Antibody Co-Administration Can Improve Systemic and Local Distribution of Antibody-Drug Conjugates to Increase In Vivo Efficacy.

Several antibody-drug conjugates (ADC) showing strong clinical responses in solid tumors target high expression antigens (HER2, TROP2, Nectin-4, and folate receptor alpha/FRα). Highly expressed tumor antigens often have significant low-level expression in normal tissues, resulting in the potential for target-mediated drug disposition (TMDD) and increased clearance. However, ADCs often do not cross-react with normal tissue in animal models used to test efficacy (typically mice), and the impact of ADC binding to normal tissue antigens on tumor response remains unclear. An antibody that cross-reacts with human and murine FRα was generated and tested in an animal model where the antibody/ADC bind both human tumor FRα and mouse FRα in normal tissue. Previous work has demonstrated that a "carrier" dose of unconjugated antibody can improve the tumor penetration of ADCs with high expression target-antigens. A carrier dose was employed to study the impact on cross-reactive ADC clearance, distribution, and efficacy. Co-administration of unconjugated anti-FRα antibody with the ADC-improved efficacy, even in low expression models where co-administration normally lowers efficacy. By reducing target-antigen-mediated clearance in normal tissue, the co-administered antibody increased systemic exposure, improved tumor tissue penetration, reduced target-antigen-mediated uptake in normal tissue, and increased ADC efficacy. However, payload potency and tumor antigen saturation are also critical to efficacy, as shown with reduced efficacy using too high of a carrier dose. The judicious use of higher antibody doses, either through lower DAR or carrier doses, can improve the therapeutic window by increasing efficacy while lowering target-mediated toxicity in normal tissue.

Synthesis and Evaluation of Camptothecin Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) that incorporate the exatecan derivative DXd in their payload are showing promising clinical results in solid tumor indications. The payload has an F-ring that also contains a second chiral center, both of which complicate its synthesis and derivatization. Here we report on new camptothecin-ADCs that do not have an F-ring in their payloads yet behave similarly to DXd-bearing conjugates in vitro and in vivo. This simplification allows easier derivatization of camptothecin A and B rings for structure-activity relationship studies and payload optimization. ADCs having different degrees of bystander killing and the ability to release hydroxyl or thiol-bearing metabolites following peptide linker cleavage were investigated.

Peptide-Cleavable Self-immolative Maytansinoid Antibody-Drug Conjugates Designed To Provide Improved Bystander Killing.

A new type of antibody-drug conjugate (ADC) has been prepared that contains a sulfur-bearing maytansinoid attached to an antibody via a highly stable tripeptide linker. Once internalized by cells, proteases in catabolic vesicles cleave the peptide of the ADC's linker causing self-immolation that releases a thiol-bearing metabolite, which is then S-methylated. Conjugates were prepared with peptide linkers containing only alanyl residues, which were all l isomers or had a single d residue in one of the three positions. A d-alanyl residue in the linker did not significantly impair a conjugate's cytotoxicity or bystander killing unless it was directly attached to the immolative moiety. Increasing the number of methylene units in the maytansinoid side chain of a conjugate did not typically affect an ADC's cytotoxicity to targeted cells but did increase bystander killing activity. ADCs with the highest in vitro bystander killing were then evaluated in vivo in mice, where they displayed improved efficacy compared to previously described types of maytansinoid conjugates.

Synthesis of Highly Potent N-10 Amino-Linked DNA-Alkylating Indolinobenzodiazepine Antibody-Drug Conjugates (ADCs).

Indolinobenzodiazepine DNA alkylators (IGNs) are the cytotoxic payloads in antibody-drug conjugates (ADCs) currently undergoing Phase I clinical evaluation (IMGN779, IMGN632, and TAK164). These ADCs possess linkers that have been incorporated into a central substituted phenyl spacer. Here, we present an alternative strategy for the IGNs, linking through a carbamate at the readily available N-10 amine present in the monoimine containing dimer. As a result, we have designed a series of N-10 linked IGN ADCs with a wide range of in vitro potency and tolerability, which may allow us to better match an IGN with a particular target based on the potential dosing needs.

A CD123-targeting antibody-drug conjugate, IMGN632, designed to eradicate AML while sparing normal bone marrow cells.

The outlook for patients with refractory/relapsed acute myeloid leukemia (AML) remains poor, with conventional chemotherapeutic treatments often associated with unacceptable toxicities, including severe infections due to profound myelosuppression. Thus there exists an urgent need for more effective agents to treat AML that confer high therapeutic indices and favorable tolerability profiles. Because of its high expression on leukemic blast and stem cells compared with normal hematopoietic stem cells and progenitors, CD123 has emerged as a rational candidate for molecularly targeted therapeutic approaches in this disease. Here we describe the development and preclinical characterization of a CD123-targeting antibody-drug conjugate (ADC), IMGN632, that comprises a novel humanized anti-CD123 antibody G4723A linked to a recently reported DNA mono-alkylating payload of the indolinobenzodiazepine pseudodimer (IGN) class of cytotoxic compounds. The activity of IMGN632 was compared with X-ADC, the ADC utilizing the G4723A antibody linked to a DNA crosslinking IGN payload. With low picomolar potency, both ADCs reduced viability in AML cell lines and patient-derived samples in culture, irrespective of their multidrug resistance or disease status. However, X-ADC exposure was >40-fold more cytotoxic to the normal myeloid progenitors than IMGN632. Of particular note, IMGN632 demonstrated potent activity in all AML samples at concentrations well below levels that impacted normal bone marrow progenitors, suggesting the potential for efficacy in AML patients in the absence of or with limited myelosuppression. Furthermore, IMGN632 demonstrated robust antitumor efficacy in multiple AML xenograft models. Overall, these findings identify IMGN632 as a promising candidate for evaluation as a novel therapy in AML.

Effects of Drug-Antibody Ratio on Pharmacokinetics, Biodistribution, Efficacy, and Tolerability of Antibody-Maytansinoid Conjugates.

Antibody-drug conjugates (ADCs) are being actively pursued as a treatment option for cancer following the regulatory approval of brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (Kadcyla). ADCs consist of a cytotoxic agent conjugated to a targeting antibody through a linker. The two approved ADCs (and most ADCs now in the clinic that use a microtubule disrupting agent as the payload) are heterogeneous conjugates with an average drug-to-antibody ratio (DAR) of 3-4 (potentially ranging from 0 to 8 for individual species). Ado-trastuzumab emtansine employs DM1, a semisynthetic cytotoxic payload of the maytansinoid class, which is conjugated via lysine residues of the antibody to an average DAR of 3.5. To understand the effect of DAR on the preclinical properties of ADCs using maytansinoid cytotoxic agents, we prepared a series of conjugates with a cleavable linker (M9346A-sulfo-SPDB-DM4 targeting folate receptor α (FRα)) or an uncleavable linker (J2898A-SMCC-DM1 targeting the epidermal growth factor receptor (EGFR)) with varying DAR and evaluated their biochemical characteristics, in vivo stability, efficacy, and tolerability. For both formats, a series of ADCs with DARs ranging from low (average of ∼2 and range of 0-4) to very high (average of 10 and range of 7-14) were prepared in good yield with high monomer content and low levels of free cytotoxic agent. The in vitro potency consistently increased with increasing DAR at a constant antibody concentration. We then characterized the in vivo disposition of these ADCs. Pharmacokinetic analysis showed that conjugates with an average DAR below ∼6 had comparable clearance rates, but for those with an average DAR of ∼9-10, rapid clearance was observed. Biodistribution studies in mice showed that these 9-10 DAR ADCs rapidly accumulate in the liver, with maximum localization for this organ at 24-28% percentage injected dose per gram (%ID/g) compared with 7-10% for lower-DAR conjugates (all at 2-6 h post-injection). Our preclinical findings on tolerability and efficacy suggest that maytansinoid conjugates with DAR ranging from 2 to 6 have a better therapeutic index than conjugates with very high DAR (∼9-10). These very high DAR ADCs suffer from decreased efficacy, likely due to faster clearance. These results support the use of DAR 3-4 for maytansinoid ADCs but suggest that the exploration of lower or higher DAR may be warranted depending on the biology of the target antigen.

A New Triglycyl Peptide Linker for Antibody-Drug Conjugates (ADCs) with Improved Targeted Killing of Cancer Cells.

A triglycyl peptide linker (CX) was designed for use in antibody -: drug conjugates (ADC), aiming to provide efficient release and lysosomal efflux of cytotoxic catabolites within targeted cancer cells. ADCs comprising anti-epithelial cell adhesion molecule (anti-EpCAM) and anti-EGFR antibodies with maytansinoid payloads were prepared using CX or a noncleavable SMCC linker (CX and SMCC ADCs). The in vitro cytotoxic activities of CX and SMCC ADCs were similar for several cancer cell lines; however, the CX ADC was more active (5-100-fold lower IC50) than the SMCC ADC in other cell lines, including a multidrug-resistant line. Both CX and SMCC ADCs showed comparable MTDs and pharmacokinetics in CD-1 mice. In Calu-3 tumor xenografts, antitumor efficacy was observed with the anti-EpCAM CX ADC at a 5-fold lower dose than the corresponding SMCC ADC in vivo Similarly, the anti-EGFR CX ADC showed improved antitumor activity over the respective SMCC conjugate in HSC-2 and H1975 tumor models; however, both exhibited similar activity against FaDu xenografts. Mechanistically, in contrast with the charged lysine-linked catabolite of SMCC ADC, a significant fraction of the carboxylic acid catabolite of CX ADC could be uncharged in the acidic lysosomes, and thus diffuse out readily into the cytosol. Upon release from tumor cells, CX catabolites are charged at extracellular pH and do not penetrate and kill neighboring cells, similar to the SMCC catabolite. Overall, these data suggest that CX represents a promising linker option for the development of ADCs with improved therapeutic properties. Mol Cancer Ther; 15(6); 1311-20. ©2016 AACR.

Development of Anilino-Maytansinoid ADCs that Efficiently Release Cytotoxic Metabolites in Cancer Cells and Induce High Levels of Bystander Killing.

Antibody anilino maytansinoid conjugates (AaMCs) have been prepared in which a maytansinoid bearing an aniline group was linked through the aniline amine to a dipeptide, which in turn was covalently attached to a desired monoclonal antibody. Several such conjugates were prepared utilizing different dipeptides in the linkage including Gly-Gly, l-Val-l-Cit, and all four stereoisomers of the Ala-Ala dipeptide. The properties of AaMCs could be altered by the choice of dipeptide in the linker. Each of the AaMCs, except the AaMC bearing a d-Ala-d-Ala peptide linker, displayed more bystander killing in vitro than maytansinoid ADCs that utilize disulfide linkers. In mouse models, the anti-CanAg AaMC bearing a d-Ala-l-Ala dipeptide in the linker was shown to be more efficacious against heterogeneous HT-29 xenografts than maytansinoid ADCs that utilize disulfide linkers, while both types of the conjugates displayed similar tolerabilities.

ErbB3-ErbB2 Complexes as a Therapeutic Target in a Subset of Wild-type BRAF/NRAS Cutaneous Melanomas.

The treatment options remain limited for patients with melanoma who are wild-type for both BRAF and NRAS (WT/WT). We demonstrate that a subgroup of WT/WT melanomas display high basal phosphorylation of ErbB3 that is associated with autocrine production of the ErbB3 ligand neuregulin-1 (NRG1). In WT/WT melanoma cells displaying high levels of phospho-ErbB3, knockdown of NRG1 reduced cell viability and was associated with decreased phosphorylation of ErbB3, its coreceptor ErbB2, and its downstream target, AKT. Similar effects were observed by targeting ErbB3 with either siRNAs or the neutralizing ErbB3 monoclonal antibodies huHER3-8 and NG33. In addition, pertuzumab-mediated inhibition of ErbB2 heterodimerization decreased AKT phosphorylation, cell growth in vitro, and xenograft growth in vivo. Pertuzumab also potentiated the effects of MEK inhibitor on WT/WT melanoma growth in vitro and in vivo. These findings demonstrate that targeting ErbB3-ErbB2 signaling in a cohort of WT/WT melanomas leads to tumor growth reduction. Together, these studies support the rationale to target the NRG1-ErbB3-ErbB2 axis as a novel treatment strategy in a subset of cutaneous melanomas.

Evaluation of Targets for Maytansinoid ADC Therapy Using a Novel Radiochemical Assay.

PURPOSE: Many antibody-drug conjugates (ADCs) become active only after antigen-mediated internalization and release of the cytotoxic agent via antibody degradation. Quantifying these processes can provide critical information on the suitability of a particular receptor target or antibody for ADC therapy by providing insight into the amount of cytotoxic agent released. We describe a simple and inexpensive radiolabel assay to monitor this process in cultured cancer cells. METHODS: Monoclonal antibodies were trace-labeled at their lysine residues by treatment with the N-hydroxysuccinimide ester of [(3)H]propionic acid. Human cancer cell cultures were treated with the labeled antibody at concentrations sufficient to saturate the targeted antigen. After washing to remove unbound antibody, cells were incubated and analyzed for antigen expression, conjugate degradation and catabolite formation. Results were compared with data obtained from similar assays run with radiolabeled antibody-[(3)H]maytansinoid conjugates ([(3)H]AMCs). To exemplify the method, studies were conducted with a panel of [(3)H]propionamide-antibodies to evaluate processing efficiency in EGFR-expressing SCCHN cell lines, and in NHL cell lines expressing the B-cell targets CD19, CD20, CD22 and CD37. RESULTS: Use of the [(3)H]propionamide-antibody assay yielded cell-mediated processing results similar to those obtained with corresponding maytansinoid ADCs. Further exploration allowed comparison of expression levels, antigen-dependent degradation, and catabolite formation across a panel of EGFR-expressing SCCHN cell lines, and for multiple targets in various B-cell cancer indications. CONCLUSIONS: The [(3)H]propionamide-antibody assay described here is a sensitive, facile method which enables rapid and robust assessment of relative antibody processing amounts for target, antibody, and cell line evaluation.

Function-blocking ERBB3 antibody inhibits the adaptive response to RAF inhibitor.

ERBB3/HER3 expression and signaling are upregulated in mutant BRAF melanoma as an adaptive, prosurvival response to FDA-approved RAF inhibitors. Because compensatory ERBB3 signaling counteracts the effects of RAF inhibitors, cotargeting ERBB3 may increase the efficacy of RAF inhibitors in mutant BRAF models of melanoma. Here, we corroborate this concept by showing that the ERBB3 function-blocking monoclonal antibody huHER3-8 can inhibit neuregulin-1 activation of ERBB3 and downstream signaling in RAF-inhibited melanoma cells. Targeting mutant BRAF in combination with huHER3-8 decreased cell proliferation and increased cell death in vitro, and decreased tumor burden in vivo, compared with targeting either mutant BRAF or ERBB3 alone. Furthermore, the likelihood of a durable tumor response in vivo was increased when huHER3-8 was combined with RAF inhibitor PLX4720. Together, these results offer a preclinical proof of concept for the application of ERBB3-neutralizing antibodies to enhance the efficacy of RAF inhibitors in melanoma to delay or prevent tumor regrowth. As ERBB3 is often upregulated in response to other kinase-targeted therapeutics, these findings may have implications for other cancers as well.

The unique neonatal NK cells: a critical component required for neonatal autoimmune disease induction by maternal autoantibody.

Human maternal autoantibodies can trigger autoimmune diseases such as congenital heart block (CHB) in the progeny of women with lupus or Sjogren's disease. The pathogenic effect of early autoantibody (autoAb) exposure has been investigated in a murine neonatal autoimmune ovarian disease (nAOD) model triggered by a unique ZP3 antibody. Although immune complexes (IC) are formed in adult and neonatal ovaries, ZP3 antibody triggers severe nAOD only in <7-day-old neonatal mice. Propensity to nAOD is due to the uniquely hyper-responsive neonatal natural killer (NK) cells that lack the inhibitory Ly49C/I receptors. In nAOD, the neonatal NK cells directly mediate ovarian inflammation and oocyte depletion while simultaneously promoting de novo pathogenic ovarian-specific T cell responses. Resistance to nAOD in older mice results from the emergence of the Ly49C/I(+) NK cells that regulate effector NK cells and from CD25(+) regulatory T cell control. In preliminary studies, FcγRIII(+) NK cells as well as the ovarian resident FcγRIII(+) macrophages and/or dendritic cells were found to be as indispensable players. Activated by ovarian IC, they migrate to lymphoid organs where NK cell priming occurs. Remarkably, the findings in nAOD are very similar to those reported for neonatal responses to a retrovirus and its cognate antibody that lead to long-lasting immunity. Studies on nAOD therefore provide insights into maternal autoAb-mediated neonatal autoimmunity, including CHB, while simultaneously uncovering new properties of the neonatal innate and adaptive responses, lethality of premature infant infection, and novel neonatal antiviral vaccine design.

Cutting edge: Ly49C/I⁻ neonatal NK cells predispose newborns to autoimmune ovarian disease induced by maternal autoantibody.

NK cells are critical in immune responses against pathogens. However, their role in autoimmunity is still controversial. In this study, we demonstrate that neonatal NK cells render newborns more susceptible to neonatal autoimmunity induced by maternal autoantibodies (neonatal autoimmune ovarian disease); thus, neonatal but not adult NK cells are pathogenic after transfer into NK cell-deficient pups. The inhibitory receptors Ly49C/I are expressed in ∼5% of neonatal and ∼50% of adult NK cells. In this study, we show that the presence of Ly49C/I⁺ adult NK cells inhibits neonatal autoimmune ovarian disease induction. Thus, the ontogenetic regulation of Ly49C/I expression determines the propensity to autoantibody-induced autoimmunity. In summary, this study provides definitive evidence of a pathogenic role of NK cells in neonatal autoimmunity and also elucidates a novel mechanism by which neonatal NK cells render newborns more susceptible to autoantibody-induced autoimmunity.

In vivo depletion of CD4+FOXP3+ Treg cells by the PC61 anti-CD25 monoclonal antibody is mediated by FcgammaRIII+ phagocytes.

Depletion of CD4(+)CD25(+)FoxP3(+) Treg using PC61 mAb (anti-murine CD25 rat IgG1) is widely used to characterize Treg function in vivo. However, the mechanism of Treg depletion remains largely unknown. Herein, we report the PC61 mAb's mechanism of action. In peripheral blood, a single injection of PC61 mAb eliminated approximately 70% of CD4(+)FoxP3(+) cells with the remaining Treg expressing low or no CD25. Functional blockade of Fcgamma receptors with 2.4G2 mAb significantly inhibited PC61 mAb activity. Furthermore, Fcgamma receptor (FcgammaR)III(-/-) mice were resistant to Treg depletion. FcgammaRIII is expressed on immune cells including NK cells and macrophages that are the major effector cells for Ab-dependent-cellular-cytotoxicity and Ab-dependent-cellular-phagocytosis, respectively. Depletion of NK cells had no effect, whereas depletion of phagocytes, including macrophages, by clodronate liposome significantly inhibited Treg depletion. Furthermore, in vitro, PC61 mAb can mediate Ab-dependent-cellular-phagocytosis of CD25(+) cells by WT or FcgammaRIIB(-/-), but not FcgammaRIII(-/-), macrophages. Altogether these data demonstrate the critical role of FcgammaRIII(+) phagocytes in mediating Treg depletion by PC61 mAb. This finding may be useful in guiding the development of human Treg targeting therapy.

The autoimmune regulator directly controls the expression of genes critical for thymic epithelial function.

The autoimmune regulator (Aire) gene plays an essential role in negative selection of T cells and deletion of autoreactive T cells in the thymus. The defect in thymic selection in Aire(-/-) mice was attributed to the repressed expression of tissue-specific Ags in the thymic epithelial cells and defective Ag presentation; however, the molecular mechanism underlying these functions has been elusive. Using the chromatin immunoprecipitation technique, we demonstrate here that Aire binds in vivo to specific DNA sequence motifs and directly regulates thymic expression of genes important for thymic functions including expression of autoantigens, cytokines, transcription factors, and posttranslational modifiers. These results unambiguously established Aire as a key transcriptional regulator of the immune system.

The role of physiological self-antigen in the acquisition and maintenance of regulatory T-cell function.

The CD4+ CD25+ regulatory T cells (Tregs) are efficient regulators of autoimmunity, but the mechanism remains elusive. We summarize recent data for the conclusion that disease-specific Tregs respond to tissue antigens to maintain physiological tolerance and prevent autoimmunity. First, polyclonal Tregs from antigen-positive donors suppress autoimmune ovarian disease (AOD) or experimental autoimmune prostatitis in day 3 thymectomized (d3tx) mice more efficiently than Tregs from antigen-negative donors. Second, Tregs of antigen-negative adult mice respond to cognate antigen in vivo and rapidly gain disease-specific Treg function. Third, in d3tx female recipients devoid of neonatal ovarian antigens, only female Tregs suppressed AOD; the male Tregs gain AOD-suppressing function by responding to the ovarian antigen in the recipients and mask the supremacy of female Tregs in AOD suppression. Fourth, when Tregs completely suppress AOD, the ovary-draining lymph node is the only location with evidence of profound and persistent (but reversible) host T-cell suppression. Fifth, from these nodes, highly potent AOD-suppressing Tregs are retrievable. We conclude that self-tolerance involves the continuous priming of Tregs by autoantigens, and in autoimmune disease suppression, the effector T-cell response is continuously negated by potent disease-specific Tregs that accumulate at the site of autoantigen presentation.

Physiologic self antigens rapidly capacitate autoimmune disease-specific polyclonal CD4+ CD25+ regulatory T cells.

Studies on CD4+ CD25+ regulatory T cells (Tregs) with transgenic T-cell receptors indicate that Tregs may receive continuous antigen (Ag) stimulation in the periphery. However, the consequence of this Ag encounter and its relevance to physiologic polyclonal Treg function are not established. In autoimmune prostatitis (EAP) of the day-3 thymectomized (d3tx) mice, male Tregs suppressed EAP 3 times better than Tregs from female mice or male mice without prostates. Importantly, the superior EAP-suppressing function was acquired after a 6-day exposure to prostate Ag in the periphery, unaffected by sex hormones. Thus, a brief exposure of physiologic prostate Ag capacitates peripheral polyclonal Tregs to suppress EAP. In striking contrast, autoimmune ovarian disease (AOD) was suppressed equally by male and female Tregs. We now provide evidence that the ovarian Ag develops at birth, 14 days earlier than prostate Ag, and that male Tregs respond to neonatal ovarian Ag in the Treg recipients to gain AOD-suppressing capacity. When d3tx female recipients were deprived of ovarian Ag in the neonatal period, AOD was suppressed by female but not by male Tregs, whereas dacryoadenitis was suppressed by both. We conclude that the physiologic autoAg quickly and continuously enhances disease-specific polyclonal Treg function to maintain self-tolerance.

Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720.

Immunotherapeutic drugs that mimic sphingosine 1-phosphate (S1P) disrupt lymphocyte trafficking and cause T helper and T effector cells to be retained in secondary lymphoid tissue and away from sites of inflammation. The prototypical therapeutic agent, 2-alkyl-2-amino-1,3-propanediol (FTY720), stimulates S1P signaling pathways only after it is phosphorylated by one or more unknown kinases. We generated sphingosine kinase 2 (SPHK2) null mice to demonstrate that this kinase is responsible for FTY720 phosphorylation and thereby its subsequent actions on the immune system. Both systemic and lymphocyte-localized sources of SPHK2 contributed to FTY720 induced lymphopenia. Although FTY720 was selectively activated in vivo by SPHK2, other S1P pro-drugs can be phosphorylated to cause lymphopenia through the action of additional sphingosine kinases. Our results emphasize the importance of SPHK2 expression in both lymphocytes and other tissues for immune modulation and drug metabolism.

Neonatal autoimmune disease: influence of CD4+ CD25+ regulatory T cells.

Although previous studies have emphasized the tolerogenic property of murine neonatal immune system, recent studies indicate that neonatal mice are prone to autoimmune disease. This chapter will summarize the evidence for neonatal propensity to autoimmune ovarian disease (AOD) and describe the new finding that autoantibody can trigger a T cell-dependent autoimmune disease in neonatal but not adult mice. Based on depletion or addition of the CD4+ CD25+ T cells, disease resistance of older mice is explicable by the emergence of CD4+ CD25+ regulatory T-cell function after day 5, whereas disease susceptibility is associated with resistance to regulation by CD4+ CD25+ T cells.

Requirements of NK cells and proinflammatory cytokines in T cell-dependent neonatal autoimmune ovarian disease triggered by immune complex.

A model of neonatal autoimmune disease has been described recently in which an epitope-specific autoantibody to murine zona pellucida 3 induces severe ovarian disease in neonatal, but not adult, mice (neonatal AOD). The autoantibody forms immune complex with endogenous ovarian zona pellucida 3, and a pathogenic CD4(+) T cell response is triggered. The basis for the predominant neonatal susceptibility has not been clarified. In this study innate immunity, including neonatal NK cells, in neonatal AOD was investigated. Neonatal spleen contained readily detectable NK1.1(+)TCRVbeta(-), but not NK1.1(+)TCRVbeta(+), cells. Ab depletion of NK1.1(+)TCRVbeta(-) cells inhibited neonatal AOD development. Moreover, in adoptive transfer of neonatal AOD, recipient disease was ameliorated when either donor or recipient NK cells were depleted. Thus, NK cells operate in both induction and effector phases of the disease. IFN-gamma was produced by neonatal NK cells in vivo, and it may be important in neonatal AOD. Indeed, ovaries with neonatal AOD expressed high levels of IFN-gamma and TNF-alpha which correlated with disease severity, and the disease was inhibited by IFN-gamma or TNF-alpha Ab. Importantly, disease was enhanced by recombinant IFN-gamma, and treatment of T cell donors with IFN-gamma Ab also significantly reduced adoptive transfer of neonatal AOD. Finally, neonatal AOD was ameliorated in mice deficient in FcgammaRIII and was enhanced in FcgammaRIIB-deficient mice. We conclude that neonatal NK cells promote pathogenic T cell response at multiple stages during neonatal autoimmune disease pathogenesis. Also operative in neonatal AOD are other mediators of the innate system, including proinflammatory cytokines and FcgammaRIII signaling.