Targeting CEACAM5-positive solid tumors using NILK-2401, a novel CEACAM5xCD47 κλ bispecific antibody.

Background: Blocking the CD47 "don't eat me"-signal on tumor cells with monoclonal antibodies or fusion proteins has shown limited clinical activity in hematologic malignancies and solid tumors thus far. Main side effects are associated with non-tumor targeted binding to CD47 particularly on blood cells. Methods: We present here the generation and preclinical development of NILK-2401, a CEACAM5×CD47 bispecific antibody (BsAb) composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). Results: NILK-2401 is a fully human BsAb binding the CEACAM5 N-terminal domain on tumor cells by its lambda light chain arm with an affinity of ≈4 nM and CD47 with its kappa chain arm with an intendedly low affinity of ≈500 nM to enabling tumor-specific blockade of the CD47-SIRPα interaction. For increased activity, NILK-2401 features a functional IgG1 Fc-part. NILK-2401 eliminates CEACAM5-positive tumor cell lines (3/3 colorectal, 2/2 gastric, 2/2 lung) with EC50 for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity ranging from 0.38 to 25.84 nM and 0.04 to 0.25 nM, respectively. NILK-2401 binds neither CD47-positive/CEACAM5-negative cell lines nor primary epithelial cells. No erythrophagocytosis or platelet activation is observed. Quantification of the pre-existing NILK-2401-reactive T-cell repertoire in the blood of 14 healthy donors with diverse HLA molecules shows a low immunogenic potential. In vivo, NILK-2401 significantly delayed tumor growth in a NOD-SCID colon cancer model and a syngeneic mouse model using human CD47/human SIRPα transgenic mice and prolonged survival. In cynomolgus monkeys, single doses of 0.5 and 20 mg/kg were well tolerated; PK linked to anti-CD47 and Fc-binding seemed to be more than dose-proportional for Cmax and AUC0-inf. Data were validated in human FcRn TG32 mice. Combination of a CEACAM5-targeting T-cell engager (NILK-2301) with NILK-2401 can either boost NILK-2301 activity (Emax) up to 2.5-fold or allows reaching equal NILK-2301 activity at >600-fold (LS174T) to >3,000-fold (MKN-45) lower doses. Conclusion: NILK-2401 combines promising preclinical activity with limited potential side effects due to the tumor-targeted blockade of CD47 and low immunogenicity and is planned to enter clinical testing.

Development and characterization of NILK-2301, a novel CEACAM5xCD3 κλ bispecific antibody for immunotherapy of CEACAM5-expressing cancers.

BACKGROUND: T-cell retargeting to eliminate CEACAM5-expressing cancer cells via CEACAM5xCD3 bispecific antibodies (BsAbs) showed limited clinical activity so far, mostly due to insufficient T-cell activation, dose-limiting toxicities, and formation of anti-drug antibodies (ADA). METHODS: We present here the generation and preclinical development of NILK-2301, a BsAb composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). RESULTS: NILK-2301 binds CD3ɛ on T-cells with its lambda light chain arm with an affinity of ≈100 nM, and the CEACAM5 A2 domain on tumor cells by its kappa light chain arm with an affinity of ≈5 nM. FcγR-binding is abrogated by the "LALAPA" mutation (Leu234Ala, Leu235Ala, Pro329Ala). NILK-2301 induced T-cell activation, proliferation, cytokine release, and T-cell dependent cellular cytotoxicity of CEACAM5-positive tumor cell lines (5/5 colorectal, 2/2 gastric, 2/2 lung), e.g., SK-CO-1 (Emax = 89%), MKN-45 (Emax = 84%), and H2122 (Emax = 97%), with EC50 ranging from 0.02 to 0.14 nM. NILK-2301 binds neither to CEACAM5-negative or primary colon epithelial cells nor to other CEACAM family members. NILK-2301 alone or in combination with checkpoint inhibition showed activity in organotypic tumor tissue slices and colorectal cancer organoid models. In vivo, NILK-2301 at 10 mg/kg significantly delayed tumor progression in colon- and a pancreatic adenocarcinoma model. Single-dose pharmacokinetics (PK) and tolerability in cynomolgus monkeys at 0.5 or 10 mg/kg intravenously or 20 mg subcutaneously showed dose-proportional PK, bioavailability ≈100%, and a projected half-life in humans of 13.1 days. NILK-2301 was well-tolerated. Data were confirmed in human FcRn TG32 mice. CONCLUSIONS: In summary, NILK-2301 combines promising preclinical activity and safety with lower probability of ADA-generation due to its format compared to other molecules and is scheduled to enter clinical testing at the end of 2023.

CD47xCD19 bispecific antibody triggers recruitment and activation of innate immune effector cells in a B-cell lymphoma xenograft model.

BACKGROUND: CD47/SIRPα axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer, particularly in hematological malignancies. In preclinical models, CD47/SIRPα blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here, we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model. METHODS: The contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry, transcriptomic analysis, and in vivo immune-cell depletion experiments. RESULTS: We showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells, monocytes, dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably, molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation, including IFNγ and IL-12b. Moreover, TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701, highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition, NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro, resulting in an increased cross-priming of tumor-specific CD8 T cells. CONCLUSIONS: The study described the mechanisms afforded by the CD47xCD19 bispecific antibody, NI-1701, at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035).

Immunological analysis of the murine anti-CD3-induced cytokine release syndrome model and therapeutic efficacy of anti-cytokine antibodies.

The aberrant release of inflammatory mediators often referred to as a cytokine storm or cytokine release syndrome (CRS), is a common and sometimes fatal complication in acute infectious diseases including Ebola, dengue, COVID-19, and influenza. Fatal CRS occurrences have also plagued the development of highly promising cancer therapies based on T-cell engagers and chimeric antigen receptor (CAR) T cells. CRS is intimately linked with dysregulated and excessive cytokine release, including IFN-γ, TNF-α, IL 1, IL-6, and IL-10, resulting in a systemic inflammatory response leading to multiple organ failure. Here, we show that mice intravenously administered the agonistic hamster anti-mouse CD3ε monoclonal antibody 145-2C11 develop clinical and laboratory manifestations seen in patients afflicted with CRS, including body weight loss, hepatosplenomegaly, thrombocytopenia, increased vascular permeability, lung inflammation, and hypercytokinemia. Blood cytokine levels and gene expression analysis from lung, liver, and spleen demonstrated a hierarchy of inflammatory cytokine production and infiltrating immune cells with differentiating organ-dependent kinetics. IL-2, IFN-γ, TNF-α, and IL-6 up-regulation preceded clinical signs of CRS. The co-treatment of mice with a neutralizing anti-cytokine antibody cocktail transiently improved early clinical and laboratory features of CRS. We discuss the predictive use of this model in the context of new anti-cytokine strategies to treat human CRS.

Targeting a membrane-proximal epitope on mesothelin increases the tumoricidal activity of a bispecific antibody blocking CD47 on mesothelin-positive tumors.

Mesothelin (MSLN) is a cell surface glycoprotein overexpressed in several solid malignancies, including gastric, lung, mesothelioma, pancreatic and ovarian cancers. While several MSLN-targeting therapeutic approaches are in development, only limited efficacy has been achieved in patients. A potential shortcoming of several described antibody-based approaches is that they target the membrane distal region of MSLN and, additionally, are known to be handicapped by the high levels of circulating soluble MSLN in patients. We show here, using monoclonal antibodies (mAbs) targeting different MSLN-spanning epitopes, that the membrane-proximal region resulted in more efficient killing of MSLN-positive tumor cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Surprisingly, no augmented killing was observed in antibody-dependent cellular phagocytosis (ADCP) by mAbs targeting this membrane-proximal region. To further increase the ADCP potential, we, therefore, generated bispecific antibodies (bsAbs) coupling a high-affinity MSLN binding arm to a blocking CD47 arm. Here, targeting the membrane-proximal domain of MSLN demonstrated enhanced ADCP activity compared to membrane-distal domains when the bsAbs were used in in vitro phagocytosis killing assays. Importantly, the superior anti-tumor activity was also translated in xenograft tumor models. Furthermore, we show that the bsAb approach targeting the membrane-proximal epitope of MSLN optimized ADCC activity by augmenting FcγR-IIIA activation and enhanced ADCP via a more efficient blockade of the CD47/SIRPα axis.

Tumor-Directed Blockade of CD47 with Bispecific Antibodies Induces Adaptive Antitumor Immunity.

CD47 serves as an anti-phagocytic receptor that is upregulated by cancer to promote immune escape. As such, CD47 is the focus of intense immuno-oncology drug development efforts. However, as CD47 is expressed ubiquitously, clinical development of conventional drugs, e.g., monoclonal antibodies, is confronted with patient safety issues and poor pharmacology due to the widespread CD47 "antigen sink". A potential solution is tumor-directed blockade of CD47, which can be achieved with bispecific antibodies (biAbs). Using mouse CD47-blocking biAbs in a syngeneic tumor model allowed us to evaluate the efficacy of tumor-directed blockade of CD47 in the presence of the CD47 antigen sink and a functional adaptive immune system. We show here that CD47-targeting biAbs inhibited tumor growth in vivo, promoting durable antitumor responses and stimulating CD8+ T cell activation in vitro. In vivo efficacy of the biAbs could be further enhanced when combined with chemotherapy or PD-1/PD-L1 immune checkpoint blockade. We also show that selectivity and pharmacological properties of the biAb are dependent on the affinity of the anti-CD47 arm. Taken together, our study validates the approach to use CD47-blocking biAbs either as a monotherapy or part of a multi-drug approach to enhance antitumor immunity.

Neutralization of IFN-γ reverts clinical and laboratory features in a mouse model of macrophage activation syndrome.

BACKGROUND: The pathogenesis of macrophage activation syndrome (MAS) is not clearly understood: a large body of evidence supports the involvement of mechanisms similar to those implicated in the setting of primary hemophagocytic lymphohistiocytosis. OBJECTIVE: We sought to investigate the pathogenic role of IFN-γ and the therapeutic efficacy of IFN-γ neutralization in an animal model of MAS. METHODS: We used an MAS model established in mice transgenic for human IL-6 (IL-6TG mice) challenged with LPS (MAS mice). Levels of IFN-γ and IFN-γ-inducible chemokines were evaluated by using real-time PCR in the liver and spleen and by means of ELISA in plasma. IFN-γ neutralization was achieved by using the anti-IFN-γ antibody XMG1.2 in vivo. RESULTS: Mice with MAS showed a significant upregulation of the IFN-γ pathway, as demonstrated by increased mRNA levels of Ifng and higher levels of phospho-signal transducer and activator of transcription 1 in the liver and spleen and increased expression of the IFN-γ-inducible chemokines Cxcl9 and Cxcl10 in the liver and spleen, as well as in plasma. A marked increase in Il12a and Il12b expression was also found in livers and spleens of mice with MAS. In addition, mice with MAS had a significant increase in numbers of liver CD68+ macrophages. Mice with MAS treated with an anti-IFN-γ antibody showed a significant improvement in survival and body weight recovery associated with a significant amelioration of ferritin, fibrinogen, and alanine aminotransferase levels. In mice with MAS, treatment with the anti-IFN-γ antibody significantly decreased circulating levels of CXCL9, CXCL10, and downstream proinflammatory cytokines. The decrease in CXCL9 and CXCL10 levels paralleled the decrease in serum levels of proinflammatory cytokines and ferritin. CONCLUSION: These results provide evidence for a pathogenic role of IFN-γ in the setting of MAS.

Use of a mouse model to identify a blood biomarker for IFNγ activity in pediatric secondary hemophagocytic lymphohistiocytosis.

Life-threatening cytokine release syndromes include primary (p) and secondary (s) forms of hemophagocytic lymphohistiocytosis (HLH). Below detection in healthy individuals, interferon γ (IFNγ) levels are elevated to measurable concentrations in these afflictions suggesting a central role for this cytokine in the development and maintenance of HLH. Mimicking an infection-driven model of sHLH in mice, we observed that the tissue-derived levels of IFNγ are actually 500- to 2000-fold higher than those measured in the blood. To identify a blood biomarker, we postulated that the IFNγ gene products, CXCL9 and CXCL10 would correlate with disease parameters in the mouse model. To translate this into a disease relevant biomarker, we investigated whether CXCL9 and CXCL10 levels correlated with disease activity in pediatric sHLH patients. Our data demonstrate that disease control in mice correlates with neutralization of IFNγ activity in tissues and that the 2 chemokines serve as serum biomarkers to reflect disease status. Importantly, CXCL9 and CXCL10 levels in pediatric sHLH were shown to correlate with key disease parameters and severity in these patients. Thus, the translatability of the IFNγ-biomarker correlates from mouse to human, advocating the use of serum CXCL9 or CXCL10 as a means to monitor total IFNγ activity in patients with sHLH.

GlycoDelete engineering of mammalian cells simplifies N-glycosylation of recombinant proteins.

Heterogeneity in the N-glycans on therapeutic proteins causes difficulties for protein purification and process reproducibility and can lead to variable therapeutic efficacy. This heterogeneity arises from the multistep process of mammalian complex-type N-glycan synthesis. Here we report a glycoengineering strategy--which we call GlycoDelete--that shortens the Golgi N-glycosylation pathway in mammalian cells. This shortening results in the expression of proteins with small, sialylated trisaccharide N-glycans and reduced complexity compared to native mammalian cell glycoproteins. GlycoDelete engineering does not interfere with the functioning of N-glycans in protein folding, and the physiology of cells modified by GlycoDelete is similar to that of wild-type cells. A therapeutic human IgG expressed in GlycoDelete cells had properties, such as reduced initial clearance, that might be beneficial when the therapeutic goal is antigen neutralization. This strategy for reducing N-glycan heterogeneity on mammalian proteins could lead to more consistent performance of therapeutic proteins and modulation of biopharmaceutical functions.

Anti-CD79 antibody induces B cell anergy that protects against autoimmunity.

B cells play a major role in the pathogenesis of many autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type I diabetes mellitus, as indicated by the efficacy of B cell-targeted therapies in these diseases. Therapeutic effects of the most commonly used B cell-targeted therapy, anti-CD20 mAb, are contingent upon long-term depletion of peripheral B cells. In this article, we describe an alternative approach involving the targeting of CD79, the transducer subunit of the B cell AgR. Unlike anti-CD20 mAbs, the protective effects of CD79-targeted mAbs do not require cell depletion; rather, they act by inducing an anergic-like state. Thus, we describe a novel B cell-targeted approach predicated on the induction of B cell anergy.

TRIM5 is an innate immune sensor for the retrovirus capsid lattice.

TRIM5 is a RING domain-E3 ubiquitin ligase that restricts infection by human immunodeficiency virus (HIV)-1 and other retroviruses immediately following virus invasion of the target cell cytoplasm. Antiviral potency correlates with TRIM5 avidity for the retrovirion capsid lattice and several reports indicate that TRIM5 has a role in signal transduction, but the precise mechanism of restriction is unknown. Here we demonstrate that TRIM5 promotes innate immune signalling and that this activity is amplified by retroviral infection and interaction with the capsid lattice. Acting with the heterodimeric, ubiquitin-conjugating enzyme UBC13-UEV1A (also known as UBE2N-UBE2V1), TRIM5 catalyses the synthesis of unattached K63-linked ubiquitin chains that activate the TAK1 (also known as MAP3K7) kinase complex and stimulate AP-1 and NFκB signalling. Interaction with the HIV-1 capsid lattice greatly enhances the UBC13-UEV1A-dependent E3 activity of TRIM5 and challenge with retroviruses induces the transcription of AP-1 and NF-κB-dependent factors with a magnitude that tracks with TRIM5 avidity for the invading capsid. Finally, TAK1 and UBC13-UEV1A contribute to capsid-specific restriction by TRIM5. Thus, the retroviral restriction factor TRIM5 has two additional activities that are linked to restriction: it constitutively promotes innate immune signalling and it acts as a pattern recognition receptor specific for the retrovirus capsid lattice.

Although IL-6 trans-signaling is sufficient to drive local immune responses, classical IL-6 signaling is obligate for the induction of T cell-mediated autoimmunity.

IL-6-mediated T cell-driven immune responses are associated with signaling occurring through the membrane-bound cognate receptor α-chain (mIL-6Rα). Once formed, IL-6-mIL-6Rα complexes induce the homodimerization and subsequent phosphorylation of the ubiquitously expressed signal-transducing protein, gp130. This signaling event is defined as classical IL-6 signaling. However, many inflammatory processes assigned to IL-6 may be mediated via binding a naturally occurring soluble IL-6Rα, which forms an agonistic complex (IL-6/soluble IL-6Rα) capable of evoking responses on a wide range of cell types that lack mIL-6Rα (IL-6 trans-signaling). To dissect the differential contribution of the two IL-6 signaling pathways in cell-mediated inflammatory processes, we pharmaceutically targeted each using two murine models of human arthritis. Whereas intra-articular neutralization of trans-signaling attenuated local inflammatory responses, the classical pathway was found to be obligate and sufficient to induce pathogenic T cells and humoral responses, leading to systemic disease. Our data illustrate that mechanisms occurring in the secondary lymphoid organs underlying arthropathies are mediated via the classical pathway of IL-6 signaling, whereas trans-signaling contributes only at the local site, that is, in the affected tissues.

Pan-CC chemokine neutralization restricts splenocyte egress and reduces inflammation in a model of arthritis.

Chemokines are key regulators of leukocyte trafficking and play a crucial role under homeostatic and inflammatory conditions. Because chemokines are involved in multiple pathologies, they represent an attractive class of therapeutic targets. However, because of the redundancy of this system, neutralizing a single chemokine may be insufficient to achieve therapeutic benefit. Our strategy was to use a Fc-fusion recombinant protein form of the poxvirus-derived viral CC chemokine inhibitor protein (vCCI-Fc) that has the ability to specifically bind to multiple CC chemokines and neutralize their activity. In this study, we demonstrate first that, in vivo, vCCI-Fc prevents CC chemokine-dependent migration of macrophages into inflamed tissue of carageenan-challenged mice. We next studied this effect of inhibiting CC chemokine activity in a model more relevant to human disease, collagen-induced arthritis. Mice receiving vCCI-Fc revealed a striking retention of splenocytes, including activated and IFN-gamma-secreting CD4(+) and CD8(+) T cells, that was associated with a concomitant decrease of cells in the draining lymph nodes. These phenomena resulted in a significant decrease in the incidence of disease and a reduction in clinical score, joint inflammation, and cartilage destruction as compared with mice receiving isotype control. Taken together, these results define a role for CC chemokines in the control of disease, as interfering with their function leads to a previously unappreciated role of controlling inflammatory cell trafficking in and out of secondary lymphoid organs.

A vector-based minigene vaccine approach results in strong induction of T-cell responses specific of hepatitis C virus.

Multiepitope-based vaccines against hepatitis C virus (HCV) were designed in the form of three minigenes encompassing four domains of the NS3, NS4 and NS5B proteins that contain multiple class I/II restricted epitopes. The polyEp-WT minigene encodes all four domains in fusion, the polyEp-C minigene encodes the same fusion but optimised for mammalian translation and the polyEp-E3 minigene has an additional endoplasmic reticulum targeting sequence. Whereas the minigenes vectorised by DNA were poorly immunogenic, adenovirus vectorisation induced strong and broader IFNgamma-ELISpot and CTL responses in HLA-A2 transgenic mice. In addition, polyEp-WT and polyEp-E3 responses were found cross-reactive in a recombinant Listeria-NS3-based surrogate challenge. This study illustrates the potency of vectorised minigenes in the field of HCV vaccine development.

Optimized vaccination regimen linked to exhaustive screening approaches identifies 2 novel HLA-B7 restricted epitopes within hepatitis C virus NS3 protein.

Broad immune responses, in particular specific for the NS3 protein and mediated by both CD8+ and CD4+T lymphocytes, are thought to play a critical role in the control of hepatitis C virus (HCV) infection. In this study, we searched for novel HLA-B*0702 NS3 restricted epitopes following an optimized NS3NS4 immunization protocol in transgenic mice expressing HLA-B*0702 molecule. Combining predicted and overlapping peptides, we identified two novel epitopes, WPA10 (aa 1111-1120) and LSP10 (aa 1153-1162), which triggered significant IFN-gamma-producing T cell frequencies and high CTL responses. Both epitopes were shown to be immunogenic when used as synthetic peptides to immunize mice. The relevance of these epitopes to humans was demonstrated, as both were able in vitro to recall specific IFN-gamma and IL10-producing cells from peripheral blood mononuclear cells of HCV infected patients. Such epitopes enlarge the pool of NS3-specific CD8+T cell epitopes available to perform immunomonitoring of HCV infection and to develop vaccines.