Using the power of innate immunoprofiling to understand vaccine design, infection, and immunity.

In the field of immunology, a systems biology approach is crucial to understanding the immune response to infection and vaccination considering the complex interplay between genetic, epigenetic, and environmental factors. Significant progress has been made in understanding the innate immune response, including cell players and critical signaling pathways, but many questions remain unanswered, including how the innate immune response dictates host/pathogen responses and responses to vaccines. To complicate things further, it is becoming increasingly clear that the innate immune response is not a linear pathway but is formed from complex networks and interactions. To further our understanding of the crosstalk and complexities, systems-level analyses and expanded experimental technologies are now needed. In this review, we discuss the most recent immunoprofiling techniques and discuss systems approaches to studying the global innate immune landscape which will inform on the development of personalized medicine and innovative vaccine strategies.

Ezh2 emerges as an epigenetic checkpoint regulator during monocyte differentiation limiting cardiac dysfunction post-MI.

Epigenetic regulation of histone H3K27 methylation has recently emerged as a key step during alternative immunoregulatory M2-like macrophage polarization; known to impact cardiac repair after Myocardial Infarction (MI). We hypothesized that EZH2, responsible for H3K27 methylation, could act as an epigenetic checkpoint regulator during this process. We demonstrate for the first time an ectopic EZH2, and putative, cytoplasmic inactive localization of the epigenetic enzyme, during monocyte differentiation into M2 macrophages in vitro as well as in immunomodulatory cardiac macrophages in vivo in the post-MI acute inflammatory phase. Moreover, we show that pharmacological EZH2 inhibition, with GSK-343, resolves H3K27 methylation of bivalent gene promoters, thus enhancing their expression to promote human monocyte repair functions. In line with this protective effect, GSK-343 treatment accelerated cardiac inflammatory resolution preventing infarct expansion and subsequent cardiac dysfunction in female mice post-MI in vivo. In conclusion, our study reveals that pharmacological epigenetic modulation of cardiac-infiltrating immune cells may hold promise to limit adverse cardiac remodeling after MI.

TNF superfamily control of tissue remodeling and fibrosis.

Fibrosis is the result of extracellular matrix protein deposition and remains a leading cause of death in USA. Despite major advances in recent years, there remains an unmet need to develop therapeutic options that can effectively degrade or reverse fibrosis. The tumor necrosis super family (TNFSF) members, previously studied for their roles in inflammation and cell death, now represent attractive therapeutic targets for fibrotic diseases. In this review, we will summarize select TNFSF and their involvement in fibrosis of the lungs, the heart, the skin, the gastrointestinal tract, the kidney, and the liver. We will emphasize their direct activity on epithelial cells, fibroblasts, and smooth muscle cells. We will further report on major clinical trials targeting these ligands. Whether in isolation or in combination with other anti-TNFSF member or treatment, targeting this superfamily remains key to improve efficacy and selectivity of currently available therapies for fibrosis.

Regulation and impact of cardiac lymphangiogenesis in pressure-overload-induced heart failure.

AIMS: Lymphatics are essential for cardiac health, and insufficient lymphatic expansion (lymphangiogenesis) contributes to development of heart failure (HF) after myocardial infarction. However, the regulation and impact of lymphangiogenesis in non-ischaemic cardiomyopathy following pressure-overload remains to be determined. Here, we investigated cardiac lymphangiogenesis following transversal aortic constriction (TAC) in C57Bl/6 and Balb/c mice, and in end-stage HF patients. METHODS AND RESULTS: Cardiac function was evaluated by echocardiography, and cardiac hypertrophy, lymphatics, inflammation, oedema, and fibrosis by immunohistochemistry, flow cytometry, microgravimetry, and gene expression analysis. Treatment with neutralizing anti-VEGFR3 antibodies was applied to inhibit cardiac lymphangiogenesis in mice. We found that VEGFR3-signalling was essential to prevent cardiac lymphatic rarefaction after TAC in C57Bl/6 mice. While anti-VEGFR3-induced lymphatic rarefaction did not significantly aggravate myocardial oedema post-TAC, cardiac immune cell levels were increased, notably myeloid cells at 3 weeks and T lymphocytes at 8 weeks. Moreover, whereas inhibition of lymphangiogenesis did not aggravate interstitial fibrosis, it increased perivascular fibrosis and accelerated development of left ventricular (LV) dilation and dysfunction. In clinical HF samples, cardiac lymphatic density tended to increase, although lymphatic sizes decreased, notably in patients with dilated cardiomyopathy. Similarly, comparing C57Bl/6 and Balb/c mice, lymphatic remodelling post-TAC was linked to LV dilation rather than to hypertrophy. The striking lymphangiogenesis in Balb/c was associated with reduced cardiac levels of macrophages, B cells, and perivascular fibrosis at 8 weeks post-TAC, as compared with C57Bl/6 mice that displayed weak lymphangiogenesis. Surprisingly, however, it did not suffice to resolve myocardial oedema, nor prevent HF development. CONCLUSIONS: We demonstrate for the first time that endogenous lymphangiogenesis limits TAC-induced cardiac inflammation and perivascular fibrosis, delaying HF development in C57Bl/6 but not in Balb/c mice. While the functional impact of lymphatic remodelling remains to be determined in HF patients, our findings suggest that under settings of pressure-overload poor cardiac lymphangiogenesis may accelerate HF development.

Myostatin in idiopathic inflammatory myopathies: Serum assessment and disease activity.

AIMS: In idiopathic inflammatory myopathies (IIM), disease activity is difficult to assess, and IIM may induce severe muscle damage, especially in immune-mediated necrotising myopathies (IMNM) and inclusion body myositis (IBM). We hypothesise that myostatin, a negative regulator of muscle mass, could be a new biomarker of disease activity and/or muscle damage. METHODS: Prospective assessment of myostatin protein level in 447 IIM serum samples (dermatomyositis [DM], n = 157; IBM, n = 72; IMNM, n = 125; and antisynthetase syndrome [ASyS], n = 93) and 59 healthy donors (HD) was performed by ELISA. A gene transcript analysis was also carried out on 18 IIM muscle biopsies and six controls to analyse myostatin and myostatin pathway-related gene expression. RESULTS: IIM patients had lower myostatin circulating protein levels and gene expression compared to HD (2379 [1490; 3678] pg/ml vs 4281 [3169; 5787] pg/ml; p < 0.0001 and log2FC = -1.83; p = 0.0005, respectively). Myostatin-related gene expression varied accordingly. Based on the Physician Global Assessment, inactive IIM patients showed higher myostatin levels than active ones. This was the case for all IIM subgroups, except IMNM where low myostatin levels were maintained (2186 [1235; 3815] vs 2349 [1518; 3922] pg/ml; p = 0.4). CONCLUSIONS: Myostatin protein and RNA levels are decreased in all IIM patients, and protein levels correlate with disease activity. Inactive ASyS and DM patients have higher myostatin levels than active patients. Myostatin could be a marker of disease activity in these subgroups. However, IMNM patients do not have significant increase in myostatin levels after disease remission. This may highlight a new pathological disease mechanism in IMNM patients.

Endothelium-Specific Deficiency of Polycystin-1 Promotes Hypertension and Cardiovascular Disorders.

BACKGROUND: Autosomal dominant polycystic kidney disease is the most frequent hereditary kidney disease and is generally due to mutations in PKD1 and PKD2, encoding polycystins 1 and 2. In autosomal dominant polycystic kidney disease, hypertension and cardiovascular disorders are highly prevalent, but their mechanisms are partially understood. METHODS: Since endothelial cells express the polycystin complex, where it plays a central role in the mechanotransduction of blood flow, we generated a murine model with inducible deletion of Pkd1 in endothelial cells (Cdh5-CreERT2;Pkd1fl/fl) to specifically determine the role of endothelial polycystin-1 in autosomal dominant polycystic kidney disease. RESULTS: Endothelial deletion of Pkd1 induced endothelial dysfunction, as demonstrated by impaired flow-mediated dilatation of resistance arteries and impaired relaxation to acetylcholine, increased blood pressure and prevented the normal development of arteriovenous fistula. In experimental chronic kidney disease induced by subtotal nephrectomy, endothelial deletion of Pkd1 further aggravated endothelial dysfunction, vascular remodeling, and heart hypertrophy. CONCLUSIONS: Altogether, this study provides the first in vivo demonstration that specific deletion of Pkd1 in endothelial cells promotes endothelial dysfunction and hypertension, impairs arteriovenous fistula development, and potentiates the cardiovascular alterations associated with chronic kidney disease.

Germinal Center T follicular helper (GC-Tfh) cell impairment in chronic HIV infection involves c-Maf signaling.

We have recently demonstrated that the function of T follicular helper (Tfh) cells from lymph nodes (LN) of HIV-infected individuals is impaired. We found that these cells were unable to provide proper help to germinal center (GC)-B cells, as observed by altered and inefficient anti-HIV antibody response and premature death of memory B cells. The underlying molecular mechanisms of this dysfunction remain poorly defined. Herein, we have used a unique transcriptional approach to identify these molecular defects. We consequently determined the transcriptional profiles of LN GC-Tfh cells following their interactions with LN GC-B cells from HIV-infected and HIV-uninfected individuals, rather than analyzing resting ex-vivo GC-Tfh cells. We observed that proliferating GC-Tfh cells from HIV-infected subjects were transcriptionally different than their HIV-uninfected counterparts, and displayed a significant downregulation of immune- and GC-Tfh-associated pathways and genes. Our results strongly demonstrated that MAF (coding for the transcription factor c-Maf) and its upstream signaling pathway mediators (IL6R and STAT3) were significantly downregulated in HIV-infected subjects, which could contribute to the impaired GC-Tfh and GC-B cell functions reported during infection. We further showed that c-Maf function was associated with the adenosine pathway and that the signaling upstream c-Maf could be partially restored by adenosine deaminase -1 (ADA-1) supplementation. Overall, we identified a novel mechanism that contributes to GC-Tfh cell impairment during HIV infection. Understanding how GC-Tfh cell function is altered in HIV is crucial and could provide critical information about the mechanisms leading to the development and maintenance of effective anti-HIV antibodies.

A follicular regulatory Innate Lymphoid Cell population impairs interactions between germinal center Tfh and B cells.

Innate Lymphoid Cells (ILCs) are immune cells typically found on mucosal surfaces and in secondary lymphoid organs where they regulate the immune response to pathogens. Despite their key role in the immune response, there are still fundamental gaps in our understanding of ILCs. Here we report a human ILC population present in the follicles of tonsils and lymph nodes termed follicular regulatory ILCs (ILCFR) that to our knowledge has not been previously identified. ILCFR have a distinct phenotype and transcriptional program when compared to other defined ILCs. Surprisingly, ILCFR inhibit the ability of follicular helper T (Tfh) cells to provide B cell help. The localization of ILCFR to the germinal centers suggests these cells may interfere with germinal center B cell (GC-B) and germinal center Tfh cell (GC-Tfh) interactions through the production of transforming growth factor beta (TGF-ß. Intriguingly, under conditions of impaired GC-Tfh-GC-B cell interactions, such as human immunodeficiency virus (HIV) infection, the frequency of these cells is increased. Overall, we predict a role for ILCFR in regulating GC-Tfh-GC-B cell interactions and propose they expand in chronic inflammatory conditions.

Lymphatic and Immune Cell Cross-Talk Regulates Cardiac Recovery After Experimental Myocardial Infarction.

OBJECTIVE: Lymphatics play an essential pathophysiological role in promoting fluid and immune cell tissue clearance. Conversely, immune cells may influence lymphatic function and remodeling. Recently, cardiac lymphangiogenesis has been proposed as a therapeutic target to prevent heart failure after myocardial infarction (MI). We investigated the effects of gene therapy to modulate cardiac lymphangiogenesis post-MI in rodents. Second, we determined the impact of cardiac-infiltrating T cells on lymphatic remodeling in the heart. Approach and Results: Comparing adenoviral versus adeno-associated viral gene delivery in mice, we found that only sustained VEGF (vascular endothelial growth factor)-CC156S therapy, achieved by adeno-associated viral vectors, increased cardiac lymphangiogenesis, and led to reduced cardiac inflammation and dysfunction by 3 weeks post-MI. Conversely, inhibition of VEGF-C/-D signaling, through adeno-associated viral delivery of soluble VEGFR3 (vascular endothelial growth factor receptor 3), limited infarct lymphangiogenesis. Unexpectedly, this treatment improved cardiac function post-MI in both mice and rats, linked to reduced infarct thinning due to acute suppression of T-cell infiltration. Finally, using pharmacological, genetic, and antibody-mediated prevention of cardiac T-cell recruitment in mice, we discovered that both CD4+ and CD8+ T cells potently suppress, in part through interferon-γ, cardiac lymphangiogenesis post-MI. CONCLUSIONS: We show that resolution of cardiac inflammation after MI may be accelerated by therapeutic lymphangiogenesis based on adeno-associated viral gene delivery of VEGF-CC156S. Conversely, our work uncovers a major negative role of cardiac-recruited T cells on lymphatic remodeling. Our results give new insight into the interconnection between immune cells and lymphatics in orchestration of cardiac repair after injury.

Adenosine deaminase-1 enhances germinal center formation and functional antibody responses to HIV-1 Envelope DNA and protein vaccines.

Adenosine deaminase-1 (ADA-1) plays both enzymatic and non-enzymatic roles in regulating immune cell function. Mutations in the ADA1 gene account for 15% of heritable severe-combined immunodeficiencies. We determined previously that ADA1 expression defines and is instrumental for the germinal center follicular helper T cell (TFH) phenotype using in vitro human assays. Herein, we tested whether ADA-1 can be used as an adjuvant to improve vaccine efficacy in vivo. In vitro, ADA-1 induced myeloid dendritic cell (mDC) maturation as measured by increased frequencies of CD40-, CD83-, CD86-, and HLA-DR-positive mDCs. ADA-1 treatment also promoted the secretion of the TFH-polarizing cytokine IL-6 from mDCs. In the context of an HIV-1 envelope (env) DNA vaccine, co-immunization with plasmid-encoded ADA-1 (pADA) enhanced humoral immunity. Animals co-immunized with env DNA and pADA had significantly increased frequencies of TFH cells in their draining lymph nodes and increased HIV-binding IgG in serum. Next, mice were co-immunized with subtype C env gp160 DNA and pADA along with simultaneous immunization with matched gp140 trimeric protein. Mice that received env gp160 DNA, pADA, and gp140 glycoprotein had significantly more heterologous HIV-specific binding IgG in their serum. Furthermore, only these mice had detectable neutralizing antibody responses. These studies support the use of ADA-1 as a vaccine adjuvant to qualitatively enhance germinal center responses and represent a novel application of an existing therapeutic agent that can be quickly translated for clinical use.

Adenosine deaminase-1 delineates human follicular helper T cell function and is altered with HIV.

Follicular helper T cells (Tfh) play critical roles instructing, and initiating T-cell dependent antibody responses. The underlying mechanisms that enhance their function is therefore critical for vaccine development. Here we apply gene array analysis identifying adenosine deaminase (ADA) as a key molecule that delineates a human Tfh helper program in proliferating circulating Tfh (cTfh) cells and Germinal Centers Tfh (GC-Tfh). ADA-1 expression and enzymatic activity are increased in efficient cTfh2-17/GC-Tfh cells. Exogenous ADA-1 enhances less efficient cTfh1 and pro-follicular Tfh PD-1+ CXCR5+ cells to provide B cell help, while pharmacological inhibition of ADA-1 activity impedes cTfh2-17/GC-Tfh function and diminished antibody response. Mechanistically, ADA-1 controls the Tfh program by influencing IL6/IL-2 production, controlling CD26 extracellular expression and could balance signals through adenosine receptors. Interestingly, dysfunctional Tfh from HIV infected-individual fail to regulate the ADA pathway. Thus, ADA-1 regulates human Tfh and represents a potential target for development of vaccine strategy.

Neonatal influenza-specific effector CTLs retain elevated CD31 levels at the site of infection and have decreased IFN-γ production.

The underlying mechanisms that regulate neonatal immune suppression are poorly characterized. CD31 (PECAM1) is highly expressed on neonatal lymphocytes and is a known modulator of TCR signaling. To further characterize the role of CD31 in the neonatal CTL response, 3-d and 7-d-old murine neonates were infected with influenza virus and compared to adults. The majority of the pulmonary viral-specific CTLs in the 3-d-old murine neonate retain CD31 expression, whereas adult CTLs have decreased CD31 expression. In addition, CD31+ neonatal viral-specific CTLs demonstrate decreased IFN-γ production, decreased proliferative capacity, and increased likelihood of death. At the peak of infection, sorted neonatal effector CTLs continue to transcribe CD31, indicating a developmental regulation of expression. To explore potential mechanisms for this reduced function, we compared the expression of the transcription factors Eomesodermin (Eomes) and T-bet; there was a significant increase in Eomes paired with a reduction in T-bet in CD31+ neonatal effector CTLs in the lung. Furthermore, in vitro stimulated neonatal CTLs significantly reduce IFN-γ production upon CD31 signaling. Altogether, these data indicate that neonatal CTLs may retain elevated levels of CD31 to maintain peripheral T cell suppression during the bridge to ex utero life.

PLD3 and PLD4 are single-stranded acid exonucleases that regulate endosomal nucleic-acid sensing.

The sensing of microbial genetic material by leukocytes often elicits beneficial pro-inflammatory cytokines, but dysregulated responses can cause severe pathogenesis. Genome-wide association studies have linked the gene encoding phospholipase D3 (PLD3) to Alzheimer's disease and have linked PLD4 to rheumatoid arthritis and systemic sclerosis. PLD3 and PLD4 are endolysosomal proteins whose functions are obscure. Here, PLD4-deficient mice were found to have an inflammatory disease, marked by elevated levels of interferon-γ (IFN-γ) and splenomegaly. These phenotypes were traced to altered responsiveness of PLD4-deficient dendritic cells to ligands of the single-stranded DNA sensor TLR9. Macrophages from PLD3-deficient mice also had exaggerated TLR9 responses. Although PLD4 and PLD3 were presumed to be phospholipases, we found that they are 5' exonucleases, probably identical to spleen phosphodiesterase, that break down TLR9 ligands. Mice deficient in both PLD3 and PLD4 developed lethal liver inflammation in early life, which indicates that both enzymes are needed to regulate inflammatory cytokine responses via the degradation of nucleic acids.

Delayed differentiation of potent effector CD8+ T cells reducing viremia and reservoir seeding in acute HIV infection.

CD8+ T cells play a critical role in controlling HIV viremia and could be important in reducing HIV-infected cells in approaches to eradicate HIV. The simian immunodeficiency virus model provided the proof of concept for a CD8+ T cell-mediated reservoir clearance but showed conflicting evidence on the role of these cells to eliminate HIV-infected cells. In humans, HIV-specific CD8+ T cell responses have not been associated with a reduction of the HIV-infected cell pool in vivo. We studied HIV-specific CD8+ T cells in the RV254 cohort of individuals initiating ART in the earliest stages of acute HIV infection (AHI). We showed that the HIV-specific CD8+ T cells generated as early as AHI stages 1 and 2 before peak viremia are delayed in expanding and acquiring effector functions but are endowed with higher memory potential. In contrast, the fully differentiated HIV-specific CD8+ T cells at peak viremia in AHI stage 3 were more prone to apoptosis but were associated with a steeper viral load decrease after ART initiation. Their capacity to persist in vivo after ART initiation correlated with a lower HIV DNA reservoir. These findings demonstrate that HIV-specific CD8+ T cell magnitude and differentiation are delayed in the earliest stages of infection. These results also demonstrate that potent HIV-specific CD8+ T cells contribute to the reduction of the pool of HIV-producing cells and the HIV reservoir seeding in vivo and provide the rationale to design interventions aiming at inducing these potent responses to cure HIV infection.

Altered Memory Circulating T Follicular Helper-B Cell Interaction in Early Acute HIV Infection.

The RV254 cohort of HIV-infected very early acute (4thG stage 1 and 2) (stage 1/2) and late acute (4thG stage 3) (stage 3) individuals was used to study T helper- B cell responses in acute HIV infection and the impact of early antiretroviral treatment (ART) on T and B cell function. To investigate this, the function of circulating T follicular helper cells (cTfh) from this cohort was examined, and cTfh and memory B cell populations were phenotyped. Impaired cTfh cell function was observed in individuals treated in stage 3 when compared to stage 1/2. The cTfh/B cell cocultures showed lower B cell survival and IgG secretion at stage 3 compared to stage 1/2. This coincided with lower IL-10 and increased RANTES and TNF-α suggesting a role for inflammation in altering cTfh and B cell responses. Elevated plasma viral load in stage 3 was found to correlate with decreased cTfh-mediated B cell IgG production indicating a role for increased viremia in cTfh impairment and dysfunctional humoral response. Phenotypic perturbations were also evident in the mature B cell compartment, most notably a decrease in resting memory B cells in stage 3 compared to stage 1/2, coinciding with higher viremia. Our coculture assay also suggested that intrinsic memory B cell defects could contribute to the impaired response despite at a lower level. Overall, cTfh-mediated B cell responses are significantly altered in stage 3 compared to stage 1/2, coinciding with increased inflammation and a reduction in memory B cells. These data suggest that early ART for acutely HIV infected individuals could prevent immune dysregulation while preserving cTfh function and B cell memory.

Reversible Reprogramming of Circulating Memory T Follicular Helper Cell Function during Chronic HIV Infection.

Despite the overwhelming benefits of antiretroviral therapy (ART) in curtailing viral load in HIV-infected individuals, ART does not fully restore cellular and humoral immunity. HIV-infected individuals under ART show reduced responses to vaccination and infections and are unable to mount an effective antiviral immune response upon ART cessation. Many factors contribute to these defects, including persistent inflammation, especially in lymphoid tissues, where T follicular helper (Tfh) cells instruct and help B cells launch an effective humoral immune response. In this study we investigated the phenotype and function of circulating memory Tfh cells as a surrogate of Tfh cells in lymph nodes and found significant impairment of this cell population in chronically HIV-infected individuals, leading to reduced B cell responses. We further show that these aberrant memory Tfh cells exhibit an IL-2-responsive gene signature and are more polarized toward a Th1 phenotype. Treatment of functional memory Tfh cells with IL-2 was able to recapitulate the detrimental reprogramming. Importantly, this defect was reversible, as interfering with the IL-2 signaling pathway helped reverse the abnormal differentiation and improved Ab responses. Thus, reversible reprogramming of memory Tfh cells in HIV-infected individuals could be used to enhance Ab responses. Altered microenvironmental conditions in lymphoid tissues leading to altered Tfh cell differentiation could provide one explanation for the poor responsiveness of HIV-infected individuals to new Ags. This explanation has important implications for the development of therapeutic interventions to enhance HIV- and vaccine-mediated Ab responses in patients under ART.

Multiformat T-cell-engaging bispecific antibodies targeting human breast cancers.

Four different formats of bispecific antibodies (bsAbs) were generated that consist of anti-Her2 IgG or Fab site-specifically conjugated to anti-CD3 Fab using the genetically encoded noncanonical amino acid. These bsAbs varied in valency or in the presence or absence of an Fc domain. Different valencies did not significantly affect antitumor efficacy, whereas the presence of an Fc domain enhanced cytotoxic activity, but triggered antigen-independent T-cell activation. We show that the bsAbs can efficiently redirect T cells to kill all Her2 expressing cancer cells, including Her2 1+ cancers, both in vitro and in rodent xenograft models. This work increases our understanding of the structural features that affect bsAb activity, and underscores the potential of bsAbs as a promising therapeutic option for breast cancer patients with low or heterogeneous Her2 expression.

A CXCR4-targeted site-specific antibody-drug conjugate.

A chemically defined anti-CXCR4-auristatin antibody-drug conjugate (ADC) was synthesized that selectively eliminates tumor cells overexpressing the CXCR4 receptor. The unnatural amino acid p-acetylphenylalanine (pAcF) was site-specifically incorporated into an anti-CXCR4 immunoglobulin G (IgG) and conjugated to an auristatin through a stable, non-cleavable oxime linkage to afford a chemically homogeneous ADC. The full-length anti-CXCR4 ADC was selectively cytotoxic to CXCR4(+) cancer cells in vitro (half maximal effective concentration (EC50 )≈80-100 pM). Moreover, the anti-CXCR4 ADC eliminated pulmonary lesions from human osteosarcoma cells in a lung-seeding tumor model in mice. No significant overt toxicity was observed but there was a modest decrease in the bone-marrow-derived CXCR4(+) cell population. Because CXCR4 is highly expressed in a majority of metastatic cancers, a CXCR4-auristatin ADC may be useful for the treatment of a variety of metastatic malignancies.

Targeting human C-type lectin-like molecule-1 (CLL1) with a bispecific antibody for immunotherapy of acute myeloid leukemia.

Acute myeloid leukemia (AML), which is the most common acute adult leukemia and the second most common pediatric leukemia, still has a poor prognosis. Human C-type lectin-like molecule-1 (CLL1) is a recently identified myeloid lineage restricted cell surface marker, which is overexpressed in over 90% of AML patient myeloid blasts and in leukemic stem cells. Here, we describe the synthesis of a novel bispecific antibody, αCLL1-αCD3, using the genetically encoded unnatural amino acid, p-acetylphenylalanine. The resulting αCLL1-αCD3 recruits cytotoxic T cells to CLL1 positive cells, and demonstrates potent and selective cytotoxicity against several human AML cell lines and primary AML patient derived cells in vitro. Moreover, αCLL1-αCD3 treatment completely eliminates established tumors in an U937 AML cell line xenograft model. These results validate the clinical potential of CLL1 as an AML-specific antigen for the generation of a novel immunotherapeutic for AML.

Bispecific small molecule-antibody conjugate targeting prostate cancer.

Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility. The efficacy of the conjugate was optimized by modifying the linker structure, relative binding orientation, and stoichiometry of the ligand. The optimized conjugate showed potent and selective in vitro activity (EC50 ~ 100 pM), good serum half-life, and potent in vivo activity in prophylactic and treatment xenograft mouse models. This semisynthetic approach is likely to be applicable to the generation of additional bispecific agents using drug-like ligands selective for other cell-surface receptors.