A First in Human, Dose Escalation and Biomarker Cohort Expansion of IMT-009 (anti CD161) in advanced cancer and Phase 1b combination with Fruquintinib in Microsatellite Stable Colorectal Cancer

A First in Human, Dose Escalation and Biomarker Cohort Expansion of IMT-009 (anti CD161) in advanced cancer and Phase 1b combination with Fruquintinib in Microsatellite Stable Colorectal Cancer Poster CT048 Susanna V. Ulahannan1, Melissa Johnson2, Jason T. Henry3, Shruti Malu4, Sudhir Manda5, Manish R. Patel6, Shivaani Kummar7,Adwitiya Kar4, Alexander Spira8, Vivian Cline9, Ana L. Costa10, Sarah Djeddi4, Diane Stebbins4, Nicholas Ferenc11, Lucy Chen11, James E. Wooldridge4, Cesar A. Perez12 1The University of Oklahoma, Stephenson Cancer Center/SCRI, Oklahoma City, OK, 2Sarah Cannon Research Institute, Nashville, TN, 3SCRI at HCA Health ONE, Denver, CO,4Immunitas Therapeutics, Boston, MA, 5TMC Health Cancer Center, Tucson, AZ,6SCRI at Florida Cancer Specialists & Research Institute, Sarasota, FL, 7Oregon Health and Science University, Portland, OR, 8Next Oncology Virginia, Fairfax, VA, 9Texas Oncology, Austin, TX,10Cogitars GmbH, Heidelberg, Germany, 11Takeda Development Center Americas, Inc., Lexington, MA,12SCRI at Florida Cancer Specialists & Research Institute, Orlando, FL 1.Marrufo AM. et al., Blocking LLT1 (CLEC2D, OCIL)-NKRP1A (CD161) interaction enhances natural killer cell-mediated lysis of triple-negative breast cancer cells. Am J Cancer Res. 2018. 8(6): 1050–1063; 2. Mathewson N. et al., Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis. Cell. 2021. 184:1281–1298. 3. Llibre A et al., LLT1 and CD161 expression in human germinal centers promotes B cell activation and CXCR4 downregulation. J Immunol 2016; 196: 2085-2094. 4.Germain C et al., Lectin-like transcript 1 is a marker of germinal center-derived B-cell non-Hodgkin’s lymphomas dampening natural killer cell functions. Oncoimmunology 2015; 4: e1026503. 5. Fusco A et al; Anti-CD161 antibody IMT-009 is a novel immunotherapeutic agent that reinvigorates T and NK cell function and anti-tumor efficacy through blocking interaction of CD161 with its ligand CLEC2D. SITC 2022 https://static1.squarespace.com/static/6164da62fcac550608373bab/t/636e4f42b3600c04654311b8/1668173640349/Anti-CD161-antibody-IMT-009.pdf. 6. Kar A et al; Abundance of KLRB1+ (CD161) T cells in anti-PD1 non-responders coupled with enhanced tumor cytotoxicity of anti-CD161 (IMT-009) with anti-PD1 makes it a rational target for combination with anti-PD-(L)1 immunotherapy. AACR 2024: https://static1.squarespace.com/static/6164da62fcac550608373bab/t/6614087a763c3b3b91bd3470/1712588951475/2024_AACR_Immunitas_Combination_of_IMT-009_with_anti_PD-1_Poster__final.pdf. 7. Bernstein M et al., Anti CD161 Antibody IMT-009 Is a Novel Immunotherapeutic Agent That Effectively Blocks the Inhibitory CLEC2D/CD161 Axis in CLEC2D+ B Cell Hematological Malignancies Reinvigorating T and NK Cell Function Leading to Anti-Tumor Benefit: ASH conference 2023 https://www.sciencedirect.com/science/article/abs/pii/S000649712309417X 8. Alvarez-Calderon F et al. Targeting of the CD161 Inhibitory Receptor Enhances T cell-mediated Immunity Against Hematological Malignancies. Blood. 2023 :blood.2023022882. 9. Sun Y. et al., Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma. Cell. 2021. 184 (2): 404-421. 10. Lao L. et al., CD8+ T cell-Dependent Remodeling of the Tumor Microenvironment Overcomes Chemoresistance. Cancer Immunol Res. 2023. 11 (3): 320-338. 11. Dasari A et al., Fruquintinib versus placebo in patients with refractory metastatic colorectal cancer (FRESCO-2): an international, multicentre, randomised, double-blind, phase 3 study. The Lancet. 2023. 40:41-53. Phase 1b IMT-009 + Fruquintinib in 2nd-4th line Microsatellite Stable (MSS) Colorectal Cancer (CRC) IMT-009 Phase 1b Translational Biomarker Studies IMT-009 was well tolerated • No DLTs, highest monotherapy treatment-related AE was Grade 2 • Well-tolerated in combination with fruquintinib Optimal dose range likely 240 mg – 1600 mg (3-20 mg/kg) • Receptor occupancy in peripheral blood appears to be 100% between 80 and 240 mg • Anti-tumor activity seen from 240 to 1600 mg Anti-tumor activity was observed as both mono- and combo-therapy • Confirmed MSS CRC partial responders with both mono- and combination therapy Presence and maturation stage of TLS may correlate with improved outcomes • Co-expression of CLEC2D and CD161 within mature TLS by multiplexed IF, suggesting that CD161⁺ T cells may be functionally suppressed via CLEC2D • Treg infiltration in tumor stroma and TLS was observed in some early progressors, providing rationale for exploring combinations that target Tregs • CXCL13 and/or CLEC2D may be TLS-related biomarkers with potential use in patient selection Translational data generated in this trial is hypotheses generating • Blocking the CD161/CLEC2D interaction using IMT-009 may restore or enhance the function of tumor-reactive CD161⁺ T cells within mature TLS, leading to improved anti-tumor T cell responses and clinical benefit. Future studies should incorporate markers to identify mature TLS in pre-treatment tissue • TLS should be further assessed for CD161, CLEC2D, and Tregs as potential selection biomarkers Conclusions Multiplexed immunofluorescence (mIF) analysis reveals co-expression of CLEC2D and CD161 within mature TLS, suggesting that CD161⁺ T cell activity may be suppressed via CLEC2D engagement in this context IMT-009 Patients with PD • Lymphoid aggregates and Immature TLS predominate, while mature TLS are scarce. • Lower CLEC2D expression in TLS suggests diminished inhibition of CD161⁺ T cells. • Increased Treg infiltration in TLS may further contribute to an immunosuppressive microenvironment. Created by Biorender.com Multiplexed immunofluorescence (mIF) analysis of pre-treatment biopsies in the Phase Ib trial suggest presence and maturation stage of tertiary lymphoid structures may correlate with improved outcomes Mature TLS in patients with clinical benefit co-express CLEC2D (within the B cell follicle) and CD161 (in the T cell region) and Regulatory T cells are higher in early and mature TLS among early and Immediate PD Association of spatial localization of Tregs (FoxP3+) within TLS and presence of CLEC2D to clinical outcome • CLEC2D expression is not detectable on B cells within early and mature TLS germinal center of early progressor patient. • CLEC2D expression is very low on all immune cells for patient with immediate PD Treg presence is high within early and mature TLS among early progressors and PD. • High levels of regulatory T cells (Tregs) within TLS may associate with poor prognosis, as Tregs suppress anti-tumor immunity within TLS by inhibiting T cells and DCs Markers associated with TLS such as CXCL13 may enrich for patients likely to benefit from combination of IMT-009 and fruquintinib Potential association between presence of Mature TLS and Clinical benefit with combination of IMT-009 and fruquintinib Immune profile of Partial Response patient in Phase I monotherapy trial supports MOA Partial Responder: 50% TILs CD161 Score = 2 40x Infiltrated Non-Responder: 10% TILs CD161 Score = 1 40x “Cold” Higher pre-Treatment CD161 IHC score in confirmed Partial Responder PR Non-R PR Non-R NK cells Non-R PR CD4+ T cells CD8+ T cells Patient with confirmed Partial Response (PR) had substantially more intra-tumoral CD161+ T and NK cells CD3+ CD4+ CD161+ cells/ mmsq CD3+ CD8+ CD161+ cells/ mmsq CD3- CD56+ CD161+ cells/ mmsq CD161 is a novel cancer immunotherapy target CD161 is a C-type lectin receptor on NK cells and memory T cells found in cancers and associated with treatment resistance. • CD161 binds to its ligand CLEC2D resulting in T and NK cell inhibition1,2. • CLEC2D is highly expressed on germinal center B cells3 in tonsils and in tertiary lymphoid structures (TLS), B cell lymphomas4, cancer cells1 and other immune cells1. • CD161 is highly expressed in a subset of tumor infiltrating lymphocytes in both solid tumors2,5,6 such as CRC, HNSCC, and NSCLC, and in hematological malignancies7,8. • Increased abundance of CD161+ T cells has been correlated to relapse in HCC tumors9 and chemotherapy resistance in breast cancer10 suggesting CD161 expression to be a potential immune evasion mechanism in patients with high unmet need. • CD161+ T cells are highly enriched in tumor than blood, and CRISPR editing2 or antibody mediated blockade of CD161 restores T cell cytotoxicity2,5,8. IMT-009 is a human, Fc-attenuated, IgG1 mAb that binds CD161 and blocks interaction with CLEC2D • IMT-009 restores T and NK cell function including tumor killing and cytokine production. Specifically, it increased human NK cell degranulation, cytokine production, and cellular killing of CLEC2D+ tumor cell targets by T cells5. • scRNAseq analysis demonstrated that treatment with IMT-009 enhanced key T cell functions including enhanced TCR signaling, T cell cytotoxicity, and interferon signaling, as well enhanced the tissue residency program of both CD4 and CD8 T cells6. • IMT-009 treatment inhibited tumor growth in humanized mouse model of CLEC2D+ tumor cells and, activated T cells from human tumor samples ex vivo, providing evidence of the role of CD161 as an inhibitory checkpoint in tumor infiltrating T cells and supporting development of IMT-009 as a novel cancer immunotherapy7. Phase I Study Design Dose Escalation IMT-009 monotherapy N=22 PD Enrichment IMT-009 monotherapy N=26 Phase 1b in MSS CRC IMT-009 + Fruquintinib N=19 6, 20, 80 mg (n=1 each) 240 mg 800 mg 1600 mg 240 mg 800 mg 1600 mg 240 mg 800 mg 1600 mg CD161+ CD161+ MSS CRC, NSCLC, HNSCC, Esophageal cancer; required to have CD161 cells in tissue biopsy prior to enrollment Multiple tumor types, no biopsy requirement MSS CRC, 2nd – 4th line; required to have CD161 cells in tissue biopsy prior to enrollment Primary Objectives • Safety and tolerability of IMT-009 as monotherapy and in combination with fruquintinib • Select dose(s) for further study Other Study Objectives • IMT-009 PK/PD, RO, Immunogenicity • Antitumor activity • Evaluate CD161 and CLEC2D as patient selection biomarkers in tumor biopsies • Explore biomarkers of activation for CD161+ T and NK cells • Explore relationships between biomarkers and preliminary clinical outcomes Presence of CD161+ T and NK cells correlate with anti- tumor response to IMT-009 in humanized mice Development of CLIA validated CD161 IHC assay for Patient Selection into Pharmacodynamic (PD) Enrichment and Phase 1b cohorts IMT-009 induced tumor regression in mice with higher infiltration of CD161+ T & NK Created by Biorender.com Baseline Characteristics of Enrolled Patients Dose Escalation (N=22) Pharmacodynamic Enrichment (N=26) Phase 1 Monotherapy (N=48) Phase 1B Cohort (N=19) Sex Female 6 (27.3%) 11 (42.3%) 17 (35.4%) 9 (47.4%) Male 16 (72.7%) 15 (57.7%) 31 (64.6%) 10 (52.6%) Age (years) n 22 26 48 19 Mean (SD) 60.3 (13.83) 59.6 (13.54) 59.9 (13.53) 54.4 (11.49) Median 63.0 60.5 61.5 54.0 Range 30-78 28-80 28-80 32-78 Number of Prior Systemic Therapies n 22 26 48 19 Mean (SD) 4.2 ( 1.68) 3.2 ( 1.42) 3.7 ( 1.60) 2.4 ( 0.68) Median 4.0 3.0 3.5 2.0 Range 2- 8 1- 6 1- 8 1- 3 RECIST v1.1 Response and Clinical Benefit Anti-tumor Activity - IMT-009 + Fruquintinib (1- 4+ cells/20X field) (5 – 19+ cells/20X field) (≥ 20+ cells/20X field) 0.0 0.5 1.0 1.5 2.0 2.5 # CD3+CD4+CD161+CXCL13+ cells/mmsq Prolonged SDEarly PDImmediate PD Phase 1 IMT-009 Monotherapy All Monotherapy Patients – Dose Escalation and Pharmacodynamic Enrichment Cohort Expansion Treatment Related, Treatment Emergent Adverse Events SOC/PT Summary Table (Monotherapy) Adverse Event Category [n (%) m] Cohort 1 - 6 mg (N=1) Cohort 2 - 20 mg (N=1) Cohort 3 - 80 mg (N=1) Cohort 4 - 240 mg (N=23) Cohort 5 - 800 mg (N=16) Cohort 6 - 1600 mg (N=6) Total (N=48) Any TEAE 0 ( 0.0%) 0 ( 0.0%) 1 (100.0%) 11 (47.8%) 5 (31.2%) 2 (33.3%) 19 (39.6%) Monotherapy IMT-009-related AEs by Dose Level Phase 1 Monotherapy Enrollment – Cancer Diagnoses Monotherapy IMT-009-related AEs (≥2 patients) RECIST v1.1 Response and Clinical Benefit Anti-tumor Activity - IMT-009 Monotherapy Pharmacokinetics and Receptor Occupancy Mean (+SD) Serum Concentration-Time Profile • The t1/2 was estimated based on single dose (Cycle 1) data only and ranged from 56.2 to 366 hours (2.3 to 15.3 days) over the 6 to 1,600 mg dose range. • Based on the t1/2, steady state is expected to be achieved by 3 or 4 cycles of dosing (i.e., 3−5 times the observed t1/2). • Interim RO analysis results predict an EC50 ranging from 90.7 to 1,946 ng/mL for certain cell populations with moderate variability • RO appears saturated at both 240 and 1600 mg Receptor Occupancy at 240 (purple) and 1,600 mg (green) Treatment-emergent AEs (≥3 patients or ≥2 patients w/Grade 3 or 4) Adverse Event Category [n (%) m] Grade 1 Grade 2 Grade 3* Grade 4* Grade 5 Total (N=19) Any TEAE (maximum grade per subject) 0 (0%) 8 (42.11%) 10 (52.63%) 1 (5.26%)* 0 (0%) 19 (100%) Fatigue 4 (21.05%) 5 (26.32%) 0 (0%) 0 (0%) 0 (0%) 9 (47.37%) Abdominal pain 3 (15.79%) 3 (15.79%) 1 (5.26%) 0 (0%) 0 (0%) 7 (36.84%) Decreased appetite 1 (5.26%) 5 (26.32%) 0 (0%) 0 (0%) 0 (0%) 6 (31.58%) Hypertension 0 (0%) 4 (21.05%) 2 (10.53%) 0 (0%) 0 (0%) 6 (31.58%) Hypokalemia 4 (21.05%) 1 (5.26%) 1 (5.26%) 0 (0%) 0 (0%) 6 (31.58%) Hypothyroidism 2 (10.53%) 4 (21.05%) 0 (0%) 0 (0%) 0 (0%) 6 (31.58%) Nausea 2 (10.53%) 4 (21.05%) 0 (0%) 0 (0%) 0 (0%) 6 (31.58%) Arthralgia 2 (10.53%) 2 (10.53%) 1 (5.26%) 0 (0%) 0 (0%) 5 (26.32%) Dehydration 1 (5.26%) 4 (21.05%) 0 (0%) 0 (0%) 0 (0%) 5 (26.32%) Dysphonia 3 (15.79%) 1 (5.26%) 0 (0%) 0 (0%) 0 (0%) 4 (21.05%) Palmar-plantar erythrodysaesthesia syndrome 4 (21.05%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 4 (21.05%) Alanine aminotransferase increased 2 (10.53%) 1 (5.26%) 0 (0%) 0 (0%) 0 (0%) 3 (15.79%) Aspartate aminotransferase increased 0 (0%) 2 (10.53%) 1 (5.26%) 0 (0%) 0 (0%) 3 (15.79%) Constipation 2 (10.53%) 1 (5.26%) 0 (0%) 0 (0%) 0 (0%) 3 (15.79%) Cough 1 (5.26%) 2 (10.53%) 0 (0%) 0 (0%) 0 (0%) 3 (15.79%) Diarrhoea 1 (5.26%) 2 (10.53%) 0 (0%) 0 (0%) 0 (0%) 3 (15.79%) Dyspnoea 1 (5.26%) 2 (10.53%) 0 (0%) 0 (0%) 0 (0%) 3 (15.79%) Fall 2 (10.53%) 0 (0%) 1 (5.26%) 0 (0%) 0 (0%) 3 (15.79%) Infusion related reaction 0 (0%) 3 (15.79%) 0 (0%) 0 (0%) 0 (0%) 3 (15.79%) Stomatitis 1 (5.26%) 2 (10.53%) 0 (0%) 0 (0%) 0 (0%) 3 (15.79%) Acute kidney injury 0 (0%) 0 (0%) 2 (10.53%) 0 (0%) 0 (0%) 2 (10.53%) * One Grade 3 arthralgia was assessed as related to IMT-009; the remaining Grade 3/4 treatment-emergent AEs were not considered by the investigator to be related to IMT-009 **Blood creatinine increased Adverse Event Category [n (%) m] Grade 1 Grade 2 Grade 3 Grade 4 Grade 5 Total (N=48) Any TEAE (maximum grade per subject) 9 (18.75%) 10 (20.83%) 0 (0%) 0 (0%) 0 (0%) 19 (39.58%) Arthralgia 1 (2.08%) 3 (6.25%) 0 (0%) 0 (0%) 0 (0%) 4 (8.33%) Nausea 3 (6.25%) 1 (2.08%) 0 (0%) 0 (0%) 0 (0%) 4 (8.33%) Fatigue 2 (4.17%) 1 (2.08%) 0 (0%) 0 (0%) 0 (0%) 3 (6.25%) Vomiting 2 (4.17%) 1 (2.08%) 0 (0%) 0 (0%) 0 (0%) 3 (6.25%) Constipation 2 (4.17%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 2 (4.17%) Diarrhoea 2 (4.17%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 2 (4.17%) Pain 1 (2.08%) 1 (2.08%) 0 (0%) 0 (0%) 0 (0%) 2 (4.17%) Pyrexia 2 (4.17%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 2 (4.17%) *Subject with 233% change is shown at 100% in both figures Cohort 4 - 240 mg (N=6) Cohort 5 - 800 mg (N=7) Cohort 6 - 1600 mg (N=6) Total (N=19) Best Overall Response (RECIST) PR 1 (16.7%) 0 (0.0%) 0 (0.0%) 1 (5.3%) SD 2 (33.3%) 6 (85.7%) 0 (0.0%) 8 (42.1%) PD 3 (50.0%) 1 (14.3%) 6 (100.0%) 10 (52.6%) Overall Response Rate (CR + PR) N, (%) 1 (16.7%) 0 (0.0%) 0 (0.0%) 1 (5.3%) CBR at month 3 N, (%) 2 (33.3%) 2 (28.6%) 1 (16.7%) 5 (26.3%) CBR at month 6 N, (%) 2 (33.3%) 1 (14.3%) 0 (0.0%) 3 (15.8%) CBR = clinical benefit rate (CR + PR + SD) Cohort 1 - 6 mg (N=1) Cohort 2 - 20 mg (N=1) Cohort 3 - 80 mg (N=1) Cohort 4 - 240 mg (N=23) Cohort 5 - 800 mg (N=16) Cohort 6 - 1600 mg (N=6) Total (N=48) Best Overall Response (RECIST) PR 0 (0.0%) 0 (0.0%) 0 (0.0%) 1 (4.3%) 0 (0.0%) 0 (0.0%) 1 (2.1%) SD 0 (0.0%) 0 (0.0%) 0 (0.0%) 2 (8.7%) 2 (12.5%) 1 (16.7%) 5 (10.4%) CBR (CR + PR + SD) at month 3 N, (%) 0 (0.0%) 0 (0.0%) 0 (0.0%) 2 (8.7%) 0 (0.0%) 1 (16.7%) 3 (6.2%) CBR = clinical benefit rate (CR + PR + SD)

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