Abstract Background: CLL-1 is a compelling therapeutic target for AML as it is highly expressed on AML tumor cells and leukemic stem cells but is not expressed on hematopoietic stem cells. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology and leverages both checkpoint disruption and immune cloaking armoring strategies to potentially improve antitumor activity. The CB-012 anti-CLL-1 CAR was developed with a fully human scFv and the CD28 costimulatory domain and is currently in development for the treatment of relapsed or refractory AML (r/r AML). Here we describe preclinical studies that supported the CB-012 IND clearance by the FDA in October 2023. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012 through prevention of GvHD, PD-1 checkpoint disruption, and suppression of allograft rejection. In vitro and in vivo studies evaluated the specificity of antigen binding, antigen-dependent activity, and toxicologic potential. Results: CB-012 demonstrated potent antigen-dependent expansion and cytotoxic activity against CLL-1+ human AML cell lines and patient-derived cells in co-cultures. In AML xenograft models, a single dose of CB-012 CAR-T cells resulted in robust tumor control, leading to significant prolongation of survival. CB-012 co-culture with multiple CLL-1-negative cell types representing vital tissues demonstrated that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated highly specific interaction with human CLL-1, with no detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised murine models. Conclusion: CB-012, the first allogeneic anti-CLL-1 CAR-T cell therapy using both checkpoint disruption and immune cloaking armoring, demonstrated specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was demonstrated in an unbiased protein-binding study and no adverse safety signals were observed from CB-012 in murine toxicology models. These preclinical studies supported the IND clearance of CB-012, which is being evaluated in the AMpLify trial, a Phase 1, first-in-human clinical trial for patients with r/r AML (NCT06128044). Citation Format: Brian Francica, Elizabeth Garner, Sai Namburi, Cian Colgan, Tristan Fowler, Devin Mutha, Art Aviles, Morena Stanaway, Raymond Guo, Zili An, Erin Kelly, Emilie Degagne, George Kwong, Leslie Edwards, Emma Jakes, McKay Shaw, Benjamin Schilling, Jeremy Huynh, Ricky Luu, Max Sidorov, Rhonda Mousali, Mikk Otsmaa, Peter Lauer, Justin Skoble, Steven Kanner. Preclinical evaluation of CB-012, an allogeneic anti-CLL-1 CAR-T cell therapy, that exhibits specific and potent toxicity in acute myeloid leukemia (AML) xenograft models [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 6323.

Background: CLL-1 is a compelling therapeutic target for acute myeloid leukemia (AML) as it is highly expressed on AML tumor cells and leukemic stem cells, but is not expressed on hematopoietic stem cells. An allogeneic anti-CLL-1 CAR-T cell therapy (CB-012) is in development for relapsed or refractory (r/r) AML. The CAR was generated with a fully human scFv targeting CLL-1 that was selected from a panel of scFvs. CB-012 was engineered with a next-generation Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology to leverage both checkpoint disruption and immune cloaking for potentially improved antitumor activity. Methods: Cas12a chRDNA guides were implemented to generate five genome edits in the manufacture of CB-012. A fully human anti-CLL-1 CAR transgene was site-specifically inserted into the TRAC gene, thereby eliminating TCR expression to reduce graft-versus-host disease. A B2M-HLA-E fusion transgene was inserted into the native B2M gene, preventing expression of all HLA class I antigens except HLA-E, to blunt both T and NK cell-mediated allograft rejection of the CAR-T cells. A knockout of the PDCD1 gene prevented PD-1 receptor expression and thus PD-L1 ligand binding to prolong antitumor activity. This multiplex genome-editing strategy was designed to enhance the antitumor activity of CB-012. In vitro and in vivo studies evaluated specificity for antigen binding, antigen-dependent activity, and preclinical safety assessments. Results: CB-012 CAR-T cells express a fully human anti-CLL-1 scFv-containing CAR construct and demonstrate potent antigen-dependent cytotoxic activity in human AML cell line co-cultures. In AML xenograft models, a single-dose administration of CB-012 CAR-T cells exerted robust tumor control, leading to significant prolongation of survival. Cell binding studies suggested that the anti-CLL-1 scFv does not exhibit tissue cross-reactivity when examined in the context of a collection of cell types representing vital tissues. In an unbiased cell surface protein microarray, the anti-CLL-1 scFv demonstrated specific interaction with human CLL-1, without detectable non-specific interactions. CB-012 CAR-T cells exhibited limited tissue infiltration and expansion in treatment naïve, immunocompromised mouse models. Conclusion: CB-012 demonstrated highly specific and potent CLL-1-targeted cytolytic activity in vitro and in vivo. Specificity of the anti-CLL-1 scFv was further demonstrated by screening in an unbiased protein-binding study and no adverse safety signals were observed in murine models. These data support advancing the development of CB-012 into a first-in-human clinical trial for patients with r/r AML.

Tumor Morphology and Volume Upon Death. A) Morphology of tumors calculated using ex vivo MRI as described. B) Calculated brain tumor volumes at the time of death as determined by ex vivo MRI (N = 3 mice/group).

TGF-β blockade does not add to RT + vaccination. TGF-β blockade with 1D11 (A) or LY2157299 (B) does not extend survival when combined with RT + rLM-OVA in mice with B16-OVA brain tumors. (N= 10 animals / group).

Brain and flank tumors are of equivalent mass. Brain and flank tumor mass was not different on day 17 when tissues were harvested for analysis. Experiment conducted x 2 with {greater than or equal to} 5 mice/group.

B16-OVA brain tumors stimulate secretion of TGF-β1 from microglia. Cytokine concentrations in the supernatants from OT-1 cells co-cultured with (A) CD11c+ APCs isolated from the spleen at a ratio of 1:5, (B) APC from brain tumor draining lymph nodes at a ratio of 1:1, (C) and CD11b+/CD45-mid microglia isolated from the brain at a ratio of 1:5.

Stimulator of interferon genes (STING) signaling has been extensively studied in inflammatory diseases and cancer, while its role in T cell responses to infection is unclear. Using Listeria monocytogenes strains engineered to induce different levels of c-di-AMP, we found that high STING signals impaired T cell memory upon infection via increased Bim levels and apoptosis. Unexpectedly, reduction of TCR signal strength or T cell-STING expression decreased Bim expression, T cell apoptosis, and recovered T cell memory. We found that TCR signal intensity coupled STING signal strength to the unfolded protein response (UPR) and T cell survival. Under strong STING signaling, Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibition also reduced apoptosis and led to a recovery of T cell memory in STING sufficient CD8 T cells. Thus, STING signaling regulates CD8 T cell memory fitness through both cell-intrinsic and extrinsic mechanisms. These studies provide insight into how IDO and STING therapies could improve long-term T cell protective immunity.

: PD-1 blockade does not add to RT + Vaccination. Adding PD-1 blockade to RT + rLM-OVA does not significantly improve survival in mice with B16-OVA brain tumors. (N = 10 animals / group)

Brain tumors are more tolerogenic than flank or lung tumors. (A) Representative FACS plots from tumor draining lymph nodes of mice with B16-OVA brain, flank, or lung tumors. (B) Summary graphs of the percentage of daughter cells producing IFN-γ recovered from the tumor draining lymph nodes of mice with B16-OVA brain, flank, or lung tumors (5 mice/group).

The safety and immunogenicity of JNJ-64041809 (JNJ-809), a live-attenuated, double-deleted Listeria monocytogenes (LADD Lm)-based immunotherapy targeting 4 relevant prostate cancer antigens, was evaluated in a phase 1 study in patients with metastatic castration-resistant prostate cancer (mCRPC). Men with progressive mCRPC who had received ≥2 prior approved therapies were enrolled. Primary study objectives were to determine the recommended phase 2 dose (RP2D) and to evaluate the safety and immunogenicity of JNJ-809. A total of 26 patients received JNJ-809 (1 × 108 CFU (n = 6); 1 × 109 CFU (n = 20)). No dose-limiting toxicities were reported, and 1 × 109 CFU was selected as the RP2D. The most common adverse events (AEs) reported were chills (92%), pyrexia (81%), and fatigue (62%). The most frequent grade ≥3 AEs were lymphopenia (27%) and hypertension (23%). Serious AEs were reported in 27% of patients including 1 patient with grade 3 intestinal obstruction. JNJ-809 transiently induced peripheral cytokines, including interferon-γ, interleukin-10, and tumor necrosis factor-α. Of the 7 patients evaluable for T cell responses at the 1 × 109 CFU dose, evidence of post-treatment antigenic responses were observed in 6 to the Listeria antigen listeriolysin O and in 5 to ≥1 of the 4 encoded tumor antigens. Best overall response was stable disease in 13/25 response-evaluable patients. The study was terminated early as data collected were considered sufficient to evaluate safety and immunogenicity. JNJ-809 has manageable safety consistent with other LADD Lm-based therapies. Limited antigen-specific immune responses were observed, which did not translate into objective clinical responses.

CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained, and the molecular programmes defining the autoimmune T cell state, are unknown. In type 1 diabetes, β-cell-specific CD8 T cells destroy insulin-producing β-cells. Here we followed the fate of β-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN), which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas, where they differentiate further and destroy β-cells. Whereas transplantation of as few as 20 autoimmune progenitors induced type 1 diabetes, as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived, and stem-like autoimmune progenitors must continuously seed the pancreas to sustain β-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes.

BACKGROUND:Immune checkpoint inhibitors are not effective for pancreatic ductal adenocarcinoma (PDAC) as single agents. Vaccine therapy may sensitize PDACs to checkpoint inhibitor treatments. Annexin A2 (ANXA2) is a pro-metastasis protein, previously identified as a relevant PDAC antigen that is expressed by a GM-CSF-secreting allogenic whole pancreatic tumor cell vaccine (GVAX) to induce an anti-ANXA2 antibody response in patients with PDAC. We hypothesized that an ANXA2-targeting vaccine approach not only provokes an immune response but also mounts anti-tumor effects.METHODS:We developed a Listeria-based, ANXA2-targeting cancer immunotherapy (Lm-ANXA2) and investigated its effectiveness within two murine models of PDAC.RESULTS:We show that Lm-ANXA2 prolonged the survival in a transplant model of mouse PDACs. More importantly, priming with the Lm-ANXA2 treatment prior to administration of anti-PD-1 antibodies increased cure rates in the implanted PDAC model and resulted in objective tumor responses and prolonged survival in the genetically engineered spontaneous PDAC model. In tumors treated with Lm-ANXA2 followed by anti-PD-1 antibody, the T cells specific to ANXA2 had significantly increased INFγ expression.CONCLUSIONS:For the first time, a listeria vaccine-based immunotherapy was shown to be able to induce a tumor antigen-specific T cell response within the tumor microenvironment of a "cold" tumor such as PDAC and sensitize the tumor to checkpoint inhibitor therapy. Moreover, this combination immunotherapy led to objective tumor responses and survival benefit in the mice with spontaneously developed PDAC tumors. Therefore, our study supports developing Lm-ANXA2 as a therapeutic agent in combination with anti-PD-1 antibody for PDAC treatment.

Tumor-specific CD8 T-cells in cancers enter a state of dysfunction characterized by the expression of inhibitory receptors and failure to produce effector cytokines and cytotoxic molecules. Here we identify the nuclear factor, Thymocyte selection-associated HMG box protein, TOX, as a master regulator of tumor-specific T-cell dysfunction. TOX is uniquely expressed in dysfunctional CD8 T-cells from mouse and human tumors but absent in functional T-cells. TOX expression is driven by continuous TCR stimulation and NFAT activity. Forced expression of TOX in functional effector T-cells was sufficient to induce a transcriptional program of dysfunction through the concerted expression of genes encoding numerous inhibitory receptors and dysfunction-associated transcription factors. Notably, TOX-deficient tumor-infiltrating T-cells did not upregulate inhibitory receptors such as PD1, LAG3, CD38, or CD39 and maintained high TCF1 expression. Surprisingly, despite their normal, “non-exhausted” phenotype, TOX-deficient T-cells failed to make effector cytokines, suggesting that loss of effector function in tumor-specific T-cells is uncoupled from inhibitory receptor expression. Furthermore, TOX-deficient T-cells failed to persist in tumors, ultimately undergoing activation-induced cell death. We propose that the TOX-induced transcriptional program of hyporesponsiveness is a physiologic negative feedback mechanism that prevents overstimulation; thus TOX is absolutely required for T-cell survival in the setting of chronic antigen stimulation as in cancers. Citation Format: Andrew C. Scott, Steven Camara, Peter Lauer, Alexandra Synder, Dmitriy Zamarin, Tyler Walther, Olivier Levy, Michael Glickman, Jonathan Kaye, Mary Philip, Andrea Schietinger. Thymocyte selection-associated HMG box protein TOX is a master regulator of tumor-specific T-cell dysfunction [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A215.

Dysregulated signaling via the epidermal growth factor receptor (EGFR)-family is believed to contribute to the progression of a diverse array of cancers. The most common variant of EGFR is EGFRvIII, which results from a consistent and tumor-specific in-frame deletion of exons 2-7 of the EGFR gene. This deletion generates a novel glycine at the junction and leads to constitutive ligand-independent activity. This junction forms a novel shared tumor neo-antigen with demonstrated immunogenicity in both mice and humans. A 21-amino acid peptide spanning the junctional region was selected, and then one or five copies of this 21-AA neo-peptide were incorporated into live-attenuated Listeria monocytogenes-based vaccine vector. These vaccine candidates demonstrated efficient secretion of the recombinant protein and potent induction of EGFRvIII-specific CD8+ T cells, which prevented growth of an EGFRvIII-expressing squamous cell carcinoma. These data demonstrate the potency of a novel cancer-specific vaccine candidate that can elicit EGFRvIII-specific cellular immunity, for the purpose of targeting EGFRvIII positive cancers that are resistant to conventional therapies.

Tumour-specific CD8 T cell dysfunction is a differentiation state that is distinct from the functional effector or memory T cell states1-6. Here we identify the nuclear factor TOX as a crucial regulator of the differentiation of tumour-specific T (TST) cells. We show that TOX is highly expressed in dysfunctional TST cells from tumours and in exhausted T cells during chronic viral infection. Expression of TOX is driven by chronic T cell receptor stimulation and NFAT activation. Ectopic expression of TOX in effector T cells in vitro induced a transcriptional program associated with T cell exhaustion. Conversely, deletion of Tox in TST cells in tumours abrogated the exhaustion program: Tox-deleted TST cells did not upregulate genes for inhibitory receptors (such as Pdcd1, Entpd1, Havcr2, Cd244 and Tigit), the chromatin of which remained largely inaccessible, and retained high expression of transcription factors such as TCF-1. Despite their normal, 'non-exhausted' immunophenotype, Tox-deleted TST cells remained dysfunctional, which suggests that the regulation of expression of inhibitory receptors is uncoupled from the loss of effector function. Notably, although Tox-deleted CD8 T cells differentiated normally to effector and memory states in response to acute infection, Tox-deleted TST cells failed to persist in tumours. We hypothesize that the TOX-induced exhaustion program serves to prevent the overstimulation of T cells and activation-induced cell death in settings of chronic antigen stimulation such as cancer.

Live-attenuatedListeria monocytogeneshas shown encouraging potential as an immunotherapy platform in preclinical and clinical settings. However, additional safety measures will enable application across malignant and infectious diseases. Here, we describe a new vaccine platform, termed Lm-RIID (L. monocytogenesrecombinase-induced intracellular death), that induces the deletion of genes required for bacterial viability yet maintains potent T cell responses to encoded antigens.

Agents that remodel the tumor microenvironment (TME), prime functional tumor-specific T cells, and block inhibitory signaling pathways are essential components of effective immunotherapy. We are evaluating live-attenuated, double-deleted Listeria monocytogenes expressing tumor antigens (LADD-Ag) in the clinic. Here we show in numerous mouse models that while treatment with nonrecombinant LADD induced some changes in the TME, no antitumor efficacy was observed, even when combined with immune checkpoint blockade. In contrast, LADD-Ag promoted tumor rejection by priming tumor-specific KLRG1+PD1loCD62L- CD8+ T cells. These IFNγ-producing effector CD8+ T cells infiltrated the tumor and converted the tumor from an immunosuppressive to an inflamed microenvironment that was characterized by a decrease in regulatory T cells (Treg) levels, a proinflammatory cytokine milieu, and the shift of M2 macrophages to an inducible nitric oxide synthase (iNOS)+CD206- M1 phenotype. Remarkably, these LADD-Ag-induced tumor-specific T cells persisted for more than 2 months after primary tumor challenge and rapidly controlled secondary tumor challenge. Our results indicate that the striking antitumor efficacy observed in mice with LADD-based immunotherapy stems from TME remodeling which is a direct consequence of eliciting potent, systemic tumor-specific CD8+ T cells.

Background. Sipuleucel T, an autologous cell-based vaccine targeting prostatic acid phosphatase (PAP), has demonstrated efficacy for the treatment of advanced prostate cancer. DNA vaccines encoding PAP and live attenuated Listeria vaccines have entered clinical trials for patients with prostate cancer, and have advantages in terms of eliciting predominantly Th1-biased immunity. In this study, we investigated whether the immunogenicity and anti-tumor efficacy of a DNA and Listeria vaccine, each encoding PAP, could be enhanced by using them in a heterologous prime/boost approach. Methods. Transgenic mice expressing HLA-A2.01 and HLA-DRB1*0101 were immunized alone or with a heterologous prime/boost strategy. Splenocytes were evaluated for MHC class I and II-restricted, PAP-specific immune responses by IFNγ ELISPOTs. Anti-tumor activity to a syngeneic, PAP-expressing tumor line was evaluated. Results. PAP-specific cellular immunity and anti-tumor activity were elicited in mice after immunization with DNA- or listeria-based vaccines. Greater CD4+ and CD8+ responses, and anti-tumor responses, were elicited when mice were immunized first with DNA and boosted with Listeria, but not when administered in the opposite order. This was found to be dependent on CD4+ T cells elicited with DNA priming, and was not due to inflammatory signals by Listeria itself or due to B cells serving as antigen-presenting cells for DNA during priming. Conclusions. Heterologous prime/boost vaccination using DNA priming with Listeria boosting may provide better anti-tumor immunity, similar to many reports evaluating DNA priming with vaccines targeting foreign microbial antigens. These findings have implications for the design of future clinical trials.

ABSTRACT Listeria monocytogenes is a Gram-positive intracellular pathogen that causes a severe invasive disease. Upon infecting a host cell, L. monocytogenes upregulates the transcription of numerous factors necessary for productive infection. VirR is the response regulator component of a two-component regulatory system in L. monocytogenes . In this report, we have identified the putative ABC transporter encoded by genes lmo1746-lmo1747 as necessary for VirR function. We have designated lmo1746-lmo1747 virAB . We constructed an in-frame deletion of virAB and determined that the Δ virAB mutant exhibited reduced transcription of VirR-regulated genes. The Δ virAB mutant also showed defects in in vitro plaque formation and in vivo virulence that were similar to those of a Δ virR deletion mutant. Since VirR is important for innate resistance to antimicrobial agents, we determined the MICs of nisin and bacitracin for Δ virAB bacteria. We found that VirAB expression was necessary for nisin resistance but was dispensable for resistance to bacitracin. This result suggested a VirAB-independent mechanism of VirR regulation in response to bacitracin. Lastly, we found that the Δ virR and Δ virAB mutants had no deficiency in growth in broth culture, intracellular replication, or production of the ActA surface protein, which facilitates actin-based motility and cell-to-cell spread. However, the Δ virR and Δ virAB mutants produced shorter actin tails during intracellular infection, which suggested that these mutants have a reduced ability to move and spread via actin-based motility. These findings have demonstrated that L. monocytogenes VirAB functions in a pathway with VirR to regulate the expression of genes necessary for virulence and resistance to antimicrobial agents.

Development of effective and durable antitumor immunity requires the activation, expansion and maintenance of function of tumor antigen-specific effector T cells. The mechanism of action of recently FDA approved monoclonal antibody (mAb) therapies which target immune checkpoints such as PD-1 and CTLA-4 and uncouple inhibitory pathways from the activation of antigen-specific T cells underlines the critical requirement for antigen-specific priming to elicit potent and long-lasting anti-tumor immunity. However, immune checkpoint blockade results in significant clinical benefit largely in malignancies with high mutational burden, where the tumor itself can initiate T cell priming, but is less effective when used as a single agent among cancers with lower mutational burden, associated typically with a low level of lymphocyte infiltration into the tumor microenvironment (TME). We are evaluating clinical immunotherapy regimens combining immune checkpoint blockade with recombinant live-attenuated double-deleted strains of the intracellular bacterium Listeria monocytogenes (LADD), based on a hypothesis that effective immunotherapy will result from agents that in combination re-polarize the TME to facilitate immune effector cell function, prime functional tumor-specific T cells in the appropriate context, and block inhibitory signaling pathways. Here we show that tumor antigen-expressing LADD therapy enhanced CD8+ T cell effector function, resulting in significant tumor eradication in several preclinical mouse models. LADD treatment in addition induced favorable changes in the TME, as shown by enhanced CD8+ T effector function, recruitment of antigen presenting cells and reduction of regulatory T cells. Treatment regimens combining LADD-based immunotherapy with PD-1 immune checkpoint blockade significantly enhanced antitumor efficacy in CT26, 4T1 and MC38 tumor models. Together these data support the rationale for integrating LADD-based immunotherapy into clinical regimens with immune checkpoint blockade on the basis that TME modification and priming of tumor Ag-specific T cells significantly enhances the activity of mAb therapies blocking T cell inhibitory pathways. Citation Format: Weiwen Deng, Weiqun Liu, Thomas Hudson, Chris S. Rae, Ed Lemmens, Anthony Desbien, Bill Hanson, Pete Lauer, Thomas W. Dubensky, Jr., Meredith Leong. Synergistic antitumor efficacy in mice with immunotherapy regimens combining recombinant live-attenuated Listeria with immune checkpoint inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B50.