0

1 0 0 0

2 0 0 0

3 0 0 0

K e y tr u d a

Tu

mo

r v

olu

me

(m

m3

)

0 2 0 4 0 6 0

0

1 0 0 0

2 0 0 0

3 0 0 0

C T X -8 3 7 1

Tu

mo

r v

olu

me

(m

m3

)

0 2 0 4 0 6 0

0

1 0 0 0

2 0 0 0

3 0 0 0

K e y tru d a + A v e lu m a b

Tu

mo

r v

olu

me

(m

m3

)

0 2 0 4 0 6 0 8 0

0

2 0

4 0

6 0

8 0

1 0 0

S u rv iv a l

D a y s a fte r tu m o r c e ll in o c u la t io n

Pe

rce

nt

su

rviv

al

C T X -8 3 7 1

K e y tru d a

A ve lu m ab

IC h Ig G 1

K e y tru d a +

A ve lu m ab

0 5 1 0 1 5 2 0 2 5

0

5 0 0

1 0 0 0

1 5 0 0

B 1 6 F 1 0 -H u P D -L 1 T u m o r V o lu m e

(D a y 2 1 c u to ff)

D a y s a fte r tu m o r c e ll in o c u la t io n

Av

era

ge

Tu

mo

r V

olu

me

(m

m3

)

Is o ty p e

A ve lu m ab

K e y tru d a

C T X -8 3 7 1

K e y tru d a +

A ve lu m ab

0 5 1 0 1 5 2 0

0

1 0 0

2 0 0

3 0 0

B 1 6 F 1 0 -H u P D -L 1 T u m o r V o lu m e

(D a y 1 5 c u to ff)

D a y s a fte r tu m o r c e ll in o c u la t io n

Av

era

ge

Tu

mo

r V

olu

me

(m

m3

)

Is o ty p e

A ve lu m ab

K e y tru d a

C T X -8 3 7 1

K e y tru d a +

A ve lu m ab

* **

**** ****

• CTX-8371 is effective against mouse syngeneic tumors, although its potency in syngeneic tumors might be underestimated

due to the very weak mouse PD-L1 cross-reactivity• Upper panel shows individual and average tumor growth curves for EMT-6 mouse breast cancer treated with CTX-8371 or isotype

control antibodies

• Lower panel shows individual and average tumor growth curves for MB49 mouse bladder carcinoma treated with CTX-8371 or isotype

control antibodies

• ****, P<0.0001, ***, P<0.001, Two-way ANOVA and Tukey's multiple comparisons test.

• CTX-8371 is effective against human xenograft tumors

• Upper panel shows individual, average tumor growth curves, survival, and body weight change of NSG mice adoptively transferred

with CMV-specific human T cells against K562 s.c. tumor challenge. Different groups of mice (n=10) were treated with CTX-8371,

Keytruda, anti-PD-1 + anti-PD-L1 monoclonal combination or isotype control antibodies.

• Lower panel shows individual, average tumor growth curves, survival, and body weight change of NSG mice adoptively transferred

with CMV-specific human T cells against Raji s.c. tumor challenge. Different groups of mice (n=8) were treated with CTX-8371,

Keytruda, Avelumab, anti-PD-1 + anti-PD-L1 monoclonal combination or isotype control antibodies.

• ****, P<0.0001, **, P<0.01, Two-way ANOVA and Tukey's multiple comparisons test; ***, P<0.001, Log-rank (Mantel-Cox) test

comparing CTX-8371 to Keytruda .

• CTX-8371 is effective against mouse tumors expressing human PD-L1

• Upper graphs show average tumor growth curves, body weight, and survival of huPD-1/PD-L1 transgenic mice bearing MC-38-hPD-L1 tumors. * 2 mice in Keytruda group and 1 mouse in control group were euthanized due to dermatitis

• Lower graphs depict individual and average tumor growth curves, survival, and body weight change of transgenic C57BL/6-huPD-

1/PD-L1 mice bearing B16-F10-huPD-L1 melanomas. Different groups of mice (n=8) were treated with CTX-8371, Keytruda,

Avelumab, Keytruda + Avelumab combination or isotype control antibodies.

• ****, P<0.0001, **, P<0.01, *, P<0.05, Two-way ANOVA and Tukey's multiple comparisons test.

• NSG Mice were co-

inoculated s.c. with 5e6

K562-A2-CMV-PDL1 and

5e6 CMV-specific T cells

(upper panels) or 5e6

Raji-A2-CMV-PDL1 and

5e6 CMV-specific T cells

(lower panels) in matrigel

• Dosing with equimolar

amounts of antibody

started at day of

inoculation and every 3

days thereafter for a total

of 5 doses

• Tumor size and body

weight were measured 2-

3 times per week, with

mice euthanized when

tumors are approaching

2000 mm3 or mice lost

20% of body weight

****

***

0 1 0 2 0 3 0 4 0

0

2 0

4 0

6 0

8 0

1 0 0

S u rv iv a l

D a y s a fte r tu m o r c e ll in o c u la t io n

Pe

rce

nt

su

rviv

al

***

0 5 1 0 1 5 2 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

K 5 6 2 T u m o r V o lu m e

D a y s a fte r tu m o r c e ll in o c u la t io n

Av

era

ge

Tu

mo

r V

olu

me

(m

m3

)

N o T ce lls

T c e lls + Is o ty p e

T c e lls + K e y tru d a

T c e lls + C T X -8 3 7 1

T c e lls + C T X -5 6 1 6

+ C T X -6 0 6 8] * *

* * * *

0 1 0 2 0 3 0 4 0

0

1 0 0 0

2 0 0 0

3 0 0 0

N o T c e lls

D a y s p o s ttre a tm e n t s ta rt

Tu

mo

r v

olu

me

(m

m3

)

0 1 0 2 0 3 0 4 0

0

1 0 0 0

2 0 0 0

3 0 0 0

Is o ty p e C o n tro l

D a y s p o s ttre a tm e n t s ta rt

Tu

mo

r v

olu

me

(m

m3

)

0 1 0 2 0 3 0 4 0

0

1 0 0 0

2 0 0 0

3 0 0 0

K e y tr u d a

D a y s p o s ttre a tm e n t s ta rt

Tu

mo

r v

olu

me

(m

m3

)

0 1 0 2 0 3 0 4 0

0

1 0 0 0

2 0 0 0

3 0 0 0

C T X -8 3 7 1

D a y s p o s ttre a tm e n t s ta rt

Tu

mo

r v

olu

me

(m

m3

)

0 1 0 2 0 3 0 4 0

0

1 0 0 0

2 0 0 0

3 0 0 0

C T X -5 6 1 6 + C T X -6 0 6 8

D a y s p o s ttre a tm e n t s ta rt

Tu

mo

r v

olu

me

(m

m3

)

CTX-8371 is effective against human xenograft tumors

CTX-8371 has potent anti-tumor activity against mouse tumors implanted in

PD-1/PD-L1 humanized mice with no apparent toxicity

EMT6

MB49

0.0001 0.001 0.01 0.1 1 100

200

400

600

800

1000

1200

1400

PD1-PDL1 bispecific testing

Ab concentration(nM)

IFN

- (

pg

/ml)

6254

6255

Keytruda

Nivolumab

1282

No Ab control

1st Gen PD-1xPD-L1bispecifics

0.0001 0.001 0.01 0.1 1 100

200

400

600

800

1000

1200

1400

PD1-PDL1 bispecific testing

Ab concentration(nM)

IFN

- (

pg

/ml)

6254

6255

Keytruda

Nivolumab

1282

No Ab control

0.0001 0.001 0.01 0.1 1 100

200

400

600

800

1000

1200

1400

PD1-PDL1 bispecific testing

Ab concentration(nM)

IFN

- (

pg

/ml)

6254

6255

Keytruda

Nivolumab

1282

No Ab control

Isotype

T cell activation

Jurkat-PD-1 + Jurkat-PD-L1

Isotype CTX-8371

0.01 0.1 1 0.01 .01 1

Jurkat-PD-1 Jurkat-PD-1/PD-L1

Ab Isotype CTX-8371 Isotype CTX-8371

nM: 0.01 1 0.01 0.1 1 0.01 0.1 1 0.01 0.1 1

Tumor growth Body weight1st Gen PD-1xPD-L1 1st Gen PD-1xPD-L1

*

1st Gen PD-1xPD-L1

*

Survival

0 5 1 0 1 5 2 0

0

2

4

6

8

1 0

B o d y w e ig h t c h a n g e

D a y s a fte r tu m o r c e ll in o c u la t io n

Bo

dy

we

igh

t %

ch

an

ge

IC h Ig G 1

A ve lu m ab

K e y tru d a

C T X -8 3 7 1

K e y tru d a +

A ve lu m ab

0 5 1 0 1 5 2 0 2 5

-5

0

5

1 0

1 5B o d y w e ig h t c h a n g e

D a y s a fte r tu m o r c e ll in o c u la t io n

% B

od

y W

eig

ht

Ch

an

ge N o T ce lls

T c e lls + IC

T c e lls + K e y tru d a

T c e lls + A v e lu m a b

T c e lls + C T X -8 3 7 1

T c e lls + C T X -5 6 1 6 + C T X -6 0 6 8

0 5 1 0 1 5 2 0

-1 0

-5

0

5B o d y w e ig h t c h a n g e

D a y s a fte r tu m o r c e ll in o c u la t io n

Bo

dy

we

igh

t %

ch

an

ge N o T ce lls

T c e lls + IC

T c e lls + K e y tru d a

T c e lls + C T X -8 3 7 1

T c e lls + C T X -5 6 1 6

+ C T X -6 0 6 8

Treatment Tumor-free/Total

IC hIgG1 0/8

CTX-8371 3/8

Keytruda 0/8

Avelumab 0/8

Keytruda + Avelumab 1/8

Treatment Tumor-free/Total

No T cells 0/8

T cells + IC 0/8

T cells + CTX-8371 2/8

T cells + Keytruda 0/8

T cells + Avelumab 2/8

T cells + a-PD-1 + a-PD-L1 2/8

Discovery of PD-1xPD-L1 synergy via a Stitchmab™ experiment