A CD6-Targeted Antibody-Drug Conjugate As a Potential Therapy for T Cell-Mediated Disorders

INTRODUCTION Pathogenic T cells cause many diseases including most autoimmune diseases and graft-versus-host disease (GVHD). Selectively targeting these pathogenic T cells while sparing the normal T cells and other tissues is a "holy grail" of therapeutics development in modern clinical immunology. So far, pan-immunosuppressive drugs such as corticosteroids are used to treat these patients, with limited efficacy and severe adverse effects. It is also well-established that these pathogenic T cells, whether auto- or alloreactive, proliferate following antigen recognition to cause tissue damage while the other normal T cells remain quiescent. Selectively targeting the proliferating T cells could be an effective strategy to develop new drugs for diseases mediated by the pathogenic T cells. Antibody-drug conjugates (ADCs) are developed by conjugating a potent toxin onto a monoclonal antibody (mAb) specific for a cancer cell surface antigen. Monomethyl auristatin E (MMAE), a synthetic mitotic toxin, is the payload in several FDA-approved ADCs, and it kills the actively dividing cancer cells by blocking the polymerization of tubulin, rapidly inducing apoptosis. These cancer cells and pathogenic T cells have one feature in common-both of them are actively proliferating, thus, this ADC approach proven successful in cancer treatment could be re-purposed to selectively kill pathogenic T cells for the treatment of T cell-mediated diseases. CD6 is a cell surface glycoprotein that is expressed at high levels on all T cells except Treg cells, a small portion of B cells and many human NK cells. CD6 is not detectable on other tissue cells, making it a highly specific target candidate for T cells. We have generated CD6 knockout mice and CD6 humanized mice, and developed anti-CD6 mAbs that treat mouse models T cell-mediated diseases. A CD6-targeted ADC (CD6-ADC) thus might be effective for treating T cell-mediated diseases by selectively eliminating the proliferating pathogenic T cells. METHODS A CD6-ADC was developed by conjugating a latent form of MMAE onto the high-affinity anti-human CD6 mAb. Its potency of selectively killing pathogenic T cells was evaluated in an antigen-specific T cell recall assay with BrdU-incorporation followed by flow cytometric analyses. To determine the toxicity of the CD6-ADC on normal quiescent T cells, naïve CD6 humanized mice were treated with the CD6-ADC or non-binding control ADC (0.5 mg/kg) by intraperitoneal injection, then numbers of circulating T cells were monitored by flow cytometry daily. To evaluate the efficacy of CD6-ADC in treating T cell-mediated autoimmune diseases, the same dose (0.5 mg/kg) of CD6-ADC, or the parental anti-CD6 mAb, or the control ADC was used to treat a model of autoimmune uveitis, and disease severities were assessed by various ocular imaging techniques including indirect ophthalmoscopy, confocal scanning laser ophthalmoscopy and optical coherence tomography. To examine the potential of the CD6-ADC in treating GVHD, the same dose (0.5mg/kg) of CD6-ADC or control ADC was tested in a model of GVHD induced in NSG mice after adoptive transfer of human PBMC. GVHD severities were assessed by flow cytometric analyses of circulating human T cells and by histopathological analyses of different tissues. RESULTS: The CD6-ADC selectively killed antigen-specific proliferating T cells in vitro. Treating naive CD6-humanized mice with this CD6-ADC (0.5 mg/kg) did not significantly eliminate normal T cells in vivo. Furthermore, systemic delivery of the same dose (0.5 mg/kg) of CD6-ADC, but not the anti-CD6 mAb alone nor the control IgG effectively reduced retinal inflammation in a preclinical model of autoimmune uveitis. The same dose of CD6-ADC, but not the control ADC, also effectively depleted activated xenogeneic T cells and prevented the development of GVHD in NSG mice after the adoptive transfer of human PBMC. CONCLUSION: These data indicate that this CD6-ADC holds promise as a new drug for treating diseases in which T cells are integrally involved in the pathogeneses such as GVHD. Figure 1 Figure 1. Disclosures Lin: Takeda Pharma: Consultancy, Research Funding.

792 Creating an immune-favorable tumor microenvironment by a novel anti-CD39/TGFβ-Trap bispecific antibody

BackgroundAdenosine and TGFβ are two key immune suppressors in tumor microenvironment (“TME”) that cause broad immune suppression resulting in resistance to current CPI immunotherapies. Cancers frequently express transforming growth factor-β (TGFβ), which drives immune dysfunction in the tumor microenvironment by inducing regulatory T cells (Tregs), inhibiting CD8+ activation and infiltration into TME, and promoting epithelial–mesenchymal transition (EMT). We observed that TGFβ induces the expression of CD39, a critical enzyme that regulates adenosine generation. CD39 is highly expressed in Tregs within TME, it drives the production of adenosine, an immunoinhibitory molecule that partly mediates Treg inhibitory function. To inhibit CD39-Adenosine and TGFβ simultaneously to create an immune favorable tumor microenvironment, we designed a bi-specific antibody targeting both CD39 and TGFβ (ES014), which aims to inhibit the generation of adenosine and iTreg in TME. The immuno-stimulating effect of ES014 was demonstrated in a PD-1-unresponsive mouse model where tumor growth was significantly inhibited after the treatment of the bi-specific antibody.MethodsThe bifunctional antibody–ligand trap ES014 was created by fusing the TGFβ receptor II ectodomain to an antibody targeting CD39. ES014 molecule could simultaneously inhibit CD39 enzymatic function to prevent extracellular ATP from degradation and neutralize autocrine/paracrine TGFβ in the target cell microenvironment. The immunological function of ES014 was studied in an in vitro Elpiscience proprietary ImmunoShine platform which includes T cell activation and apoptosis assay, iTreg differentiation and suppression assay, NK cell activation assay and DC maturation activity. The in vivo efficacy of ES014 was investigated in a human PBMC engrafted cancer model.ResultsWe demonstrated that ES014 bispecific antibody can inhibit CD39 enzymatic activity and neutralizes TGFβ-induced effect, resulting in greater T cell activation and suppression of Treg differentiation. Interestingly, we found ES014 molecule demonstrated a unique mechanism by significantly protecting effector T cell from anti-Fas induced apoptosis or activation induced cell death (AICD) that is not observed in monotherapy or combo treatment. The ES014 molecule is more effective in inhibiting tumor growth as compared with anti-CD39 antibody or TGFβ-trap in a human PBMC engrafted in vivo model.ConclusionsWe find that by simultaneously targeting CD39 and TGFβ by a novel bi-specific molecule ES014, a more immune-favorable TME and synergistic anti-tumor effects can be achieved. Our pre-clinical data demonstrate that ES014 counteracts TGFβ-mediated inhibitory effect and adenosine induced immune tolerance and has a unique ability to protect T cell from apoptosis. ES014 demonstrated strong efficacy in in vivo tumor growth inhibition.

607 Regional delivery of a TLR9 agonist to boost checkpoint inhibitor responsiveness in liver metastases

BackgroundClass C TLR9 agonists, CpG oligodeoxynucleotides (ODNs) enhance responsiveness to anti-PD1 therapy in solid tumors through favorable modulation of the tumor microenvironment (TME) [1]. Recently, we reported that regional delivery of a TLR9 agonist eliminated myeloid derived suppressor cells (MDSC) and promoted pro-inflammatory/anti-tumorigenic M1 macrophage programming in the TME of liver metastases (LM) [2]. Further, we found enhanced TLR9 activation in LM following regional TLR9 agonist infusion compared to the systemic treatment. We hypothesize that regional delivery of a TLR9A into LM will enhance the responsiveness to systemically infused anti-PD1 therapy.MethodsIn this study, we treated mice with established MC38-CEA-Luc LM with ODN-2395 (30µg/mouse) regionally with or without anti PD-1 antibody (250µg/mouse) intraperitoneally.ResultsControl of LM growth (Figure 1) was significantly higher with combinatorial treatment as compared to anti-PD1 (p<0.01) or PBS treatments (p<0.05). To study the impact of TLR9 activation on human MDSC, we treated healthy donor PBMCs with ODN-2395 or SD101. We found that both reduced the hu-MDSC (CD11b+CD33+HLADR-) population in a dose-dependent manner with an increase in PD-L1 expression as determined by flow cytometry (FC) analysis (Figure 2). Moreover, by using Luminex, demonstrated that ODN-2395 and SD101 enhanced expression of IL 29, IFNα, and NFκB, along with downstream cytokines IL 6 and IL 10. To investigate the effect of SD101 in modulating the differentiation of huMDSC from huPBMC, we treated huPBMC with IL6+GM-CSF in the presence or absence of SD101. By FC analysis, we found that SD-101 blocked huMDSC development induced by IL6+GM-CSF, preferentially limited the more immunosuppressive monocytic MDSC subtype, and drove M1 macrophage polarization. Treatment of SD101 only once for 48hrs was sufficient to inhibit huMDSC differentiation for two weeks.Abstract 607 Figure 1Combinatorial treatment of CPI and ODN’s reduces tPV = portal vein; IP = intraperitoneal.Eight to twelve weeks old male C57/BL6 mice were challenged intra-splenic with 0.5e6 MC38-CEA-Luc cells for a week. Bioluminescence value was determined by IVIS on D0, and mice were randomized accordingly and treated with 30 µg/mouse ODN2395 via PV with or without 250 µg/mouse anti-PD1 antibody via IP on D0, D+3 and D+6. PBS served as the vehicle (Veh) control and administered via PV. Fold change of the tumor burden was calculated based on D0 baseline bioluminescence. Tumor progression was analyzed unpaired t test among groups. (*p <0.05).Abstract 607 Figure 2Human PBMC treated with ODN2395 and SD101 reducesCtrl = control; MDSC = ODN = oligodeoxynucleotide’ PBMC = peripheral blood monocytes.Human PBMC were isolated from the Leukoreduction system chamber. 1e6/ml PBMCs were treated with increasing concentrations (0.04–10 µM) SD101, ODN2395 along with ctrl ODN5328 (1µM) for 48 hours. Panels A and B: MDSC population and their corresponding PD–L1 expression were evaluated (n=12). Four donors with three replicates were used. Data represented as mean ± SEM.ConclusionsBoth the in vitro and in vivo findings suggest that regional TLR9 stimulation in a model of LM improves responsiveness to systemic anti-PD-1 therapy through elimination of MDSC, and the effect on huMDSC was confirmed in vitro. Increased PDL-1 expression in response to TLR9 stimulation among MDSC may further enhance the anti-PD-1 effect. Therefore, combing regional infusions of a TLR9 agonist with systemic anti-PD-1 agents may be a promising approach for liver tumor treatment.ReferencesWang, S., et al., Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A, 2016. 113(46): p. E7240-E7249.Ghosh CC, H.K., O’Connell K, Laporte J, Guha P, Cox B, Jaroch D, Katz SC, Regional administration of class C CpG Oligodeoxynucleotides results in superior intrahepatic TLR9 activation and immunomodulation compared to systemic infusion, Abstract: AACR Annual Meeting. 2021.

873 S-531011, a novel anti-human CCR8 antibody: anti-tumor responses through depletion of tumor-infiltrating CCR8-positive Tregs

BackgroundRegulatory T cells (Tregs) are suppressive immune cells required for the maintenance of immune homeostasis, but tumor-infiltrating Tregs are known to suppress the antitumor immune system and promote tumor progression. Therefore, selective reduction of tumor-infiltrating Tregs is anticipated to reinvigorate antitumor immunity without inducing autoimmunity. S-531011 is a novel anti-human IgG1 antibody targeting human CCR8 (C-C motif chemokine receptor 8) which is selectively expressed in tumor-infiltrating Tregs, with both in vitro antibody dependent cellular cytotoxicity (ADCC) against CCR8-expressing cells and neutralizing activity against CCL1-CCR8 signaling. Here, to evaluate antitumor activities and safety aspects of S-531011, we conducted non-clinical pharmacology studies of S-531011 using human CCR8 knock-in (KI) mice and human tissues.MethodsS-531011 was administrated to CT26WT tumor-bearing hCCR8-KI mice, and the effect on the presence of tumor-infiltrating CCR8+ Treg and tumor growth were evaluated. We also investigated the antitumor efficacy of S-531011 in combination with anti-mouse PD-1 antibody. Next, human lung cancer tissues and human NK-cells were co-cultured, and the ex vivo ADCC against tumor-infiltrating Tregs by S-531011 was verified. We also incubated human peripheral blood-derived mononuclear cells (PBMC) from healthy individuals with S-531011 to investigate the effects on the proportion of Tregs in human PBMC.ResultsIntravenous administration of S-531011 to CT26WT tumor-bearing hCCR8-KI mice significantly reduced tumor-infiltrating CCR8+ Tregs and markedly suppressed tumor growth. Furthermore, the combined therapy of S-531011 with anti-mouse PD-1 antibody showed greater anti-tumor effect than monotherapy without any apparent side effects. Ex vivo ADCC studies using human lung cancer tissues and FCM analysis of CCR8 expression in tumor-infiltrating Tregs suggested that most of the tumor-infiltrating CCR8+ Tregs were depleted by S-531011. On the other hand, S-531011 didn’t reduce Tregs in human PBMC.ConclusionsS-531011 is a promising drug which has a strong antitumor effect by depleting tumor-infiltrating CCR8+ Tregs, as a not only monotherapy but also combination therapy with other immune checkpoint inhibitors.Ethics ApprovalThe present study was approved by the Institutional Ethics Committee of Osaka University Hospital (approved number: 13266-15) and Shionogi Co., Ltd. (approved number: 021-003). Animal studies were approved by the Institutional Animal Care and Use Committee (approved number: S20093D, S20197D and S20198D).

Use of human PBMC to analyse the impact of obesity on lipid metabolism and metabolic status: a proof-of-concept pilot study

Peripheral blood mononuclear cells (PBMC) are widely used as a biomarker source in nutrition/obesity studies because they reflect gene expression profiles of internal tissues. In this pilot proof-of-concept study we analysed in humans if, as we previously suggested in rodents, PBMC could be a surrogate tissue to study overweight/obesity impact on lipid metabolism. Pre-selected key lipid metabolism genes based in our previous preclinical studies were analysed in PBMC of normoglycemic normal-weight (NW), and overweight-obese (OW-OB) subjects before and after a 6-month weight-loss plan. PBMC mRNA levels of CPT1A, FASN and SREBP-1c increased in the OW-OB group, according with what described in liver and adipose tissue of humans with obesity. This altered expression pattern was related to increased adiposity and early signs of metabolic impairment. Greater weight loss and/or metabolic improvement as result of the intervention was related to lower CPT1A, FASN and SREBP-1c gene expression in an adjusted linear mixed-effects regression analysis, although no gene expression recovery was observed when considering mean comparisons. Thus, human PBMC reflect lipid metabolism expression profile of energy homeostatic tissues, and early obesity-related alterations in metabolic at-risk subjects. Further studies are needed to understand PBMC usefulness for analysis of metabolic recovery in weigh management programs.

Defining stable reference genes in HIV latency reversal experiments

Quantification of cell associated HIV RNA (ca-RNA) is one of the most important and commonly used methods to evaluate the performance of latency-reversing agents (LRAs). Copies of HIV RNA measured by qPCR, are often normalized to the input RNA or cell number. However, these could be affected by biological variability and/or technical errors, which can be avoided by using an internal reference gene. To obtain reliable data, it is essential to select stable reference genes (RGs) of which the expression is not influenced by biological variability, the type of cells, or the LRAs used. However, to date, no study has carefully evaluated RG stability following LRA exposure. We analyzed the stability of six widely used RGs (GAPDH, TBP, YWHAZ, UBE2D2, HPRT1 and RPL27A) in human PBMC and CD4+ T cells. LRA exposure significantly influenced the stability of these RGs. Overall, TBP, UBE2D2, and RPL27A were the most stable RGs in all tested conditions. TBP was generally the most stable RG whereas GAPDH varied the most. Finally, we evaluated the impact of applying different RG normalizers to host genes and HIV ca-RNA data. Altered results were observed both in host and HIV gene expression when unstable RGs were used. Our data underline the importance of testing the stability of RGs utilized to evaluate LRA-induced HIV ca-RNA expression. To our knowledge, this is the first careful evaluation of the stability of RGs after LRA exposure and will significantly contribute to the quality of data analysis in regard to gene expression. IMPORTANCE Latency-reversing agents (LRAs) are ubiquitously used in the “shock-and-kill” HIV cure strategy and their performance is often evaluated by ex-vivo quantification of cell associated HIV RNA. HIV RNA, measured by qPCR, is often normalized to internal reference genes, but the expression of these genes should not be influenced by the experimental settings. We found that treatment of human PBMC and CD4+ T cells with LRAs significantly altered the expression of several commonly used reference genes, such as GAPDH. Finally, we evaluate the impact of different reference genes on normalization of host genes and HIV cell associated RNA expression and demonstrated that using unstable reference genes dramatically altered experimental outcome. Our data highlight the importance of using reference genes that are unaffected by LRAs under study to correctly evaluate host gene and cell associated HIV RNA expression induced by latency-reversing agents.