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

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 Poster #1375 Adwitiya Kar1, Cynthia Hession1 , Tim Nieuwenhuis2, Michelle Duan3, Matthew A. Huggins1, Elizabeth Scanlon1, Siamak Tabrizi1 , Matthew Bernstein1, Andy Tu4, Michael Ross1, Annalisa D’ Andrea1, Ming ‘Tommy’ Tang1,5 and Shruti Malu1,5 1Immunitas Therapeutics, Waltham, MA; 2Astra Zeneca, Waltham, MA; 3Novartis, Cambridge, MA, 4Immunai, New York, NY , 5Co-corresponding The IMT-009-101 is a Phase 1/2a, First-in-Human (FIH), Open-Label, Dose-Escalation and Dose Expansion Study of the Monoclonal Antibody IMT-009 in Patients with Advanced Solid Tumors or Lymphomas (Clinical trial.gov identifier: NCT05565417). It is currently enrolling in the dose escalation and expansion phase. Contact: [email protected] Figure 3. (A) scRNAseq analysis reveals broad expression of KLRB1 in comparison to PDCD1 in MSS CRC. Data reanalyzed from Zhang L. et al15. (B) scRNAseq in MSS CRC identifies population of KLRB1+ PDCD1+ exhausted CD8 T cells with high cytotoxicity and KLRB1+ CD4 cytotoxic T cells. Data from Pelka K. et al16 re-analyzed by Immunitas. Background Hypothesis for therapeutically combining unique pathways of CD161 and PD1 in Immuno -oncology High KLRB1 expression and KLRB1 + T cell abundance in anti - PD1 refractory MSS CRC and in anti -PD1 non responder patients in multiple solid tumors IMT -009 mediated activation of antigen specif ic CD8 T cells leads to enhanced killing of tumor target cells IMT -009 in combination with anti -PD1 enhances CD8 T cell cytotoxicity in a Raji HLA -A2/PD -L1 lymphoma model system with endogenous CLEC2D and overexpression of PD -L1 Treatment of fresh NSCLC tumor with IMT -009 leads to enhanced activation of tumor inf iltrating lymphocytes Figure 1. Mechanisms of CLEC2D/CD161 mediated inhibition in the TME. CLEC2D ligand on tumor cells/immune cells interacts with CD161 to inhibit T cell and NK cell function. Transcriptomic changes induced in T cells upon IMT -009 treatment include enhanced TCR signaling, Cytotoxicity, NK cell like activity and Interferon signaling Created with BioRender.com Figure 4. Antigen-expanded CD8 T cells were co-cultured with antigen-primed Raji lymphoma cell lines at E:T ratio of 2:1 or 4:1 (E: CD8 T cells; T: Raji cells). Tumor cell death was measured by flow cytometry 48 hours post co-culture. IMT009 treatment reverses CLEC2D/CD161 mediated inhibition and enhanced CD8 T cell mediated cytotoxicity. Figure 6. PBMCs from specific donors were expanded on MHC class I HLA-A2 restricted viral peptides (A) or the covid spike peptide (B) and set up in a cytotoxicity assay with HLA-A2 and PDL1 overexpressing Raji tumor cells expressing endogenous CLEC2D. The co-culture was treated with either Pembrolizumab (anti-PD1) (5mg/ml) or IMT-009 (11mg/ml) or combination. No treatment and combination of isotypes were used as controls. 48 hrs post co-culture live CD19+ Raji tumor cells was determined by flow cytometry providing % measure of cytotoxicity. Both antibodies show increased cytotoxicity over controls with additive effect in the combination treatment. (** p<0.01) Created with BioRender.com Conclusions Figure 7. (A) UMAP visualization of clusters from 10X genomics scRNA-seq analysis of a co-culture of antigen specific T cells with Raji tumor cells with or without treatment of IMT-009. Colors indicate subtypes of CD4, CD8 and Raji tumor cells. Equal number of cells are depicted. (B) IMT009 treatment led to increase in % CD4 and CD8 T cells and a decrease in Raji tumor cell population indicating blocking of CD161 pathway leads to T cell activation and proliferation and enhanced tumor cell killing. Figure 9. (A) Heat map showing functional enrichment in key biological processes after IMT-009 treatment using NanoString’s pan cancer IO 360 panel pathways. These include antigen specific proximal TCR activation and signaling, distal PI3K_Akt signaling and NF-KB activation. Control internal reference genes remain insignificant with IMT009 treatment.(B,C) GSEA enrichment analysis of genes related to key signaling pathways in CD4 T cells and CD8 T cells upon IMT-009 treatment. Log2 Fold changes in gene expression compared to no Ab control in IMT009 treated CD4 T cells Log2 Fold changes in gene expression compared to no Ab control in IMT009 treated CD8 T cells Figure 11. (A) Heat map showing functional enrichment in key biological processes across different treatment conditions using NanoString’s pan cancer IO 360 panel pathways. Significant enrichment is observed with either treatments in pathways involved in TCR activation and signaling. (B) Treatment with combination compared to either drugs alone enhances mean expression of IFNG, TNF, and GZMB in CD8 T cells (p<0.01 between anti PD1 and combination) underscoring enhanced T cell activation in combination with anti-PD1 (pembrolizumab). Created with BioRender.com Figure 5. (A) Surgically resected NSCLC tumor was dissociated into single cells and analyzed for expression of CD161 and PD1 in CD4 and CD8+ T cells. (B) Dissociated tumor cells were then treated with IMT-009 (11mg/ml). T cell activation was determined by measuring cytokines in culture supernatants by MSD 3 days post-treatment. IMT-009 enhanced T cell activating cytokine production. Figure 2. Trends of High KLRB1 expression and KLRB1+ CD4 (A, B) and CD8 T cell (C, D) abundance in anti-PD1 non-responsive patient tumors were observed. KLRB1 expression in tumor infiltrating CD4 and CD8 T cells was re-analysed from the following datasets: Yost K. et al12 (A), Bassez A. et al13 (B,D) and Zhang Y. et al14 (C). scRNAseq analysis of CD8 T cells and Raji tumor cells co - culture reveals increase in % KLRB1 + CD8 and CD4 T cells upon treatment with IMT -009 alone scRNAseq analysis of T cells upon treatment with IMT -009 in combination with anti -PD1 shows fur ther enrichment in T cell activation and cytotoxicity gene signatures Figure 8. (A) UMAP visualization of KLRB1+ clusters of T cells between no treatment and IMT009 treatment shows increased KLRB1+ T cells. Equal number of cells are depicted. (B) Quantification of percent increase in KLRB1+ CD4 T cells and KLRB1+ CD8 T cells after IMT-009 treatment. Figure 10. Respective volcano plots show key genes that are most upregulated (red) with IMT-009 treatment. Tissue resident memory (Trm) markers such as ITGA1, CXCR6; NK cell markers such as KLRK1, KLRC1, KLRD1, GNLY and cytotoxic genes such as GZMB and GZMA are among the most statistically significant differentially expressed genes in CD8 T cells supporting enhanced cytotoxicity in presence of IMT-009. Functional activity of IMT-009 • IMT-009 is a fully human anti-CD161 antibody that restores T and NK cell function including tumor killing and cytokine production • In this study, we show that IMT-009 can activate T cells in human tumor samples providing evidence of CD161’s role as an inhibitory checkpoint in tumor infiltrating T cells. • scRNAseq analysis shows that treatment with IMT-009 enhanced key T cell functions including enhanced TCR signaling, T cell cytotoxicity and interferon signaling. Furthermore, there is an increase in tissue residency program of both CD4 and CD8 T cells. Rationale for combination of IMT-009 with anti-PD1 • CD161 and PD1 are unique pathways of immunomodulation in cancer. • scRNAseq analysis of tumors from anti-PD1 responders and non-responders reveals higher abundance of KLRB1+ T cells (gene encoding for CD161) in non-responders. • Anti-PD1 non-responding tumor types such as MSS CRC show broader expression of KLRB1 than PDCD1. KLRB1+ cells show high expression of a cytotoxicity module and low expression of an exhaustion module as compared to PDCD1+ cells • We further show that combination of anti-CD161 (IMT-009) and anti-PD1 can enhance T cell mediated tumor cell killing in vitro. Table 1. Differentiating characteristics of the CD161 and PD1 pathways A. B. C. CD4 T CD8 T A. B. log2FC log2FC CD161 (KLRB1), a C type lectin related transmembrane protein, and its ligand CLEC2D make a key immunomodulatory pathway in both T and NK cells that suppresses their activation and cytotoxic function1,2. Immunitas has developed a fully human anti CD161 monoclonal antibody called IMT-009 that can restore both NK mediated cytotoxicity and polyfunctionality of CD4 and CD8 T cells. We have also shown monotherapy activity of IMT-009 in an in vivo humanized mouse model of B cell lymphoma that is unresponsive to anti-PD-(L)1. Furthermore, we have shown that IMT-009 enhances NK mediated antibody dependent cellular cytotoxicity (ADCC) in a Raji Lymphoma model that endogenously expresses CLEC2D. CD161 is highly expressed in a subset of tumor infiltrating lymphocytes in both solid tumors such as CRC, HNSCC, and NSCLC and in hematological malignancies. Increased abundance of CD161+ T cells has been correlated to relapse in HCC tumors3 and chemotherapy resistance in breast cancer4 suggesting CD161 expression to be a potential immune evasion mechanism in patients with high unmet need. IMT-009 is currently being tested in a Phase 1/2a monotherapy clinical trial. The data described here provides support for potential efficacy of IMT-009 in combination with anti-PD-(L)1 therapy in patients who have previously progressed on, or are refractory to, anti-PD-(L)1 therapy: ▪ scRNA seq analysis using publicly available data for anti-PD(L)1 treated patients shows higher abundance of KLRB1+ T cells in non responder patients compared to responders ▪ KLRB1 is expressed more broadly in anti PD1 non-responsive tumor types such as MSS CRC than PDCD1 ▪ Combination of IMT-009 with anti-PD1 enhances antigen specific T cell mediated cytotoxicity in vitro ▪ Transcriptomic changes upon treatment with IMT-009 and combination of anti-PD1 show further increase in T cell activation and cytotoxicity. p<0.01 CD161 PD1 Expression profile • Expressed on NK cells, T cells, Th17, Innate like T cells including Tc1, MAIT and γδ T cells5 • In cancer, CD161 is expressed by clonally expanding, cytotoxic memory CD8 T cells that have tissue resident phenotype2,6 • CD161+ CD4 T cells are associated with enhanced secretion of IL-17a, IL-22, IFN-g. • Minimal expression by FoxP3+ Tregs5 • PD-1 is upregulated on almost all T cells post TCR activation and inhibits effector function of T cells • PD1 expression is correlated to T cell exhaustion • Upregulated transiently on NK cells Ligand • CLEC2D is the only known ligand for CD1615 • It is expressed on germinal center B cells, activated T cells, NK cells, macrophages and dendritic cells5 • B cell lymphomas express high levels of CLEC2D7 • Solid tumors express variable levels of CLEC2D • PD-L1 and PD-L2 are the two known ligands8 • PD-L1 is constitutively expressed in a wide range of cells, including hematopoietic and non- hematopoietic cells such as tumor • PD-L2 expression is restricted to professional antigen-presenting cells • Expression of both PD-L1 and PD-L2 is regulated by Interferons Signaling • CD161 interacts and activates acid sphingomyelinase (ASM) that produces the second messenger molecule ceramide9. This regulates calcium influx and mTOR/STAT310 signaling affecting NK and T cell function • Cytoplasmic domain has ITIM and ITSM motifs11 • Upon interaction with PD-L1, recruitment of SHP phosphatases to the phosphorylated tyrosine residues to these motifs leads to inhibition of signaling downstream of CD28 and TCR affecting T cell activation Targeting relevance in cancer • Enhanced cytotoxicity, tissue homing, memory phenotype, broad expression in TME and dual inhibition of both T and NK cells make CD161/CLEC2D as an important immunomodulatory pathway in cancer • Anti-CD161 antibody IMT009 is in Phase I clinical trail for hematological and solid tumor malignancies ( NCT05565457). • Anti PD(L)1 have been FDA approved for treatment of a variety of solid tumors. • Certain tumors such as MSS colorectal cancer do not respond to anti PD1 therapy and have high unmet need • Acquired resistance to anti-PD1 treatment is also prevalent and supports rationale for targeting the immune evasion mechanisms 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. Sun Y. et al., Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma. Cell. 2021. 184 (2): 404-421. 4. Lao L. et al., CD8+ T cell-Dependent Remodeling of the Tumor Microenvironment Overcomes Chemoresistance. Cancer Immunol Res. 2023. 11 (3): 320-338; 5. Braud VM et al. LLT1-CD161 Interaction in Cancer: Promises and Challenges. Front Immunol. 2022. 13; 13:847576; 6. Alvarez-Calderon F et al. Targeting of the CD161 Inhibitory Receptor Enhances T cell-mediated Immunity Against Hematological Malignancies. Blood. 2023 :blood.2023022882; 7. 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(8): e1026503; 8. Keir ME et al. PD-1 and its ligands in tolerance and immunity. Annu. Rev. Immunol. 2008. 26, 677–704; 9. Pozo D. et al. CD161 (human NKR-P1A) signaling in NK cells involves the activation of acid sphingomyelinase. J Immunol. 2006. 176(4):2397-406; 10. Bai A et al. CD39 and CD161 modulate Th17 responses in Crohn's disease. J Immunol 2014. 193; 3366-3377; 11. Riley JL. PD-1 signaling in primary T cells. Immunol Rev 2009. 229(1): 114-125; 12. Yost K. et al. Clonal replacement of tumor-specific T cells following PD-1 blockade. Nat Med. 2019. 25(8): 1251–1259; 13. Bassez A. et al. A single-cell map of intratumoral changes during anti-PD1 treatment of patients with breast cancer. Nature. 2021. 27, 820–832; 14. Zhang Y. et al. Single-cell analyses reveal key immune cell subsets associated with response to PD-L1 blockade in triple-negative breast cancer. Cancer Cell. 2021. 39(12):1578-1593.e8; 15. Zhang L. et al. Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer. 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