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Background Toll-like receptor (TLR) pathways play a crucial role in mounting potent innate immune responses against invading pathogens, as well as the subsequent engagement of adaptive immunity. Innate immune activation via the TLR9 pathway has potential for treating cancer as demonstrated clinically with TLR9 agonists administered intra-tumorally in melanoma patients.1 We developed a novel toll-like receptor agonist antibody conjugate (TRAAC) platform to systemically deliver a differentiated, targeted TLR9 agonist (T-CpG) for immune activation. The activation of TLR9 pathways can be directed systemically towards specific immune cell populations and tumor microenvironment via antibodies binding to various immune cell receptors. Using multiple TRAACs targeting either immune cells including plasmacytoid DCs (pDCs), myeloid and B lymphocytes, or tumor specific antigens, we evaluated immune modulatory phenotypes, therapeutic potentials, as well as safety and tolerability of this platform in pre-clinical settings. Methods TRAACs were generated using site-specific conjugation. In vitro activity of immune- and tumor-targeted antibody-CpG conjugates was evaluated using human PBMCs. Anti-tumor efficacy and mechanistic assessment of B lymphocyte and myeloid cell-targeted antibody-CpG conjugates were conducted in syngeneic tumor models. Pharmacokinetic (PK), pharmacodynamic and exploratory toxicity evaluations were performed in non-human primates (NHP). Results T-CpG is comprised of monomeric CpG-containing oligonucleotides optimized for potency and stability as an antibody conjugate. TRAACs targeting immune cells enable directed TLR9 activation leading to potent cytokine production and cellular activation that is superior to free CpG. This targeted immune activation also elicits a cascade of downstream modulation of non-targeted immune cells. When administrated systemically in multiple syngeneic models, murine TRAACs targeting either immune cells or tumor antigens exhibited potent, durable, and dose-dependent anti-tumor activity as a single agent and in combination with T-cell checkpoint inhibitors (CPIs). A single peripheral dose of either B lymphocyte or myeloid targeted-CpG evoked both innate and adaptive immune responses within the tumor microenvironment as demonstrated by NanoString analysis. The observed immunomodulatory phenotypes are consistent with those elicited by direct intra-tumoral CpG delivery. Following repeated intravenous doses in NHP, TRAACs demonstrated targeted receptor occupancy, antibody-like PK, and favorable tolerability profile. Conclusions Pre-clinical evaluation of a novel platform comprised of antibodies conjugated to a differentiated TLR9 agonist demonstrated targeted immune activation, potent anti-tumor activity as single agent and in combination with CPIs and favorable tolerability profiles in NHPs. Such antibody-CpG conjugates have the potential for clinical development as systemically delivered therapeutics providing powerful innate and adaptive anti-tumor immunity across multiple tumor types. Reference Hamid O, Ismail R, Puzanov I. Intratumoral Immunotherapy—Update 2019. The Oncol2019; 25:343–359.

The restricted expression pattern of B-cell maturation antigen (BCMA) makes it an ideal tumor-associated antigen (TAA) for the treatment of myeloma. BCMA has been targeted by both CD3 bispecific antibody and antibody–drug conjugate (ADC) modalities, but a true comparison of modalities has yet to be performed. Here we utilized a single BCMA antibody to develop and characterize both a CD3 bispecific and 2 ADC formats (cleavable and noncleavable) and compared activity both in vitro and in vivo with the aim of generating an optimal therapeutic. Antibody affinity, but not epitope was influential in drug activity and hence a high-affinity BCMA antibody was selected. Both the bispecific and ADCs were potent in vitro and in vivo, causing dose-dependent cell killing of myeloma cell lines and tumor regression in orthotopic myeloma xenograft models. Primary patient cells were effectively lysed by both CD3 bispecific and ADCs, with the bispecific demonstrating improved potency, maximal cell killing, and consistency across patients. Safety was evaluated in cynomolgus monkey toxicity studies and both modalities were active based on on-target elimination of B lineage cells. Distinct nonclinical toxicity profiles were seen for the bispecific and ADC modalities. When taken together, results from this comparison of BCMA CD3 bispecific and ADC modalities suggest better efficacy and an improved toxicity profile might be achieved with the bispecific modality. This led to the advancement of a bispecific candidate into phase I clinical trials.

Targeting the CD47-SIRPα myeloid checkpoint pathway represents a novel therapeutic approach to enhance anti-cancer immunity. CD47 is a widely expressed cell surface protein that functions as a marker of self. Signal regulatory protein-α (SIRPα) is an ITIM-containing inhibitory receptor of CD47 on myeloid cells. Tumor cells up-regulate CD47 expression to evade immune surveillance. Clinical trials targeting CD47 to inhibit SIRPα interaction have shown promising results (N Lakhani et al, 2018 SITC #P335). Unlike CD47, which is expressed ubiquitously, SIRPα has limited expression, mainly on myeloid cells including macrophages, dendritic cells, and neutrophils, and neurons. Targeting SIRPα circumvents the need to overcome the CD47 sink that is necessary for CD47 targeting molecules. Antibodies to SIRPα that bind mouse, human and cynomolgus monkey SIRPα with high affinity, were evaluated for their effects on macrophage function, anti-tumor activity and normal blood cells. Humanized antibody to SIRPα was generated to bind both human SIRPα allele V1 and V2 with high affinity (pM) and cross-react with cynomolgus and mouse SIRPα. Anti-SIRPα antibody in combination with targeted anti-cancer antibodies enhanced antibody-dependent cellular phagocytosis (ADCP) of tumor cell lines by human monocyte derived macrophages. In preclinical xenograft models of SIRPα positive and negative malignant cell lines, administration of anti-SIRPα antibody enhanced anti-tumor activity of rituximab and cetuximab via ADCP. The impact of SIRPα blockade on anti-tumor immunity was assessed in immunocompetent tumor mouse models. SIRPα antibodies enhanced anti-tumor activity of checkpoint inhibitors in syngeneic tumor models. Combination therapy with SIRPα antibodies and checkpoint inhibitors in tumor models showed eradication of tumors, acquired memory immune response upon tumor rechallenge and decreased lung metastasis. The cellular mechanisms driving efficacy include dendritic cell activation, reduction of myeloid suppression and enhancement of T cell effector profile. Exploratory safety and PK/PD profiles of anti-SIRPα antibodies were assessed in cynomolgus monkeys. Antibodies to SIRPα demonstrated a typical antibody PK and complete SIRPα target occupancy. There were no adverse clinical observations, no changes in hematological parameters and no impact on serum chemistries. In summary, anti-SIRPα antibodies combined with targeted anti-cancer antibodies or checkpoint inhibitors integrate innate and adaptive immune responses to promote anti-tumor activity in preclinical models. Together with the favorable safety profile in cynomolgus monkeys, these data support the development of SIRPα antibodies for solid tumors and hematological malignancies. Citation Format: Tracy C. Kuo, Amy Chen, Ons Harrabi, Steven E. Kauder, Emma R. Sangalang, Laura Doyle, Sangeetha Bollini, Bora Han, Janet Sim, Jaume Pons, Hong I. Wan. Antibodies to Sirpα enhance innate and adaptive immune responses to promote anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 562.

Genetic variation in the IL-7 receptor-α ( IL-7R ) gene is associated with susceptibility to human type 1 diabetes (T1D). Here we investigate the therapeutic efficacy and mechanism of IL-7Rα antibody in a mouse model of T1D. IL-7Rα antibody induces durable, complete remission in newly onset diabetic mice after only two to three injections. IL-7 increases, whereas IL-7Rα antibody therapy reduces, the IFN-γ–producing CD4 + (T H 1) and IFN-γ–producing CD8 + T cells. Conversely, IL-7 decreases and IL-7Rα antibody enhances the inhibitory receptor Programmed Death 1 (PD-1) expression in the effector T cells. Programmed Death 1 blockade reversed the immune tolerance mediated by the IL-7Rα antibody therapy. Furthermore, IL-7Rα antibody therapy increases the frequency of regulatory T cells without affecting their suppressor activity. The durable efficacy and the multipronged tolerogenic mechanisms of IL-7Rα antibody therapy suggest a unique disease-modifying approach to T1D.

Site-specific conjugation improves therapeutic index of antibody drug conjugates with high drug loading

Vascular inflammation, infusion reactions, glomerulopathies, and other potentially adverse effects may be observed in laboratory animals, including monkeys, on toxicity studies of therapeutic monoclonal antibodies and recombinant human protein drugs. Histopathologic and immunohistochemical (IHC) evaluation suggests these effects may be mediated by deposition of immune complexes (ICs) containing the drug, endogenous immunoglobulin, and/or complement components in the affected tissues. ICs may be observed in glomerulus, blood vessels, synovium, lung, liver, skin, eye, choroid plexus, or other tissues or bound to neutrophils, monocytes/macrophages, or platelets. IC deposition may activate complement, kinin, and/or coagulation/fibrinolytic pathways and result in a systemic proinflammatory response. IC clearance is biphasic in humans and monkeys (first from plasma to liver and/or spleen, second from liver or spleen). IC deposition/clearance is affected by IC composition, immunomodulation, and/or complement activation. Case studies are presented from toxicity study monkeys or rats and indicate IHC-IC deposition patterns similar to those predicted by experimental studies of IC-mediated reactions to heterologous protein administration to monkeys and other species. The IHC-staining patterns are consistent with findings associated with generalized and localized IC-associated pathology in humans. However, manifestations of immunogenicity in preclinical species are generally not considered predictive to humans.

Two 6-month repeat-dose toxicity studies in cynomolgus monkeys illustrated immune complex–mediated adverse findings in individual monkeys and identified parameters that potentially signal the onset of immune complex–mediated reactions following administration of RN6G, a monoclonal antibody (mAb). In the first study, 3 monkeys exhibited nondose-dependent severe clinical signs accompanied by decreased erythrocytes with increased reticulocytes, neutrophilia, monocytosis, thrombocytopenia, coagulopathy, decreased albumin, azotemia, and increased serum levels of activated complement products, prompting unscheduled euthanasia. Histologically, immunohistochemical localization of RN6G was associated with monkey immunoglobulin and complement components in glomeruli and other tissues, attributable to immune complex disease (ICD). All 3 animals also had anti-RN6G antibodies and decreased plasma levels of RN6G. Subsequently, an investigational study was designed and conducted with regulatory agency input to detect early onset of ICD and assess reversibility to support further clinical development. Dosing of individual animals ceased when biomarkers of ICD indicated adverse findings. Of the 12 monkeys, 1 developed anti-RN6G antibodies and decreased RN6G exposure that preceded elevations in complement products, interleukin-6, and coagulation parameters and decreases in albumin and fibrinogen. All findings in this monkey, except for antidrug antibody (ADA), reversed after cessation of dosing without progressing to adverse sequelae typically associated with ICD.

Item does not contain fulltext The interleukin-7 receptor alpha chain (IL-7Ralpha) gene was identified as a top non-major histocompatibility complex-linked risk locus for multiple sclerosis (MS). Recently, we showed that a T helper 1 (T(H)1)-driven, but not a T(H)17-driven, form of MS exhibited a good clinical response to interferon-beta (IFN-beta) therapy. We now demonstrate that high serum levels of IL-7, particularly when paired with low levels of IL-17F, predict responsiveness to IFN-beta and hence a T(H)1-driven subtype of MS. We also show that although IL-7 signaling is neither necessary nor sufficient for the induction or expansion of T(H)17 cells, IL-7 can greatly enhance both human and mouse T(H)1 cell differentiation. IL-7 alone is sufficient to induce human T(H)1 differentiation in the absence of IL-12 or other cytokines. Furthermore, targeting IL-7/IL-7Ralpha is beneficial in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Mice treated with IL-7Ralpha-blocking antibodies before or after onset of paralysis exhibited reduced clinical signs of EAE, with reduction in peripheral naive and activated T cells, whereas central memory T, regulatory T, B, and natural killer cell populations were largely spared. IL-7Ralpha antibody treatment markedly reduced lymphocyte infiltration into the central nervous system in mice with EAE. Thus, a serum profile of high IL-7 may signify a T(H)1-driven form of MS and may predict outcome in MS patients undergoing IFN-beta therapy. Blockade of IL-7 and the IL-7Ralpha pathway may have therapeutic potential in MS and other autoimmune diseases.

Notch signaling is deregulated in T-cell acute lymphoblastic leukemia (T-ALL) and advanced solid tumors, making it an attractive target for oncology drug development. In this present report, we describe a novel mouse monoclonal antibody, Notch1 mAb, that specifically binds to the negative regulatory region (NRR) of human Notch1 receptor. Using a T-ALL cell line, HPB-ALL, that harbors mutations in Notch1 heterodimerization and PEST domains, we demonstrated that Notch1 mAb blocked Notch signaling by reduction of Notch1 intracellular domain (NICD) and down-regulation of Notch target genes, Hes-1 and cMyc. Notch1 mAb caused cell growth inhibition of HPB-ALL and several other T-ALL cell lines, via induction of cell cycle arrest and apoptosis. Notch1 mAb treatment resulted in robust NICD reduction and marked antitumor efficacy in HBP-ALL xenograft model. Additional mechanism-of-action studies revealed inhibition of tumor cell proliferation and induction of apoptosis in HPB-ALL tumors, suggesting that the anti-tumor activity of Notch1 mAb may be mediated by its direct effects on tumor cell growth or survival. Furthermore, this antibody led to a significant reduction in leukemia progenitor cells (LPCs) baring NOTCH1 mutation in bioluminescent humanized T-ALL LPC mouse models. In addition to its inhibitory effect on mutant Notch1, Notch1 mAb also displayed robust neutralization activity on wild type Notch1 and caused tumor growth inhibition in breast cancer models MDA-MB-231 and MX-1 by targeting the wild type receptor in these tumor types. Interestingly, using a gamma secretase inhibitor PF-03084014, we showed that Notch1 mAb and PF-03084014 elicited similar degree of biology responses in HPB-ALL cells. Our results indicate that antibodies that bind to NRR can act as potent inhibitors of Notch1 signaling and provide opportunities for development of novel cancer therapeutics for T-ALL and solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1765. doi:10.1158/1538-7445.AM2011-1765