Expanding the Therapeutic Potential of anti-PD-1 and anti-CTLA-4 Therapy with Innovative Fc Engineering and Rationale Combinations for the Treatment of Solid Tumors Antoine Tanne, Sylvia Vincent, Elena Paltrinieri, Sudesh Pawaria, Vidur Patel, Marques Marilyn, Margaret Wilkens, Shalu Kharkwal, Daniel Levey, David Savitsky, Jennifer Buell, Dhan Chand 1 1 Agenus Inc. or subsidiary thereof (current or former employee), Lexington, MA AGEN1181 ms has improved therapeutic potential against immunogenic tumors by enhancing Treg depletion and T cell priming Hypothesis: Optimizing Fc - FcgR co-engagement enhances the activity of anti-CTLA-4 antagonist antibodies 1,2 . CTLA-4 antibodies with increased binding affinities to activating Fcg receptors FcgRIV (CD16-2, mouse) or FcgRIIIA (CD16a, human) augment T cell priming by improving the quality of the immune synapse between a T cell and an antigen presenting cell (APC). Table 1: anti-CTLA-4 Fc Isotype Fc mutations Blocking Properties FcgR Binding Characteristics Human Parental IgG1 - + Low Fc-Enhanced AGEN1181 IgG1.DLE S239D.A330L.I332E + > FcgRIIIA binding (“Fc enhanced”) Murine Surrogates Parental 9D9 mIgG2b - + Low AGEN1181 ms Fc-Enhanced 9D9 mIgG2b.DLE S241D.A332L.I334E + > FcgRIV binding Fcүr binding characteristics of studied antibodies 1. Ligand blockade & CD28- dependent T cell activation 2. Depletion of intratumoral regulatory T cells (Tregs) 3. Effectory T cell priming and memory formation FcγR-independent FcγR-dependent Combination with anti-PD-1 and Focal Radiation promotes significant tumor control Fc-enhanced CTLA-4 promotes tumor control in combination with Tumor Antigen Vaccine + QS-21 to enhance tumor control Fc-enhanced CTLA-4 combines with Adoptive T Cell Therapy (ACT) to promote robust tumor control AGEN1181 expands the therapeutic reach and activity of CTLA-4 therapy by improved binding to both low and high affinity FcγRIIIA allele Combination with Alpha-GalCer as iNKT triggering therapy promotes robust tumor clearance in the lung Fc-enhanced CTLA-4, AGEN1181, combines with anti-PD-1 or anti-TIGIT to further enhance T cell responsiveness Concurrent treatment promotes curative responses in combination with HSC70 tumor antigen vaccine + QS-21 Agenus Inc. or subsidiary thereof (current or former employee), Lexington, MA 02421, USA. Correspondence Antoine Tanne [email protected] Dhan Chand [email protected] Presented at The American Association for Cancer Research (AACR), June 22 - 24, 2020, Virtual Meeting Figure 1: BALB/c and C57Bl6 mice with established CT26 and MC38 tumors received a single injection intraperitoneally (i.p.) of anti- CTLA4 clone 9D9 Parental or Fc-Enhanced or mIgG2a isotype control. A-B: Tumor growth are represented as average per group and single mice to emphasize the increased potency of the AGEN1181 “surrogate” C-D: CD8 Teff / CD4 Treg ratio changes over time is shown as average per group to emphasize the increased ability of the Fc-Enh. Antibody to deplete Treg and expend Teff. Briefly, tumor were resected and were phenotyped by flow cytometry the effector CD8 T (CD3+ CD8+ CD44+) Vs CD4 regulatory T (CD3+ CD4+ FoxP3+) ratio was calculated at different time points post treatment. E-F: The antitumoral memory response was assessed in complete responder (CR) mice treated with the Fc Enh. Mice were bilaterally re-challenged with 100K primary tumor CT26 and MC38 and non relevant tumors EMT 6 or TC1 . Figure 5: C57Bl6 were challenged s.c. with B16F1::OVA cells. Mice were preconditioned with early treatment (D3) using AGEN1181 anti-CTLA-4 Fc-Enhanced “surrogate” and anti-PD-1 or the corresponding isotype control. Eight days after tumor implantation, tumor-bearing mice were treated intravenously with OT1-specific transgenic CD8+ T cells (ACT) or PBS. A: Tumor volumes were measured every 3-4 days and represented as average per group (N=10) B: Representation of the overall survival per treatment group. Sequential treatment with Fc-enhanced anti-CTLA-4 and PD1 promotes curative responses in combination with ACT Figure 6: C57Bl6 were challenged s.c. with B16F1::OVA cells. Tumor-bearing mice were subcutaneously treated with the Agenus preclinical heat shock protein vaccine targeting MHC-I/II ovalbumin, and MHC-I TRP2 epitopes (HSC70-OVA–TRP2) administered with Agenu’s QS21 Stimulon and CpG adjuvant. Mice were treated i.p. with anti-CTLA-4 and anti-PD-1 or corresponding isotype control antibodies A: Average tumor volumes per group (n=10/group) B: survival curves and C: treatment response evaluation per treatment group (CR: Complete Responder; PR: partial responder defined by relapsing pattern). Conclusions AGEN1181 Fc-enhanced anti-CTLA-4 demonstrates superior single agent and broader IO & SOC combination activity than conventional CTLA-4 mAbs: • Engages multiple mechanisms of action to promote optimal anti-tumor immunity • Promotes curative responses in checkpoint resistant preclinical cancer models in combination with anti-PD-1 and Vaccine + QS-21, Adoptive T cell therapy, radiation therapy and iNKT activating Alpha-GalCer adjuvant therapy • Enhances T cell responsiveness in combination with anti-PD-1 or anti-TIGIT mAbs • Expands the therapeutic reach and activity of CTLA-4 therapy by improved binding to both the low and high affinity FcyRIIIA and in turn expands the therapeutic reach of anti-CTLA-4 to an additional 40% of patients 1,3,4 who express the low affinity FcyRIIIA allele and respond poorly to first generation anti-CTLA-4 mAbs. AGEN1181 ms Fc-Enhanced CTLA4 combines with adjuvant therapies AGEN1181 ms Fc-Enhanced CTLA4 & PD1 triple combination with ACT and Vaccines are curative in IO refractory models Figure 2: C57BL/6 mice were injected i.p. with the staphylococcal enterotoxin B (SEB) superantigen together with anti-CTLA-4 parental , Fc-Enhanced AGEN1181 “surrogate” or isotype control antibodies. SEB-specific (TCR Vb8) CD8 + T cells (CD3+ CD8+ CD44+), their replicative status (Ki67) and their functional profile (GrZb) were evaluated by flow cytometry on day 6; demonstrating an increase potency of the Fc-Enh. antibody to prime antigen specific effector CD8 T cells in a T reg depletion independent mechanism (not shown) . N=4 mice/group, and data are representative of at least three independent experiments. Figure 3: C57Bl6 were challenged s.c. with B16F1::OVA cells. At day 10 tumor-bearing mice were treated intravenously with OT1-specific transgenic CD8+ T cells (ACT) or PBS . CTLA4 treated groups received three doses of i.p weekly injections of Fc-Enhanced AGEN1181 “surrogate” or the reference molecule starting at D10 A: Tumor volumes were measured every 3-4 days and represented as average per group (N=10) B: Representation of the overall survival per treatment group. C: Tumor infiltrating CD8 & CD4 Teff / CD4 Treg ratio evaluation D: Host and ACT Tumor infiltrating OT1 Tet + CD8 Teff / CD4 Treg ratio evaluation. Briefly, tumor were resected and phenotyped by flow cytometry. Effector CD8 T OT1 Tet +/- (CD3+ CD8+ CD44+ PD1+ GzB+) Vs CD4 regulatory T (CD3+ CD4+ FoxP3+) ratio was calculated for Host (CD45.1) and ACT (CD45.2). Figure 4: C57Bl6 were injected s.c. with B16F1::OVA cells. Challenged mice were subcutaneously treated with Agenus preclinical heat shock protein vaccine targeting MHC-I/II ovalbumin epitopes, (HSC70-OVA OTI/II) administered with Agenu’s QS21 Stimulon and CpG adjuvant. Mice were treated i.p. with anti-CTLA-4 and anti-PD-1 or corresponding isotype control antibodies A: Tumor volumes were measured every 3-4 days and represented as average per group (N=10) B: survival curves per treatment group are shown. C: In an independent study tumor were resected after euthanasia at day 24 to prepare CD45+ enriched single cell solution and phenotyped by flow cytometry. Activated Eff. Cd8 T cells +/- Tetramer (OT1) (CD3+ CD8+ CD44+ PD1+ GzB+) and Eff. CD4 T cells (CD3+ CD4+ CD44+) were characterized Vs host CD4 Tregs (CD3+ CD4+ FoxP3+) Figure 7: C57BL6 mice, were challenged s.c. with B16F10 cells. Tumor bearing mice received no radiation or a single-dose 10 Gy radiation treatment administered using a Small Animal Radiation Research Platform. Mice were subsequently treated i.p with anti-PD-1 and AGEN1181 anti-CTLA-4 Fc Enhanced “surrogate” or the corresponding isotype control . A-B: Tumor growth are represented as average per group and single mice to emphasize the increased efficacy of the triple combo RT with Fc-enhanced and PD1. Figure 9: A: IL-2 production by human PBMCs stimulated with a suboptimal concentration of SEA peptide together with increasing concentrations of Fc-enhanced anti-CTLA-4 AGEN1181, parental anti-CTLA-4 AGEN1884 (IgG1) or isotype control alone or in combination with 10 μg/ml anti-PD-1 (Pembrolizumab). B: IL-2 production (day 4) by human PBMCs stimulated with a suboptimal concentration of SEA peptide together with 10 μg/ml of Fc-enhanced anti-CTLA-4, AGEN1181, alone or in combination with 5 μg/ml of anti-TIGIT mAb. AGEN1181, an Fc-Enhanced Anti-CTLA-4 Antibodies Engage Multiple Mechanisms of Action to Promote T Cell-Mediated Anti-Tumor Immunity Background AGEN1181 ms Fc Enhanced anti CTLA4 combines with ACT & Vaccines in CPM refractory tumor models AGEN1181 outperforms conventional IgG1 CTLA-4 mAbs 922 Poster # AGEN1181 ms demonstrates superior efficacy 10 15 20 25 0 500 1000 1500 Days post implantation Tumor volume (mm 3 ) Isotype Parental Fc Enhanced Anti-CTLA-4 0 10 20 CTLA4 Parental 0 10 20 CTLA4 Fc Enhanced 10 15 20 25 0 500 1000 1500 2000 Tumor volume (mm 3 ) Isotype Fc Enhanced Parental Anti-CTLA-4 0 10 20 CTLA4 Parental 0 10 20 CTLA4 Fc Enhanced In vivo s.c. 100K CT26 D8 50-80 mm 3 Single dose: 0.5mg/kg CTLA4 In vivo s.c. MC38 D8 50-80 mm 3 Single dose: 0.5mg/kg CTLA4 0 48 96 144 192 240 0 20 40 60 80 CD8 T eff /T reg ratio Hours post treatment T eff /T reg ratio 0 50 100 150 200 250 0 20 40 60 80 CD8 T eff /T reg ratio Hours post treatment T eff /T reg ratio 0 20 40 0 500 1000 1500 2000 60 80 100 CR rechalenge Days post primary implantation Tumor volume (mm 3 ) CT26 (left) CR 7/8 EMT6 (right) CR 0/8 0 10 20 30 40 0 500 1000 1500 50 60 70 80 CR rechalenge Days post implantation Tumor volume (mm 3 ) MC38 (left ) CR=3/3 TC1 (Right) CR=0/3 0 20 40 60 80 100 TCR Vb8 CD8 T cells (% effector) ** 0 10 20 30 40 Ki-67 % of TCR + CD8 T cells **** 0 20 40 60 80 GrzB GrzB + of TCR + CD8 T cells **** In vivo Murine SEB Assay SEB 150ug i.p. Single dose: 5 mg/kg CTLA4 Parental Fc Enhanced Parental Fc Enhanced Anti-CTLA-4 Isotypes AGEN1181 ms demonstrates a superior CD8 effector T cell responses enhancing ability In vivo s.c. 150K B16F1::Ova D10: 120-150 mm 3 OT1 ACT (1Dose i.v.1M) CTLA4 (3 Weekly Doses i.p. 2.5 mg/kg) 0 20 40 60 0 1000 2000 3000 Days post implantation Tumor Volume (mm 3 ) NT + Isotype ACT + Isotype ACT + Parental ACT + Fc Enhanced 10 30 50 70 0 20 40 60 80 100 Study Days Percent survival ACT + Fc Enhanced ACT + Parental ACT + Isotype NT + Isotype * Host T-CD8 Host T-CD4 0 2 4 6 8 10 25 50 Activated Eff. T cells / T regs Ratio / Tregs NT+Isotype ACT+Isotype ACT+Parental ACT+Fc Enhanced ACT Host Host+ACT 0.0 0.2 0.4 0.6 Eff. CD8+ OT1 Tet + /Tregs Ratio /Tregs NT+Isotype ACT+Isotype ACT+Parental ACT+Fc Enhanced **** **** AGEN1181 ms unleashes the ACT antitumor effect AGEN1181 ms significantly increases the effector T cells to T Reg ratio Tet+ Act. Eff. CD8 Tet- Act. Eff. CD8 Act. Eff. CD4 0 2 4 6 8 10 CD8/Tregs Ratio / Tregs NT+Isotype Vaccine+Isotype Vaccine + Parental Vaccine+ Fc Enhanced In vivo s.c. 50K B16F1::Ova D3 OVA Vax (3 Weekly Doses)+Qs21-CpG D3 CTLA4 (3 Weekly 2.5mg/kg Doses Doses) D3 PD1 ( 6 Bi-Weekly 10mg/kg Doses) AGEN1181 ms increases the frequency of tumor-specific effector T cell responses In vivo s.c. 100K B16F1::Ova D7: 60-80 mm 3 D7 OVA Vax (3 Weekly Doses )+Qs21-CpG D3 CTLA4 (3 Weekly 2.5mg/kg Doses) 0 10 20 30 40 50 60 70 0 500 1000 1500 2000 2500 Days post tumor challenge Tumor Volume (mm 3 ) NT + Isotype Vaccine + Isotype Vaccine + Parental Vaccine + Fc Enhanced 0 50 100 0 50 100 Days post tumor challenge Percent survival NT + Isotype Vaccine + Isotype Vaccine + Parental Vaccine + Fc Enhanced 1/9 CR 3/10 CR AGEN1181 ms unleashes the antitumor vaccine effect In vivo s.c. 150K B16F1::Ova D8 OT1 ACT (1 Dose 1M ) D3 CTLA4 (3 Weekly Doses i.p. 2.5 mg/kg) D3 PD1 (6 Bi-Weekly Doses i.p. 10 mg/kg ) 0 20 40 60 0 1000 2000 3000 Days Post Tumor Challenge Tumor Volume (mm 3 ) NT + Isotypes ACT + Isotypes ACT + Fc Enhanced & PD-1 NT + Fc Enhanced & PD-1 0 20 40 60 0 50 100 Days Post Tumor Challenge Percent survival NT + Fc Enhanced & PD-1 ACT + Fc Enhanced & PD-1 ACT + Isotypes NT + Isotypes In vivo s.c. 50K B16F1::Ova D3 OVA-TRP2 Vax (3 Weekly Doses )+Qs21-CpG D3 CTLA4 (3 Weekly Doses i.p. 2.5 mg/kg) D3 PD1 (6 Bi-Weekly Doses i.p. 10 mg/kg ) 0 20 40 60 80 0 1000 2000 3000 4000 5000 Days post tumor challenge Tumor Volume mm 3 NT + Isotypes Vaccine + Isotypes NT + Fc Enhanced & PD-1 Vaccine + Fc Enhanced & PD-1 0 20 40 60 80 0 50 100 Days post tumor challenge Percent survival NT+Isotype Isotype+ TAA Vax NT+Fc Enhanced & PD1 Vaccine+ Fc Enhanced & PD1 Vax Iso. Vax Fc Enh. & PD-1 0 50 100 Percent Animals CR PR NT a GalCer NT Fc Enh.& PD-1 a GalCer Fc Enh. & PD-1 0 10 20 30 40 % iNKT of Live CD45 + B220 - Liver **** * **** NT a GalCer NT Fc Enh.& PD-1 a GalCer Fc Enh. & PD-1 0 2 4 6 % iNKT of Live CD45 + B220 - Spleen ns NT a GalCer NT Fc Enh.& PD-1 a GalCer Fc Enh. & PD-1 0 2 4 6 8 % iNKT of Live CD45 + B220 - Lungs *** *** **** 0 100 200 300 Nymber of metastases Control Control + Fc enhanced CTLA-4 + PD-1 aGalCer aGalCer + PD-1 aGalCer + Fc enhanced CTLA-4 aGalCer + Fc enhanced CTLA-4 + PD-1 In vivo i.v. 500K B16F1::Ova D2 aGAL-Cer ( 1 Dose i.p. 0.2 mg/kg) D3 CTLA4 (3 Weekly Doses i.p. 2.5 mg/kg) D3 PD1 (6 Bi-Weekly Doses i.p. 10 mg/kg) Figure 8: C57BL6 mice, were injected i.v. with B16F1::OVA cells. Challenged mice were treated i.p. with the iNKT activator lipid aGalCer or vehicle control. Mice were subsequently treated i.p with anti-PD-1 and AGEN1181 anti-CTLA-4 Fc Enhanced “surrogate”. or the corresponding isotype control . A: Nodule counting to evaluate Pulmonary Disease burden per group (N=8) B: Phenotyping of the lung, spleen and liver infiltrating leukocytes by flow cytometry focusing on the iNKT infiltration. Isotype AGEN1181 anti-TIGIT AGEN1181 anti-TIGIT 0 5 10 15 20 25 IL-2 Secretion (fold change) In vitro PBMC SEA Assay AGEN1181 and anti-PD-1 potentiate T cell signaling -3 -2 -1 0 1 2 0 2000 4000 6000 8000 Antibody μg/ml [log] IL-2 (pg/ml) Isotype control AGEN1884 (parental) AGEN1181 (Fc enh.) Isotype control AGEN1884 (parental) AGEN1181 (Fc enh.) + Anti-PD-1 AGEN1181 and anti-TIGIT potentiate T cell signaling In-vitro Cell Binding Assay AGEN1884 : IgG1 AGEN 1181: Fc enhanced IgG1 In-vitro SEA Assay AGEN1884 : IgG1 AGEN 1181: Fc enhanced IgG1 Figure 10: A: Binding profiles of the human Fc engineered antibody AGEN1181 Vs the parental AGEN1884 and Fc silent variants of the anti-CTLA4 blocking antibody to cells stably expressing hFcγRIIIA V/V haplotype, hFcγRIIIA F/F haplotype. Binding was assessed by flow cytometry, mean fluorescence intensity normalized according to standard methods and the ratio of the increase binding Vs parental was calculated. B: Evaluation of IL-2 production by human PBMCs donors that are homozygous for the high affinity hFcγRIIIA V/V haplotype, or the low affinity hFcγRIIIA F/F haplotype and stimulated with staphylococcal enterotoxin A (SEA) peptide together with increasing concentrations of anti-CTLA-4 hIgG1: AGEN1884, antiCTLA-4 Fc-enhanced hIgG1: AGEN1181 or an hIgG1 Fc-enhanced (Fc E) isotype control antibody. Polymorphism in Fcg receptor was determined by PCR followed by Sanger sequencing. AGEN1181 ms reshapes the tumor immune microenvironment and induces a durable memory response On-Going Trial Combination of AGEN1181 with Agenus's balstilimab (anti-PD-1) is advancing in the clinic (NCT03860272). A B C D C A B D C A B A B A B C A B E F In vivo s.c. 200K B16F10 D7 (100mm 3 ) D7 10 Gy radiation (1 Dose) D8 CTLA4 (5 Bi Weekly Doses i.p. 10 mg/kg) D8 PD1 (5 Bi Weekly Doses i.p. 10 mg/kg) 0 10 20 30 40 0 1000 2000 3000 4000 Days Post Tumor Challenge Tumor volume (mm 3 ) Isotype Control anti-PD-1 Fc-enhanced CTLA-4 Fc-enhanced CTLA-4 + anti-PD-1 Isotype Control anti-PD-1 Fc-enhanced CTLA-4 Fc-enhanced CTLA-4 + anti-PD-1 + Focal Radiation (10Gy) 0 10 20 30 0 1000 2000 3000 4000 Focal Radiation + Isotype Control Tumor volume (mm 3 ) 0 10 20 30 Focal Radiation + anti-PD1 0 10 20 30 Focal Radiation + Fc-enhanced anti-CTLA-4 0 10 20 30 40 Triple combination therapy -3 -2 -1 0 1 2 0 10 20 30 40 50 Antibody [ug/mL] Fold change (binding) -3 -2 -1 0 1 2 0 50 100 150 Antibody [μ g/mL] Fold change (binding) IgG1 variant Isotype control AGEN1181 FcγRIIIA V/V158 FcγRIIIA F/F158 -3 -2 -1 0 1 2 0 5000 10000 15000 Antibody (log μ g/mL) IL-2 pg/mL AGEN1181 IgG1 variant Isotype control -3 -2 -1 0 1 2 0 5000 10000 15000 Antibody (log μg/mL) IL-2 pg/mL FcγRIIIA V/F158 FcγRIIIA F/F158 F - low affinity allele V - high affinity allele A B References: 1 Waight et al. Cancer Cell 2018 2 Danbee et al. PNAS 2018 3 Vargas et al. Cancer Cell 2018 4 Romano et al. PNAS 2015 B A A B For a more detailed presentation: This version includes corrections to an error in the original presentation contained on the legend for Figure 10B.