Analysis of dendritic cells in response to mCLN-617

T cell-engaging antibody constructs (TCEs) have emerged as a potent modality to treat cancer and autoimmune diseases. Twelve TCEs have been approved by the FDA and EMA for the treatment of hematological malignancies or solid tumors. Despite the varying designs and binding properties, they all lead to robust single-agent efficacy and approvals in refractory or relapsed leukemia, lymphomas, multiple myeloma (MM), small cell lung cancer, EpCAM-expressing cancers or uveal melanoma. Where comparisons can be deduced, TCEs appear to achieve response rates like those obtained with CAR-T cell therapies. Given the success of the first generation of TCEs, considerable attempts are underway to further improve upon this modality. With the goal of expanding TCEs into other malignant and autoimmune indications, and to further enhance efficacy and improve safety, a multitude of novel TCEs are currently in preclinical and clinical development. Here we review the current approaches to developing next-generation TCEs that can widen the therapeutic index, address heterogeneous target expression, and thereby potentially improve efficacy and safety.

IT versus IV injection of mCLN-617

Background The CDKN2A gene encodes two canonical tumor suppressors, p16INK4A and p14ARF, which safeguard cells from malignant transformation by inducing cell cycle arrest and apoptosis in response to aberrant growth signals. Paradoxically, many cancers overexpress these proteins when downstream effectors that enforce negative feedback regulation are lost or inactivated. For example, p14ARF, which regulates p53 activation, is aberrantly expressed in more than 50% of tumors with inactivating p53 mutations. Here, we evaluated the feasibility of targeting dysregulated p16INK4A and p14ARF expression using TCR-T cell therapeutics.Methods We analyzed a panel of p16INK4A- and p14ARF-derived peptides for HLA-A*02:01-associated presentation and recognition by CD8+ T cells. Antigen-specific T cell receptors were isolated from healthy donor repertoires and expressed in primary T cells to assess specificity, functional avidity, tumor recognition, and safety using in vitro T cell functional assays, in vivo tumor models, and an in vivo safety model.Results We identified a unique and well-presented p14ARF epitope that was consistently detected in the HLA-A*02:01-associated immunopeptidome of cancer biopsies but not in normal tissues. High-avidity ARF-specific TCRs were isolated from the peripheral repertoire of healthy donors, and TCR-transduced T cells mediated potent tumor cell killing in vitro and in vivo in preclinical models. Furthermore, targeting p14ARF-expressing cells did not result in detectable on-target toxicity in an in vivo safety model.Conclusions These findings demonstrate the feasibility of targeting dysregulated tumor suppressor proteins with TCR-T cell therapeutics and identify p14ARF as a promising target for future therapies.

Dose level and schedule evaluation for the treatment of CT26 tumors

Gating scheme for flow cytometry data analysis

T cell engagers (TCEs) are antibody-based, bispecific or multi-specific constructs that can reprogramme T cells to eliminate target cells expressing a defined surface antigen. Originally developed for cancer therapy, TCEs are now being investigated for the treatment of autoimmune diseases, with promising initial results. The interest in using TCEs for autoimmune diseases is rapidly growing given their comparable potency with cellular therapies, combined with the advantages of biologics, including ease of manufacturing, off-the-shelf availability, better safety and more convenient delivery. Here we review the history of TCEs, focus on distinct aspects of the mechanism of action of TCEs and explain design principles. We also discuss key challenges for future TCE development in autoimmunity, including enhanced safety, high convenience and complete target cell elimination. Finally, we provide an overview of the preclinical and clinical development landscape and give an outlook on next-generation TCEs that are optimized to treat a wide variety of autoimmune diseases.

Serum IL2 and IL12 levels measured up to 72 hours after injection with single cytokine fusion or mCLN-617

Minimal effects on Treg and NK cells

BACKGROUND:Major histocompatibility complex class I-related protein A and B (MICA/B) are ligands for the natural killer group 2 member D (NKG2D) receptor and are broadly expressed on tumor cells but minimally on normal tissues. When cytotoxic NKG2D-expressing immune cells engage MICA/B, the ligand-expressing cells are targeted for lysis. Cancer cells can evade NKG2D-mediated destruction by shedding MICA/B from their cell surface via proteases present in the tumor microenvironment. CLN-619 is a humanized IgG1 monoclonal antibody (mAb) which binds MICA/B and inhibits shedding resulting in accumulation of MICA/B on the tumor cell surface. CLN-619 may thereby have therapeutic effects in a broad range of malignancies by re-establishing the MICA/B-NKG2D axis to enable NKG2D-mediated, as well as Fc-gamma receptor-mediated, tumor cell lysis. METHODS:CLN-619 was characterized for binding epitope and affinity, effects on surface and soluble levels of MICA/B, and in vitro tumor cell killing. In mouse models, the mAb was tested for tumor growth inhibition. The contribution of the Fc-gamma (Fcγ) 1 domain to CLN-619 activity was also assessed. RESULTS:CLN-619 bound with high affinity to the alpha-3 domain of MICA/B without encumbering the interaction with NKG2D on natural killer cells. CLN-619 increased the level of cell surface expression of MICA/B and concomitantly decreased the levels of soluble MICA/B in cell culture assays. Treatment of cancer cell lines with CLN-619 induced antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. CLN-619 resulted in potent inhibition of tumor growth in multiple xenograft models and increased survival of mice in a disseminated cancer model. CONCLUSIONS:CLN-619 inhibited the shedding of MICA/B to effectively restore cytotoxic signaling pathways in immune cells. Potent antitumor activity of CLN-619 as a monotherapy was observed in several preclinical models. Activity of CLN-619 required a functional Fcγ1 domain, suggesting the requirement of simultaneous engagement of NKG2D and cluster of differentiation 16A (CD16A) on immune cells for optimal cytotoxicity. The preclinical data reported here support the assessment of CLN-619 in patients with cancer.

Jennifer S. Michaelson, Chief Scientific Officer at Cullinan Oncology, and Patrick A. Baeuerle, scientific advisor to Cullinan Oncology and honorary professor in immunology at Ludwig Maximilians University Munich, discuss the use of CD19-specific T cell–engaging antibody therapies (TCEs) as therapeutics for autoimmune diseases.

As powerful activators of the immune system, cytokines have been extensively explored for treating various cancers. But despite encouraging advances and some drug approvals, the broad adoption of cytokine therapies in the clinic has been limited by low response rates and sometimes severe toxicities. This in part reflects an inefficient biodistribution to tumors or a pleiotropic action on bystander cells and tissues. Here, we first review these issues and then argue for the intratumoral delivery of engineered cytokine fusion proteins that have been optimized for tumor retention as a potential solution to overcome these limitations and realize the potential of cytokines as highly effective therapeutics for cancer.

Abstract CLN-619 is a humanized IgG1 monoclonal antibody that targets MICA and MICB (MICA/B) and is currently in phase 1 clinical development in cancer patients (NCT05117476). MICA/B serve as activating signals on target cells for recognition by the NKG2D receptor, which is expressed on NK cells and a subset of T cell populations. MICA/B expression is induced in response to stressed conditions, thereby enabling NKG2D-mediated elimination of target cells. On NK cells, NKG2D is one of many receptors in the complex network of activating and inhibitory receptors, whereby NKG2D pathway activation results in cytokine production, enhancement of ADCC and target cell death. On CD8 T cells, the NKG2D axis plays a costimulatory role in lowering the threshold for lysis upon TCR engagement, and may also drive direct CD8-mediated killing following prior TCR activation. MICA/B is expressed broadly on a range of solid and hematological malignancies. However, tumor cells evade NKG2D-mediated elimination by shedding MICA/B ligand from the cell surface via proteases present in the tumor microenvironment (TME). CLN-619 functions by binding to MICA/B and preventing shedding, thereby increasing MICA/B cell surface expression to restore the NKG2D-MICA/B axis to promote tumor cell killing by NK cells and T cells. Additionally, CLN-619 can mediate ADCC and ADCP by NK cells and macrophages. In preclinical xenograft models, doses as low as 0.03 mg/kg inhibited tumor outgrowth. We investigated the effects of CLN-619 on a variety of primary immune cells including NK cells, T cells and macrophages. In the evaluation of NK cells, we measured CLN-619 modulation of cytokine production and cytotoxicity in the presence of MICA/B expressing target cells. In addition, we demonstrated the contribution of Fc-mediated functions of CLN-619. In the context of T cells, we explored the requirement of TCR co-stimulation or prior antigen exposure in the activation of the NKG2D pathway by CLN-619. In the context of macrophages, we measured the ability of CLN-619 to mediate ADCP against MICA/B-expressing target cells. These data highlight the potential of CLN-619 to engage multiple immune cell types within the TME, which may lead to greater and broader efficacy compared to IO therapies targeting a single immune cell type. Citation Format: Kerry A. Whalen, Catherine C. Henry, Kavya Rakhra, Kristan Meetze, Jennifer S. Michaelson, Patrick A. Baeuerle. CLN-619, a clinical stage MICA/B-specific hIgG1 monoclonal antibody, engages multiple immune effector cells to promote anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5297.

Abstract Despite clinical evidence of antitumor activity, the development of cytokine therapies has been hampered by a narrow therapeutic window and limited response rates. Two cytokines of high interest for clinical development are interleukin 2 (IL2) and interleukin 12 (IL12), which potently synergize to promote the activation and proliferation of T cells and NK cells. However, the only approved human IL2 therapy, Proleukin, is rarely used in the clinic due to systemic toxicities, and no IL12 product has been approved to date due to severe dose-limiting toxicities. Here, we describe CLN-617, a first-in-class therapeutic for intratumoral (IT) injection that co-delivers IL2 and IL12 on a single molecule in a safe and effective manner. CLN-617 is a single-chain fusion protein comprised of IL2, leukocyte-associated immunoglobulin-like receptor 2 (LAIR2), human serum albumin (HSA), and IL12. LAIR2 and HSA function to retain CLN-617 in the treated tumor by binding collagen and increasing molecular weight, respectively. We found that IT administration of a murine surrogate of CLN-617, mCLN-617, eradicated established treated and untreated tumors in syngeneic models, significantly improved response to anti-PD1 checkpoint therapy, and generated a robust abscopal response dependent on cellular immunity and antigen cross-presentation. CLN-617 is being evaluated in a clinical trial in patients with advanced solid tumors (NCT06035744).

Background IL-2 and IL-12 synergistically mediate anti-tumor immunity but trigger severe adverse events. CLN-617 is comprised of IL-2, leukocyte-associated immunoglobulin-like receptor 2 (LAIR2), human serum albumin (HSA), and IL-12, and is designed for intratumoral (IT) administration and retention via LAIR2-mediated collagen binding. Here, we investigate the mechanism of action of a murine CLN-617 surrogate (mCLN-617) and elucidate the benefits of co-delivery of IL-2 and IL-12 in a single molecule. Methods In vivo studies were conducted with mCLN-617 (IL12-LAIR1-MSA-IL2) in B16F10 and MC38 syngeneic tumor models. Cytokines were measured by Mesoscale Discovery, and cells analyzed by flow cytometry and T cell receptor (TCR) sequencing from blood and tumor samples. Results Following mCLN-617 treatment, complete responses with no significant body weight loss were observed in checkpoint-refractory tumor models, including B16F10, CT26, and MC38. In mice implanted with two MC38 tumors, 100% of mCLN-617-treated tumors and 40% of non-treated tumors were eradicated, demonstrating a robust abscopal effect. When combined with systemically delivered anti-PD1 antibody, 100% of non-treated tumors also showed a complete response. Efficacy of mCLN-617 in both the treated and non-treated tumors was partially dependent on CD4+ and CD8+ T cells, IFNγ, and BATF3+ dendritic cells (DCs). mCLN-617 treatment was associated with >5-fold increase in CD8:Treg ratio and significant expansion of DCs in both tumors, and expansion of tumor-associated T cell clonotypes in peripheral blood. A combination of IL-2 and IL-12 co-administered on separate molecules (LAIR1-MSA-IL2 and IL12-MSA-LAIR1) showed efficacy similar to mCLN-617 in B16F10 tumor-bearing mice with >90% tumor growth inhibition in the injected tumor. However, the combination of individual agents triggered >10% body weight loss while mCLN-617 led to no body weight loss, suggesting IL-2 and IL-12 were delivered more safely on a single molecule. IT injection of MC38 tumors with mCLN-617 triggered a 15-fold reduction in IFNγ production in serum compared to IL12-MSA-LAIR1, demonstrating a safety advantage of mCLN-617 and suggesting IL-12 in mCLN-617 was functionally de-tuned. Robust immune infiltrates in both treated and non-treated tumors were observed upon mCLN-617 treatment, compared with LAIR1-MSA-IL2 or LAIR1-MSA-IL2 alone. Conclusions mCLN-617 drove a robust abscopal effect mediated by antigen presentation and systemic cellular immunity. Co-delivery of IL-2 and IL-12 in a single molecule optimally balanced safety and efficacy. CLN-617 is currently in Phase 1 dose escalation in patients with advanced solid tumors.