Human Knock-in Research Articles

Human MHC Class II and Invariant Chain Knock-in Mice Mimic Rheumatoid Arthritis with Allele Restriction in Immune Response and Arthritis Association.

Transgenic mice expressing human major histocompatibility complex class II (MHCII) risk alleles are widely used in autoimmune disease research, but limitations arise due to non-physiologic expression. To address this, physiologically relevant mouse models are established via knock-in technology to explore the role of MHCII in diseases like rheumatoid arthritis. The gene sequences encoding the ectodomains are replaced with the human DRB1*04:01 and 04:02 alleles, DRA, and CD74 (invariant chain) in C57BL/6N mice. The collagen type II (Col2a1) gene is modified to mimic human COL2. Importantly, DRB1*04:01 knock-in mice display physiologic expression of human MHCII also on thymic epithelial cells, in contrast to DRB1*04:01 transgenic mice. Humanization of the invariant chain enhances MHCII expression on thymic epithelial cells, increases mature B cell numbers in spleen, and improves antigen presentation. To validate its functionality, the collagen-induced arthritis (CIA) model is used, where DRB1*04:01 expression led to a higher susceptibility to arthritis, as compared with mice expressing DRB1*04:02. In addition, the humanized T cell epitope on COL2 allows autoreactive T cell-mediated arthritis development. In conclusion, the humanized knock-in mouse faithfully expresses MHCII, confirming the DRB1*04:01 alleles role in rheumatoid arthritis and being also useful for studying MHCII-associated diseases.

Analyses of the autism-associated neuroligin-3 R451C mutation in human neurons reveal a gain-of-function synaptic mechanism.

Mutations in many synaptic genes are associated with autism spectrum disorders (ASD), suggesting that synaptic dysfunction is a key driver of ASD pathogenesis. Among these mutations, the R451C substitution in the NLGN3 gene that encodes the postsynaptic adhesion molecule Neuroligin-3 is noteworthy because it was the first specific mutation linked to ASDs. In mice, the corresponding Nlgn3 R451C-knockin mutation recapitulates social interaction deficits of ASD patients and produces synaptic abnormalities, but the impact of the NLGN3 R451C mutation on human neurons has not been investigated. Here, we generated human knockin neurons with the NLGN3 R451C and NLGN3 null mutations. Strikingly, analyses of NLGN3 R451C-mutant neurons revealed that the R451C mutation decreased NLGN3 protein levels but enhanced the strength of excitatory synapses without affecting inhibitory synapses; meanwhile NLGN3 knockout neurons showed reduction in excitatory synaptic strengths. Moreover, overexpression of NLGN3 R451C recapitulated the synaptic enhancement in human neurons. Notably, the augmentation of excitatory transmission was confirmed in vivo with human neurons transplanted into mouse forebrain. Using single-cell RNA-seq experiments with co-cultured excitatory and inhibitory NLGN3 R451C-mutant neurons, we identified differentially expressed genes in relatively mature human neurons corresponding to synaptic gene expression networks. Moreover, gene ontology and enrichment analyses revealed convergent gene networks associated with ASDs and other mental disorders. Our findings suggest that the NLGN3 R451C mutation induces a gain-of-function enhancement in excitatory synaptic transmission that may contribute to the pathophysiology of ASD.

Inhibition of microRNA-33b specifically ameliorates abdominal aortic aneurysm formation via suppression of inflammatory pathways

Abdominal aortic aneurysm (AAA) is a lethal disease, but no beneficial therapeutic agents have been established to date. Previously, we found that AAA formation is suppressed in microRNA (miR)-33-deficient mice compared with wild-type mice. Mice have only one miR-33, but humans have two miR-33 s, miR-33a and miR-33b. The data so far strongly support that inhibiting miR-33a or miR-33b will be a new strategy to treat AAA. We produced two specific anti-microRNA oligonucleotides (AMOs) that may inhibit miR-33a and miR-33b, respectively. In vitro studies showed that the AMO against miR-33b was more effective; therefore, we examined the in vivo effects of this AMO in a calcium chloride (CaCl2)-induced AAA model in humanized miR-33b knock-in mice. In this model, AAA was clearly improved by application of anti-miR-33b. To further elucidate the mechanism, we evaluated AAA 1 week after CaCl2 administration to examine the effect of anti-miR-33b. Histological examination revealed that the number of MMP-9-positive macrophages and the level of MCP-1 in the aorta of mice treated with anti-miR-33b was significantly reduced, and the serum lipid profile was improved compared with mice treated with control oligonucleotides. These results support that inhibition of miR-33b is effective in the treatment for AAA.

Abstract 628: Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies

Abstract Background: On the basis of efficacy in mouse tumor models, multiple CD137 (4-1BB) agonist agents are being preclinically and clinically developed. The costimulatory molecule CD137 is inducibly expressed as a transmembrane or as a soluble protein (sCD137). Moreover, the CD137 cytoplasmic signaling domain is a key part in approved Chimeric Antigen Receptors (CARs). Reliable pharmacodynamic biomarkers for CD137 ligation and co-stimulation of T cells will facilitate clinical development of CD137 agonists in the clinic. Methods: We used human and mouse CD8 T cells undergoing activation to measure CD137 transcription and protein expression levels determining both of the membrane-bound and soluble forms. In tumor-bearing mice plasma sCD137 concentrations were monitored upon treatment with agonist anti-CD137 mAbs. Human CD137 knock-in mice were treated with clinical-grade agonist anti-human CD137 (Urelumab). Sequential plasma samples were collected from the first patients intratumorally treated with Urelumab in the INTRUST clinical trial. Anti-Mesothelin CD137-encompassing CAR-transduced T cells were stimulated with mesothelin coated microbeads. sCD137 was measured by sandwich ELISAs and Luminex and flow cytometry was used to monitor CD137 surface expression. Results: CD137 co-stimulation upregulates transcription and protein expression of CD137 itself including sCD137 in human and mouse CD8 T cells. Immunotherapy with anti-CD137 agonist mAb resulted in increased plasma sCD137 in mice bearing syngeneic tumors. sCD137 induction is also observed in human CD137 knock-in mice treated with Urelumab and in mice transiently humanized with T cells undergoing CD137 co-stimulation inside subcutaneous implanted Matrigel® plugs. CD137 signaling domain-containing CAR T cells readily released sCD137 upon antigen recognition in a manner favored by the CD137 signaling domain. Patients treated intratumorally with low dose Urelumab showed increased plasma concentrations of sCD137. Conclusion: sCD137 in plasma and CD137 surface expression can be used as quantitative parameters dynamically reflecting therapeutic co-stimulatory activity elicited by agonist CD137-targeted agents. Citation Format: Javier Glez-Vaz, Arantza Azpilikueta, Irene Olivera, Assunta Cirella, Álvaro Teijeira, Carmen Ochoa, Maite Álvarez, Iñaki Eguren, Carlos Luri-Rey, Miguel F. Sanmamed, Ignacio Melero. Soluble CD137 as a dynamic biomarker to monitor agonist CD137 immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 628.

Abstract 5518: Single cell RNA sequencing revealed that the mechanism of the synergistic efficacy of STA551 and immune checkpoint inhibitor results from the induction of highly activated T cells in tumor

Abstract Background: CD137 (4-1BB) is a co-stimulatory molecule which exerts activation signals in multiple immune cell subsets. However, agonist antibodies targeting CD137 have not been clinically successful due to severe systemic toxicity. Therefore, we generated STA551, a tumor selective CD137 agonist antibody. STA551 binds to CD137 only in the presence of ATP, which is known to be present at high levels in tumor tissue, and activates immune cells such as T cells. Previous studies have shown that STA551 exhibits antitumor efficacy without inducing a systemic immune response in a murine model. Combination studies with anti-PD-L1 antibody have also shown synergistic efficacy without inducing a systemic immune response. However, it was unclear why they exhibit strong combination effects. In this study, we elucidated the mechanism of STA551 and anti-PD-L1 antibody efficacy through comprehensive analysis of gene expression using single cell RNA sequencing. Method & Result: In a colon 38-bearing human CD137 knock-in mouse model, combination therapy with STA551 and anti-PD-L1 antibody showed potent anti-tumor efficacy. From immunohistochemistry of tumor tissue, increased numbers of CD8+ T cells and elevated expression of PD-L1 molecules were observed. To perform a detailed mechanistic analysis, tumor tissue was collected after antibody administration and CD3 positive and CD3 negative fractions were separated and gene expression was comprehensively analyzed using single cell RNA-seq. Next, the fraction expressing Cd8a gene was extracted from CD3 positive fraction and clustered. One cluster was found to be markedly increased in STA551 and anti-PD-L1 antibody combination compared to vehicle or monotherapy treatment. Differential gene expression analysis revealed that the cluster was enriched for genes related to cytokines and chemokines. This result revealed that the combination of STA551 and anti-PD-L1 antibody induced highly activated CD8+T cells. Conclusion: The combination of STA551 and PD-L1 showed synergistic anti-tumor efficacy in a colon38-bearing human CD137 knock-in mouse model. Detailed mechanistic analysis showed that the combination of STA551 and PD-L1 induced highly activated CD8+T cells expressing chemokines and cytokines compared to monotherapy. This cluster also express chemokines, suggesting that other immune cells have been recruited. These results suggest that even the activation of only tumor tissue, which is a characteristic of STA551, can induce a strong immune response. Citation Format: Ryo Uchikawa, Kazuya Takakuwa, Hirofumi Mikami, Shoichi Metsugi, Kenji Taniguchi, Yoshinori Narita, Mika Kamata-Sakurai. Single cell RNA sequencing revealed that the mechanism of the synergistic efficacy of STA551 and immune checkpoint inhibitor results from the induction of highly activated T cells in tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5518.

Abstract 1996: Intravenous administration of the small molecule STING agonist CRD5500 elicits potent anti-tumor immune responses in cold tumors

Abstract Background: Innate immune signaling mediated through STING (Stimulator of Interferon Genes) generates a Type I interferon (Type 1 IFN) signal that increases T cell infiltration into cold or non-inflamed tumors leading to significant regression1. CRD5500 is a small molecule STING agonist that has demonstrated excellent systemic tolerability in primate GLP toxicology studies and is scheduled for IND filing with the US FDA in the first half of 2022. The compound binds to STING at a unique location that is outside the dimeric cleft that is the natural ligand binding site2. While this first-in class agent cross-reacts with cynomolgus monkey STING and activates all five major variants of human STING with similar potency, it does not cross-react with rodent STING. Here we demonstrate the potent anti-tumor activity of CRD5500 administered intravenously in a range of immunologically cold murine tumors implanted in human STING knock-in mice. Methods: The functional potency of STING across species was evaluated in STING over-expressing HEK293T cells and PBMCs. Human STING KI mice (Genoway) were used to monitor anti-tumor effects of CRD5500 dosed IV or IT against the following tumors: B16F10 (melanoma), PANO2 (pancreatic), C1498 (acute myeloid leukemia) and LL2 (lung). Intravenous pharmacokinetic and pharmacodynamic evaluation was carried out in cynomolgus monkeys. Results: CRD5500’s activated the different variants of human STING with EC50 values that ranged from 10-70 nM in an pIRF3 reporter assay. Treatment of hSTING transfected cells, human tumor cell lines or hPBMC with CRD5500 led to dose dependent activation of STING. Further, CRD5500 treatment of human DCs and PBMCs led to the induction of inflammatory cytokines such as IFNβ and TNFα. Intravenous administration of CRD5500 in human STING knock-in mice caused tumor regression in several cold tumor models such as B16F10, PANO2, LL2, C1498. Further, CRD5500 demonstrated the abscopal effect in B16F10 tumors when administered via intratumoral route. The compound was well tolerated at high doses in the primate GLP study when administered intravenously and caused dose and exposure dependent increases in CXCL10. Conclusion: The ability of CRD5500 to generate impressive anti-tumor immune responses in cold tumors as a single agent coupled with its excellent IV safety profile in non-human primates make it well suited for FIH investigations planned for mid-2022 under a US FDA IND.

Abstract 5557: PMC-309, a highly selective anti-VISTA antibody reverses immunosuppressive TME to immune-supportive TME

Abstract Purpose: We investigated the pharmacology of PMC-309 in ex vivo or in vivo characterization for the development of the preclinical study. Experimental procedures: Target selectivity: it was measured by SPR assay. Human VISTA expressing CHO k1 cells or CHO k1 mock cells were used in cell binding assay. Target cell analysis: human peripheral blood mononuclear cells (PBMC) containing lymphocyte and myeloid lineage cells were used for target cells analysis with flow cytometry. In vitro T cell activity: human PBMC was employed for the evaluation of T cell activity (IRB #1041107-201703-BR-002-02) and was stimulated with or w/o PMC-309 in the presence of anti-CD3/28 Antibody. In vivo study: MC38 bearing human VISTA knock-in (KI) mice were employed for the assessment of anti-tumor activity of PMC-309. The tumor infiltrated immune cells: Immune cells in the TME were evaluated by immunohistochemistry (IHC) or flow cytometry (FACS) analysis. Statistics: All data were presented as the mean ± SEM and analyzed using one-way ANOVA followed by Dunnett’s multiple comparison tests in Graph pad prism®. ***p<0.001, **p<0.01 and *p<0.05 compared to Control group. Summary of data: PMC-309 binding to VISTA expressing cells is highly selective and the selectivity is maintained even in the low pH conditions that mimic TME. PMC-309 enhances the secretion of IFN-gamma, TNF-alpha, and IL-2 in Mixed Lymphocyte Reaction (MLR) settings. In addition, PMC-309 promoted monocyte differentiation into M1 macrophage that stimulates proinflammatory cytokine secretion of T cells. For the in vivo study, PMC-309 was intravenously administrated in VISTA-KI mice. The tumor growth rate was suppressed in accompanied by a synergistic effect with an anti-PD1 antibody. The anti-tumor activity was associated with enhanced T cell activation, increased secretion of pro-inflammatory cytokines, and increased penetration of cytotoxic T cells, but lowing immune-suppressive MDSC cells into TME as demonstrated with IHC analysis. Conclusions: PMC-309 is a fully human anti-VISTA antibody that targets human VISTA specifically. PMC-309 enhanced both T cell activation and monocyte activation when monocytes were cultured with or without T cells by targeting VISTA. PMC-309 increased the number of T cell infiltration while a decrease of MDSCs in the TME. PMC-309 in combination with chemotherapy or other IO drugs could address highly medical unmet needs from patients with drug resistance to currently available IO treatment options. Citation Format: Cheon Ho Park, Sang Soon Byun, Jin Yong An, Hyerim Han, Weon Sup Lee. PMC-309, a highly selective anti-VISTA antibody reverses immunosuppressive TME to immune-supportive TME [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5557.

Abstract 3180: Targeting LRG1 to normalize tumor vasculature and enhance therapeutic efficacy

Abstract In recent years improving tumor vascular function, to render the tumour microenvironment less permissive and to improve delivery of therapeutics, has gained traction due to a growing body of supportive evidence. Identifying suitable targets that are tractable, ubiquitous and safe, however, has proven to be more challenging. Almost a decade ago we reported that a secreted glycoprotein, leucine-rich alpha-2-glycoprotein 1 (LRG1), was induced in ocular neovascular complications and contributed to the formation of dysfunctional neovessels1. More recently, we have reported that LRG1 is expressed in experimental and human tumors, and that its inhibition with a function-blocking antibody improves outcome in multiple primary2 and metastatic3 models of cancer. Crucially, we found that LRG1 blockade normalizes tumor vessels and enhances the efficacy of cisplatin, adoptive T cell and checkpoint inhibitor therapy, and that a humanized version of our blocking antibody named Magacizumab effectively inhibits tumour growth both alone and as an antibody-drug-conjugate4.Using animal models and in vitro assays we present here further data in support of LRG1 as a promising target for the treatment of solid cancers. We have further investigated the effects of LRG1 blockade on immune cell infiltration using flow cytometric and immunohistochemical analysis. In subcutaneous B16 melanoma-bearing mice treated with a PD1 checkpoint inhibitor, antibody blockade of LRG1 significantly enhanced the infiltration of CD3+ and CD8+ T cells, with the latter exhibiting a more activated phenotype as evidenced by higher GrzB, reduced PD1hi expression and increased proliferation. We also observed a reduction in the Tregs:Th ratio and a higher number of MHCII+ cells. These outcomes were also observed in LLC tumors alongside a reduction in the number of infiltrated neutrophils. In preliminary studies, where we investigated LRG1 blockade in the Rag1 mouse, we observed no effect suggesting that the downstream mode of action is mediated by impacting the immune system. Finally, to test the development of our humanized anti-LRG1 antibody Magacizumab, that only recognizes human LRG1, we demonstrated its efficacy in B16F0 tumors grown in a human LRG1 knock-in mouse. These studies provide compelling evidence that LRG1 is a novel, legitimate and potentially efficacious target for the treatment of various human solid cancers.

Abstract LB534: Development of GB2101, a novel anti-CCR8 antibody for cancer treatment

Abstract Background: Regulatory T cells (Tregs) play a crucial role in suppressing immune response and maintaining hemostasis and immune tolerance. Tregs are enriched in the tumor microenvironment (TME) and suppress anti-tumor immunity. Therefore, specific depletion of Tregs in the TME is expected to reduce anti-tumor immune suppression without affecting hemostasis in the periphery. The C-C motif chemokine receptor 8 (CCR8), highly expressed on Tregs from TME but significantly lower expressed on Tregs from peripheral blood, provides an ideal target for preferentially deletion of Tregs within tumors. GB2101 is a novel anti-CCR8 monoclonal antibody generated from hybridoma. The data from in vitro and in vivo studies indicated that GB2101 is a good PCC molecule with great potential for clinical development. Methods: GB2101 was generated by immunization of CCR8 gene plus CCR8 over-expressing cells in mice followed by hybridoma technology. The binding of the antibody to CCR8-expressing cells was detected by flow cytometry. CCL1 ligand blocking was assessed by flow cytometry and calcium flux assay. Antibody-dependent cell-mediated cytotoxicity (ADCC) function was evaluated by Jurkat-CD16A-NFAT reporter system. In vivo anti-tumor efficacy was assessed in syngeneic model using human CCR8 knock-in mice. Results: GB2101 displayed potent binding to cells expressing CCR8. GB2101 efficiently blocked CCL1 binding and CCL1-induced signals in CCR8 expressing cells. ADCC activity against target cells was observed and afucosylated version of GB2101 improved ADCC response. GB2101 significantly inhibited tumor growth in syngeneic model. Conclusions: GB2101 is a novel monoclonal antibody specifically binding to CCR8 with high affinity, and efficiently blocked CCL1-CCR8 pathway and suppressed tumor growth in vivo. Therefore, it is a very promising preclinical candidate that may be used in the treatment of multiple tumor types. Citation Format: Qinglin Du, Yiqi Cao, Xueyan Yang, Jing Lu, Fei Peng, Wenming Zhou, Liwen Liang, James Li, Shuhua Han. Development of GB2101, a novel anti-CCR8 antibody for cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB534.

Abstract 2090: Humanized IL36R mice provide a preclinical tool for the evaluation of therapeutic drugs targeting IL36R

Abstract The IL36 family of cytokines (IL36a, IL36b, and IL36g) is part of the IL-1 superfamily and triggers proinflammatory signaling through the IL36 receptor complex. This heterodimer complex is formed by the IL36 Receptor (IL1RL2) and IL-1R co-receptor accessory protein (IL-1RAcP). IL36R is present on a diverse variety of cell types, including the surface of dendritic cells, CD4+ T cells, and epithelial cells in various tissues. IL36 signaling can exert widespread inflammation, and its activity is implicated in several inflammatory diseases affecting epithelial tissues, including inflammatory bowel disease, rheumatoid arthritis, and psoriasis. Thus, identification of novel therapeutics targeting this pathway are of significant clinical interest. To provide a preclinical platform for efficacy assessments of novel anti-human IL36R drugs, we generated a humanized hIL36R knock-in mouse model. In this model, a chimeric IL1RL2 coding sequence, which is composed of the human IL1RL2 extracellular region and the mouse Il1rl2 cytoplasmic region, was inserted after the 5’UTR of mouse Il1rl2 gene using CRISPR-Cas9-based gene editing technology. The mouse cytoplasmic region was maintained to ensure intact downstream signaling. Polymerase chain reaction assessment of mRNA isolated from the lung tissue of edited homozygous humanized IL36R mice confirmed the absence of mouse Il1rl2 transcripts. Finally, to confirm the expression of human IL36R protein, immunohistochemistry was performed on the small intestine and skin tissue of humanized mice subjected to a chemical induction of psoriasis. Psoriasis-like symptoms were achieved by application of imiquimod cream on the back skin of the mice for 5 consecutive days. As expected, IL36R protein expression was detected in both tissues isolated from the homozygous humanized mice. These results indicate the successful generation of humanized IL36R mice, and suggest that this model will be an effective tool for future in vivo efficacy evaluation of immunomodulatory drugs targeting human IL36R. Citation Format: Xiaofei Zhou, Jiahui Chang, Jenna Frame, Chong Li, Zhifang Bai, Yuelei Shen. Humanized IL36R mice provide a preclinical tool for the evaluation of therapeutic drugs targeting IL36R [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2090.

Abstract 2899: Targeting regulatory T cells with HFB101110, a novel anti-human CCR8 antibody for the treatment of solid tumors

Abstract Regulatory T cells (Tregs) contribute to immunosuppression within the tumor microenvironment and have been associated with poor outcomes in a range of cancers. Targeted depletion of tumor-infiltrating Tregs (TITRs) is an attractive therapeutic strategy with the potential to enhance antitumor immunity in patients who do not respond to current treatments. However, such approaches have been limited to date by a lack of markers that are highly specific for TITRs. CCR8 is a chemokine G protein-coupled receptor (GPCR) that has recently been shown to be specifically expressed on TITRs as compared to Tregs in the periphery or other T cells within tumors. Here, we report the development and characterization of HFB101110, a humanized monoclonal antibody against CCR8 with potent and specific antibody-dependent cellular cytotoxicity (ADCC) activity. HFB101110 specifically recognizes an epitope on the N-terminus extracellular domain of CCR8 and does not recognize the closely related chemokine receptor CCR4. HFB101110 acts through a dual mechanism of action, by both depleting CCR8+ cells via ADCC and blocking binding of the CCL1 chemokine to its receptor CCR8. Blockade by HFB101110 inhibited calcium flux and chemotaxis induced by the interaction between CCL1 and CCR8. Furthermore, HFB101110 showed potent single agent anti-tumor activity associated with depletion of intratumoral Tregs in a human CCR8 knock-in mouse model. HFB101110 mediated specific ex vivo killing of Tregs from primary patient tumor samples both in the presence, and in some cases the absence, of allogeneic NK cells. HFB101110 showed favorable pharmacokinetic properties and a favorable developability profile, and was well-tolerated in both wild-type mice and cynomolgus monkeys. HFB101110 is currently being developed as a novel immunotherapy for the treatment of solid tumors coupled with a patient biomarker strategy derived from HiFiBiO’s Drug Intelligent Science (DIS™) single-cell immune profiling platform. Citation Format: Roshan M. Kumar, Julie Prigent, Hombline Poullain, Ayrin Kok, Carine George, Sami Ellouze, Yun-Yueh Lu, Rebecca Silver, Clarisse Monchecourt, Ross B. Fulton, Qian Zhang, Nicola Beltraminelli, Bernhard Moser, Francisco Adrian, Liang Schweizer. Targeting regulatory T cells with HFB101110, a novel anti-human CCR8 antibody for the treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2899.

Abstract 3590: ZL-1218, a novel anti-CCR8 antibody, exerts potent antitumor effect by depleting intratumoral regulatory T cells

Abstract Despite the promise of immunotherapy for cancer treatment, nearly 80% of patients fail to respond to checkpoint inhibitor (CPI) therapy. Regulatory T cells (Tregs), which inhibit immune responses in the tumor microenvironment via multiple suppressive mechanisms, have been proposed to play a key role in those patients who are not responding to CPI therapy. Therefore, targeted depletion of Tregs should promote more effective antitumor immunity. CCR8 is a chemokine receptor that is selectively expressed on highly activated human tumor-resident Tregs, and these intratumoral CCR8+ Tregs have been shown to drive immunosuppression that leads to poor prognosis for cancer patients. Here, we demonstrate that CCR8 is highly expressed on intratumoral FoxP3+ Treg cells in multiple cancers and is absent on other major immune cell populations in tumor microenvironment including effector T cells, conventional CD4 T cells, B cells, NK cells, some FoxP3+ cells, and myeloid cells. Importantly, no CCR8 protein expression was observed on any peripheral human leukocyte subset examined. These results provide strong rationale for targeting CCR8 as a cancer immunotherapy by selectively depleting the most suppressive intratumoral Treg cells. We have developed a humanized therapeutic antibody, ZL-1218, that binds to human CCR8 with high affinity and specificity and can induce potent ADCC activity enabling strong NK cell-mediated killing of CCR8-expressing Tregs. We show that in human CCR8 knock-in mouse models bearing syngeneic tumors, ZL-1218 reduces intratumoral Treg cells and thus elicits significant tumor growth inhibition in a dose-dependent manner. We have recently explored the potential for ZL-1218 in combination immunotherapy, examining the enhanced antitumor activity when ZL-1218 is combined with anti-PD-1. Using human dissociated tumor samples, we further observed that different tumor types may induce different CCR8 expression levels on intratumoral Tregs leading to multiple, distinct CCR8+ subsets in various indications. We are currently exploring the significance of these distinct populations and the impact of ZL-1218-mediated depletion of both CCR8 high- and CCR8 low-expressing subsets in multiple indications. Together, these data support the advancement of ZL-1218 into clinical evaluation as a novel therapeutic candidate to treat human solid tumors. Citation Format: Jing Zhang, Wenhua Shi, Min Chen, Xinchuan Dai, Hongshui Liu, Shou Li, Lina Wang, Bee-Chun Sun, Monica You, Vivian Morton, Qiuping Ye, Lishan Kang, Bing Wan, Peter Brams, David I. Bellovin. ZL-1218, a novel anti-CCR8 antibody, exerts potent antitumor effect by depleting intratumoral regulatory T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3590.

Abstract 6008: Effective depletion of tumor-infiltrating Tregs by a novel anti-CCR8 antibody (LM-108): Addressing resistance associated with immune checkpoint inhibitors

Abstract Immune checkpoint inhibitors (ICIs) have generated durable clinical responses in multiple tumor types, but their therapeutic efficacy is limited by development of primary or acquired resistance. The tumor microenvironment (TME) contains many factors influencing ICI responses. The ratio of effector T-cells (Teffs) and regulatory T-cells (Tregs) in tumors has been shown to be associated with the efficacy of ICIs in murine models, indicating that increasing Teffs or decreasing Tregs in tumor may prevent development of resistance to immunotherapy. There is a high and restricted expression of CC motif chemokine receptor 8 (CCR8) on tumor-infiltrating Tregs, whereas they are negligibly expressed on Teffs. Deletion of tumor-infiltrating Tregs via murine anti-CCR8 surrogates has shown effective anti-tumor activity in tumor models.LM-108 is a novel humanized anti-CCR8 monoclonal antibody with engineered IgG1 that enhances antibody-dependent cell-mediated cytotoxicity (ADCC). The expression of CCR8 and Foxp3 on tumor-infiltrating lymphocytes (TILs) was analyzed using published single-cell sequencing data. The binding affinity of LM-108 was assessed by flow cytometry using CCR8 high- and low-expressing cell lines. In vitro measurements of ADCC and antibody-dependent cellular phagocytosis (ADCP) mediated by LM-108 were conducted in CCR8 overexpressing HEK293, CHO-K1, and Jurkat cells. Furthermore, in vivo anti-tumor activity of LM-108 as monotherapy and in combination with anti-PD-1 therapy was evaluated in syngeneic models and PD-1-resistant tumor models with human CCR8 knock-in mice. Analysis of published single-cell sequencing data showed that in comparison with CCR4, CCR8 gene expression was specifically overlapped with Foxp3 in TILs of breast cancer. Fluorescence-activated cell sorting (FACS) assay revealed that LM-108 specifically binds with high affinity to human CCR8 in a dose-dependent manner. ADCC and ADCP effects induced by LM-108 were observed in HEK293 and CHO-K1 cells with different levels of CCR8 expression. In vivo data of CT26 syngeneic model showed that LM-108 murine surrogate antibody alone or in combination with anti-PD-1 antibody significantly inhibited tumor growth. Treatment with LM-108 effectively depleted tumor-infiltrating Tregs accompanied by increase in CD8/Treg ratio in tumors, leading to significant anti-tumor activity in MC38 model. Finally, LM-108 showed a synergistic therapeutic efficacy in PD-1 resistant models in combination with anti-PD-1 antibody. To conclude, LM-108 is a novel Fc-optimized CCR8 antibody that selectively depletes tumor infiltrating Tregs thereby improving anti-tumor immune response either as monotherapy or combination therapy. Thus, LM-108 can be a promising therapeutic approach to overcome ICI resistance in cancer patients. Citation Format: Jie Luo, Wentao Huang, Junwei Yang, Jin Li, Yifan Li, Da Fei, Xia Qin, Runsheng Li. Effective depletion of tumor-infiltrating Tregs by a novel anti-CCR8 antibody (LM-108): Addressing resistance associated with immune checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6008.

Abstract 2869: ADG138, a novel HER2×CD3 POWERbody™ integrating bispecific TCE with precision masking to control cytokine release syndrome and on-target off-tumor toxicity for single agent and combination therapies in HER2-expressing solid tumors

Abstract Bispecific T-cell engagers (TCEs) have shown therapeutic promise in treating hematologic cancers. However, their utility in treating solid tumors has yet to be effectively demonstrated due to on-target off-tumor toxicity and systemic cytokine release syndrome (CRS). To explore the full potential of TCEs as a powerful modality of immunotherapy for solid tumors, we developed ADG138, a novel HER2×CD3 POWERbody™ TCE by applying our SAFEbody® precision masking technology to double mask the binding interfaces in both ADG138 arms using covalently linked designer peptides in order to limit on-target off-tumor toxicities in normal tissues and to avert systemic CRS. In the tumor microenvironment (TME), the double masked ADG138 prodrug can be activated for concomitant in situ binding to the targeted cancer cells by the HER2 arm and to T cells by the CD3 arm to selectively enhance T cell responses against tumor cells specifically. Nonclinical studies demonstrated that when activated, ADG138 was highly potent in inducing T cell mediated cytotoxicity of both high and low HER2-expressing tumor cells in vitro, whereas in its double masked prodrug form ADG138 exhibited high masking efficiency with greatly reduced HER2 binding, T cell activation, and T cell mediated cytotoxicity. In in vivo human PBMC-engrafted tumor xenograft models, ADG138 induced robust tumor regression of both high and low HER2-expressing tumors in mice. Furthermore, combination of ADG138 with our species cross-reactive immuno-oncology therapeutics mediated strong synergistic antitumor activities in HER2 positive syngeneic solid tumor models in human CD3e knock-in mice. The double masked ADG138 prodrug also exhibited excellent safety profiles and normal pharmacokinetics in exploratory toxicology studies in cynomolgus monkeys, achieving over a 300-fold higher tolerated dose in striking contrast with its parental TCE that lacks masking. These nonclinical studies demonstrated that ADG138, a novel double masked HER2×CD3 POWERbody TCE, exhibited an impressively high therapeutic index relative to its parental non-masked TCE in HER2 high and low expressing solid tumors. This provides strong support for advancing the ADG138 prodrug into clinical development for treating HER2-expressing solid tumors as a single agent and in combination with other immune stimulatory agents. Citation Format: Guizhong Liu, Yan Li, Zhengxi Dai, Jiagui Qu, Jianfeng Shi, Qi Zhao, Linhai Qu, Aaron N. Nguyen, Songmao Zheng, Jiangchun Xu, Felix Du, Peter Luo. ADG138, a novel HER2×CD3 POWERbody™ integrating bispecific TCE with precision masking to control cytokine release syndrome and on-target off-tumor toxicity for single agent and combination therapies in HER2-expressing solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2869.

Abstract 2583: Utilization of a novel double knock-in humanized mouse model to evaluate the efficacy of ipilimumab and pembrolizumab against EMT-6 mammary carcinoma tumors

Abstract The cancer immunotherapy field has seen an increasing demand for identifying and characterizing pre-clinical models to evaluate the efficacy of the checkpoint inhibitors such as, ipilimumab and pembrolizumab. PBMC- or CD34-humanized mouse models are invaluable tools to assess effector functions of human T-cells to immune check point inhibitors (ICI). However, these models offer limited understanding of the overall potential therapeutic activity as they lack a complete repertoire of human immune cells required to elicit a full immune response. Thus, the development of multigenic humanized knock-in models provide a unique approach to evaluate the efficacy of ICI specific for human cancer targets in a fully immunocompetent organism. Here we describe the response of the EMT-6 murine breast cancer tumor model to ipilimumab and pembrolizumab in a novel hPDCD1/hCTLA4-KI double knock-in humanized BALB/c mouse model. EMT-6 tumors were implanted subcutaneously in the flank of hPDCD1/hCTLA4-KI mice. Three days post-implant we initiated treatment with ipilimumab or pembrolizumab alone and in combination. We observed significant ipilimumab anti-tumor activity, whereas pembrolizumab did not show efficacy despite effective T-cell PD-1 blockade. Unexpectedly, sudden respiratory distress occurred in animals receiving the 4th dose of combination therapy and the treatment was discontinued. To assess the immune phenotypic profiles in response to ipilimumab and/or pembrolizumab therapies, we performed multi-parameter flow cytometry analysis of peripheral blood, spleens and tumors. Additionally, we performed histopathological evaluation of lung tissues to assess potential neutrophil infiltration-mediated anaphylaxis in the mice treated with the combination treatment. The preliminary results highlight the value of the hPDCD1/hCTLA4-KI double knock-in humanized Balb/c model as a tool to evaluate human-specific immune-checkpoint based modalities. These models represent some of the difficulties faced in treating refractory tumors as well as reproducing some of the adverse effects seen in the clinic. Citation Format: Murray Stackhouse, Mariah Kuta, Joseph Kolb, Sydney Scatigno, Amber Blackwell, Chassidy Hall, Christopher Dowdy, Payel Sil, Diana Gietl, Steve Festin, Paula L. Miliani De Marval. Utilization of a novel double knock-in humanized mouse model to evaluate the efficacy of ipilimumab and pembrolizumab against EMT-6 mammary carcinoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2583.

Blockade of mIL-6R alleviated lipopolysaccharide-induced systemic inflammatory response syndrome by suppressing NF-κB-mediated Ccl2 expression and inflammasome activation.

Systemic inflammatory response syndrome (SIRS) is characterized by dysregulated cytokine release, immune responses and is associated with organ dysfunction. IL‐6R blockade indicates promising therapeutic effects in cytokine release storm but still remains unknown in SIRS. To address the issue, we generated the human il‐6r knock‐in mice and a defined epitope murine anti‐human membrane‐bound IL‐6R (mIL‐6R) mAb named h‐mIL‐6R mAb. We found that the h‐mIL‐6R and the commercial IL‐6R mAb Tocilizumab significantly improved the survival rate, reduced the levels of TNF‐α, IL‐6, IL‐1β, IFN‐γ, transaminases and blood urea nitrogen of LPS‐induced SIRS mice. Besides, the h‐mIL‐6R mAb could also dramatically reduce the levels of inflammatory cytokines in LPS‐treated THP‐1 cells in vitro. RNA‐seq analysis indicated that the h‐mIL‐6R mAb could regulate LPS‐induced activation of NF‐κB/Ccl2 and NOD‐like receptor signaling pathways. Furthermore, we found that the h‐mIL‐6R mAb could forwardly inhibit Ccl2 expression and NLRP3‐mediated pyroptosis by suppressing NF‐κB in combination with the NF‐κB inhibitor. Collectively, mIL‐6R mAbs suppressed NF‐κB/Ccl2 signaling and inflammasome activation. IL‐6R mAbs are potential alternative therapeutics for suppressing excessive cytokine release, over‐activated inflammatory responses and alleviating organ injuries in SIRS.

Abstract P1-08-05: Expression of the human APOE4 genotype modulates Doxorubicin cardiotoxicity in mice

Abstract Background: The human apolipoprotein E (APOE) gene has three major allelic variants: APOE2, -E3 and -E4. Carriers of the E4 allele are at increased risk for Alzheimer’s and other age-related neurodegenerative disorders. Recently, APOE4 has been associated with risk for cancer-related cognitive decline following chemotherapy with Doxorubicin (DOX), a frequent component of adjuvant therapy for breast cancer. To date, the mechanisms for APOE4 neurotoxicity are unknown, but may greater vulnerability to oxidative stress. DOX also causes dose-dependent cardiotoxicity, but a role for APOE4 in heart damage has not been previously demonstrated. Objective: To determine whether APOE4 predisposes to DOX-induced damage to the myocardium. Methods: C57Bl/6 mice (5-7 mo and 14-18 mo, male and female) with human APOE3 or APOE4 homozygous knock-in (=hAPOE3 and hAPOE4, respectively) were subjected to a single IP injection of saline (Control) or DOX (10 mg/kg, or 5 weekly injections of 5 mg/kg, =DOX), and monitored between 3-45 days afterward. Left ventricular systolic function was quantitated using echocardiography (Vevo 3100) at baseline, during and at the end of each study. TUNEL assay was used to identify apoptotic cells. Collagen was imaged using Masson’s Trichrome staining. Myocardial protein and RNA were extracted from the left ventricle and was analyzed by immunoblotting, realtime PCR and RNASeq on an Illumina NexGen platform.Results: Aged female APOE4 mice had more myocardial collagen at baseline (8.93±1.23 vs. 6.06±1.44%). Baseline cardiac function were similar at baseline in the 2 mouse strains. Three days after DOX, apoptotic myocytes were more abundant in hAPOE4 vs hAPOE3 hearts (1.2 vs. 4.6 TUNEL+ cells per section, n= 5, p<0.05). After DOX treatment, hAPOE4 mice had greater declines in left ventricular ejection fraction (EF, 46.9 vs 62.1%, p = hAPOE4 vs hAPOE3, p < 0.001). Quantitative PCR, RNASeq and protein studies revealed multiple differences in the cardiac transcriptome between hAPOE3 and hAPOE4 mice both at baseline and after DOX treatment. Baseline expression of MEF2D, a key driver of the myocardial stress response, was increased in hAPOE4 vs APOE3, driven by a 3500x (p = 4.76E-17) in transcript variant 1. Robust differences were seen in expression of c-Jun, JunB, and myosin heavy chain genes Myh6 and Myh7 at baseline and following DOX treatment at day 3. Growth Arrest And DNA Damage Inducible Alpha(GADD45A) gene expression was ~2x higher in hAPOE4 mice at baseline, but was similar to hAPOE3 mice by day 14 after DOX. Protein levels of caspase-1, an essential regulator of inflammation and the innate immune response, increased with age in hAPOE3 mice. In contrast, levels of casp-1 were lower in hAPOE4 and did not increase with age (p= 0.002 for APOE4 vs APOE3 at 17 mo). Conclusions: These findings support a model in which the APOE4 allele increases vulnerability to DOX-induced cardiac damage through mechanisms that involve significant and complex alterations in the response to oxidative stress and/or damage signaling. Studies are ongoing to further elucidate transcriptional differences conferred by APOE4, to analyze substrates downstream of caspase-1 affected by APOE4, and to determine the extent to which APOE4 may contribute to DOX toxicity in patient populations. Citation Format: Guannan Wang, G. William Rebeck, Olga Rodriguez, Marc E. Lippman, Nanette H. Bishopric. Expression of the human APOE4 genotype modulates Doxorubicin cardiotoxicity in mice [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-05.

ERBB3-dependent AKT and ERK pathways are essential for atrioventricular cushion development in mouse embryos.

ERBB family members and their ligands play an essential role in embryonic heart development and adult heart physiology. Among them, ERBB3 is a binding partner of ERBB2; the ERBB2/3 complex mediates downstream signaling for cell proliferation. ERBB3 has seven consensus binding sites to the p85 regulatory subunit of PI3K, which activates the downstream AKT pathway, leading to the proliferation of various cells. This study generated a human ERBB3 knock-in mouse expressing a mutant ERBB3 whose seven YXXM p85 binding sites were replaced with YXXA. Erbb3 knock-in embryos exhibited lethality between E12.5 to E13.5, and showed a decrease in mesenchymal cell numbers and density in AV cushions. We determined that the proliferation of mesenchymal cells in the atrioventricular (AV) cushion in Erbb3 knock-in mutant embryos was temporarily reduced due to the decrease of AKT and ERK1/2 phosphorylation. Overall, our results suggest that AKT/ERK activation by the ERBB3-dependent PI3K signaling is crucial for AV cushion morphogenesis during embryonic heart development.

Abstract 497: Human CD73 knock-in mice facilitate evaluation of in vivo efficacy of anti-human CD73 cancer immunotherapies

Abstract In recent years, immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment. However, most patients with solid cancers do not respond to the current ICIs against PD-1/PD-L1 or CTLA-4 alone. Stimulating an effective anti-tumor immune response may require countering additional immunosuppressive pathways in the tumor microenvironment (TME). Adenosinergic pathways are critical to maintain normal immune system homeostasis, and tumors could exploit them to avoid immune attack. CD73 (NT5E) is a surface enzyme that catalyzes the final hydrolysis of AMP to adenosine, which impairs immune function through adenosine receptors like A2AR (see our poster on humanized A2AR mice) on immune cells. Some tumors overexpress CD73 driven by hypoxia in the TME, which associates with poor prognosis in some cases. It is also expressed on the surface of multiple immune cells that infiltrate tumors, including T-cells, B-cells, NK cells, macrophages, and myeloid-derived suppressor cells. Several anti-CD73 antibodies have started early stage clinical trials as a treatment for advanced cancers. Combined anti-CD73, anti-PD-1, and anti-CTLA4 therapies synergize to reduce tumor burden and extend survival. Thus, there is a need for pre-clinical models to evaluate the safety and efficacy of CD73-targeted therapeutics before they enter human trials. Biocytogen has generated a human CD73 knock-in (KI) mouse (B-hCD73) and a human CD73-expressing MC38 cell line (MC38-hCD73) for both in vitro functional validation and in vivo efficacy evaluation of CD73-specific antibodies. Human CD73 (hCD73) whole coding sequence was inserted following exon 2 of the mouse Cd73 gene. B-hCD73 mice did not exhibit any immune or developmental abnormalities compared to wild-type mice, but splenic Tregs of B-hCD73 mice and not wild-type expressed hCD73. Anti-hCD73 antibodies bound well only to splenocytes of hCD73 KI mice and also blocked the enzyme activity of CD73. Using our MC38-hCD73 tumor model, we showed robust efficacy of anti-hCD73 antibodies in inhibiting tumor growth in vivo. Furthermore, in PD-1/PD-L1/CD73 triple KI humanized mice implanted with MC38 tumors, combined CD73 and PD-1/PD-L1 blockade showed greater tumor inhibition than monotherapies. Taken together, CD73 humanized mice and their variants are useful tools for in vivo efficacy evaluation of candidate human CD73 antibodies and combination therapies being considered for clinical trials. Citation Format: Chengzhang Shang, Huilin Li, Jing Zhang, Eugene Lin, Luke (Zhaoxue) Yu, Yuelei Shen. Human CD73 knock-in mice facilitate evaluation of in vivo efficacy of anti-human CD73 cancer immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 497.

Abstract 529: Characterization of a humanized TLR8 mouse model and its application in immuno-oncology drug development

Abstract Background: TLR8 belongs to the Toll-like receptor family, serving as endosomal sensors of uridine rich single strand viral or endogenous RNA and several types of small molecule agonist. In humans, TLR8 is primarily expressed on monocytes, macrophages and myeloid dendritic cells (DCs), while in murine it was originally reported that TLR8 has no function due to the lack of five amino acids that recognize the endogenous RNA ligand. Increasing evidence suggests that mouse TLR8 may function differently from its human counterpart, including ligand binding and signaling through pathways other than NK-κB. Therefore, a humanized TLR8 mouse model is pivotal for mechanistically understanding TLR8 innate immune regulation and pharmacologically targeting TLR8 in various malignancies and inflammatory diseases. Methods: Exon 3 of mouse Tlr8 was replaced with the corresponding human region to generate humanized TLR8 knock-in mice. hTLR8+/- mice (n=5) and age matched naïve C57BL/6 mice were maintained until 27 weeks of age. Liver, spleen, kidney and pancreas were collected for pathological characterization. Serum cytokines levels were analyzed by MSD, and hTLR8 expression pattern and immune cells were analyzed in the spleen by flow cytometry. Comparisons of anti-PD-1 response in TLR8 knock-in mice and naïve C57BL/6 mice bearing various syngeneic tumors were also conducted. Results: We systemically characterized the phenotypic changes between age-matched 27-week-old C57BL/6 and hTLR8 mice. A high percentage of hTLR8 expression was detected on CD11b+ F4/80+ macrophages, CD11b+Gr-1+ myeloid cells and CD11c+ DCs. Hepatomegaly, splenomegaly, and nephromegaly were observed in aged hTLR8 mice. Histology analysis revealed a prominent portal inflammation with fibrosis and bile duct hyperplasia in liver; a decreased cellularity in periarteriolar lymphoid sheath and increase of histiocytes in the red pulp of spleen; and inflammatory immune cell infiltration in the kidneys. Analysis of serum cytokines showed a significant increase of KC/GRO (CXLCL1), TNF-α and IL-10 in the peripheral blood of the aged hTLR8 mice. PBMC from hTLR8 mice responded to a specific RNA-ligand based human TLR8 agonist (ORN-8L) in vitro. We are now profiling different syngeneic and genetically engineered murine tumor models to fully characterize the potential adjuvant-like function of hTLR8 in different tumor models. Conclusion: Expression of humanized TLR8 leads to auto-activation of TLR8 signaling in mice. This pivotal model system enables us to study the function of human TLR8 and pharmacology of its agonists and antagonists in vivo. These knock-in mice may also serve as an interesting host strain for syngeneic tumor studies, potentiating immune responses to therapies. Citation Format: Hongjuan Zhang, Ruilin Sun, Annie X. An, Henry Q.x Li, Davy Xuesong Ouyang. Characterization of a humanized TLR8 mouse model and its application in immuno-oncology drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 529.