Nanobodies and chemical cross-links advance the structural and functional analysis of PI3Kα.

Nanobodies and chemical cross-linking were used to gain information on the identity and positions of flexible domains of PI3Kα. The application of chemical cross-linking mass spectrometry (CXMS) facilitated the identification of the p85 domains BH, cSH2, and SH3 as well as their docking positions on the PI3Kα catalytic core. Binding of individual nanobodies to PI3Kα induced activation or inhibition of enzyme activity and caused conformational changes that could be correlated with enzyme function. Binding of nanobody Nb3-126 to the BH domain of p85α substantially improved resolution for parts of the PI3Kα complex, and binding of nanobody Nb3-159 induced a conformation of PI3Kα that is distinct from known PI3Kα structures. The analysis of CXMS data also provided mechanistic insights into the molecular underpinning of the flexibility of PI3Kα.

Development of Neutralizing Multimeric Nanobody Constructs Directed against IL-13: From Immunization to Lead Optimization.

IL-13 is a pleiotropic cytokine mainly secreted by Th2 cells. It reacts with many different types of cells involved in allergy, inflammation, and fibrosis, e.g., mastocytes, B cells, and fibroblasts. The role of IL-13 in conditions involving one or several of these phenotypes has therefore been extensively investigated. The inhibition of this cytokine in animal models for various pathologies yielded highly promising results. However, most human trials relying on anti-IL-13 conventional mAbs have failed to achieve a significant improvement of the envisaged disorders. Where some studies might have suffered from several weaknesses, the strategies themselves, such as targeting only IL-13 using conventional mAbs or employing a systemic administration, could be questioned. Nanobodies are recombinant Ag-binding fragments derived from the variable part of H chain-only Abs occurring in Camelidae. Thanks to their single-domain structure, small size (≈15 kDa), good stability, and solubility, they can be engineered into multispecific constructs for combined therapies or for use in new strategies such as formulations for local administration, e.g., pulmonary administration. In this study, we describe the generation of 38 nanobodies that can be subdivided into five CDR3 families. Nine nanobodies were found to have a good affinity profile (KD = 1-200 nM), but none were able to strongly inhibit IL-13 biological activity in vitro (IC50 > 50 µM: HEK-Blue IL-13/IL-4 cells). Multimeric constructs were therefore designed from these inhibitors and resulted in an up to 36-fold improvement in affinity and up to 300-fold enhancement of the biological activity while conserving a high specificity toward IL-13.

Novel half-life extended anti-MIF nanobodies protect against endotoxic shock.

Sepsis-leading to septic shock-is the leading cause of death in intensive care units. The systemic inflammatory response to infection, which is initiated by activated myeloid cells, plays a key role in the lethal outcome. Macrophage migration inhibitory factor (MIF) is an upstream immunoregulatory mediator, released by myeloid cells, that underlies a common genetic susceptibility to different infections and septic shock. Accordingly, strategies that are aimed at inhibiting the action of MIF have therapeutic potential. Here, we report the isolation and characterization of tailorable, small, affinity-matured nanobodies (Nbs; single-domain antigen-binding fragments derived from camelid heavy-chain Abs) directed against MIF. Of importance, these bioengineered Nbs bind both human and mouse MIFs with nanomolar affinity. NbE5 and NbE10 inhibit key MIF functions that can exacerbate septic shock, such as the tautomerase activity of MIF (by blocking catalytic pocket residues that are critical for MIF's conformation and receptor binding), the TNF-inducing potential, and the ability of MIF to antagonize glucocorticoid action. A lead NbE10, tailored to be a multivalent, half-life extended construct (NbE10-NbAlb8-NbE10), attenuated lethality in murine endotoxemia when administered via single injection, either prophylactically or therapeutically. Hence, Nbs, with their structural and pharmacologic advantages over currently available inhibitors, may be an effective, novel approach to interfere with the action of MIF in septic shock and other conditions of inflammatory end-organ damage.-Sparkes, A., De Baetselier, P., Brys, L., Cabrito, I., Sterckx, Y. G.-J., Schoonooghe, S., Muyldermans, S., Raes, G., Bucala, R., Vanlandschoot, P., Van Ginderachter, J. A., Stijlemans, B. Novel half-life extended anti-MIF nanobodies protect against endotoxic shock.

NIRF-Molecular Imaging with Synovial Macrophages-Targeting Vsig4 Nanobody for Disease Monitoring in a Mouse Model of Arthritis.

Nanobody against V-set and Ig domain-containing 4 (Vsig4) on tissue macrophages, such as synovial macrophages, could visualize joint inflammation in multiple experimental arthritis models via single-photon emission computed tomography imaging. Here, we further addressed the specificity and assessed the potential for arthritis monitoring using near-infrared fluorescence (NIRF) Cy7-labeled Vsig4 nanobody (Cy7-Nb119). In vivo NIRF-imaging of collagen-induced arthritis (CIA) was performed using Cy7-Nb119. Signals obtained with Cy7-Nb119 or isotope control Cy7-NbBCII10 were compared in joints of naive mice versus CIA mice. In addition, pathological microscopy and fluorescence microscopy were used to validate the arthritis development in CIA. Cy7-Nb119 accumulated in inflamed joints of CIA mice, but not the naive mice. Development of symptoms in CIA was reflected in increased joint accumulation of Cy7-Nb119, which correlated with the conventional measurements of disease. Vsig4 is co-expressed with F4/80, indicating targeting of the increasing number of synovial macrophages associated with the severity of inflammation by the Vsig4 nanobody. NIRF imaging with Cy7-Nb119 allows specific assessment of inflammation in experimental arthritis and provides complementary information to clinical scoring for quantitative, non-invasive and economical monitoring of the pathological process. Nanobody labelled with fluorescence can also be used for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Hierarchical and Redundant Roles of Activating FcγRs in Protection against Influenza Disease by M2e-Specific IgG1 and IgG2a Antibodies.

The ectodomain of matrix protein 2 is a universal influenza A virus vaccine candidate that provides protection through antibody-dependent effector mechanisms. Here we compared the functional engagement of Fcγ receptor (FcγR) family members by two M2e-specific monoclonal antibodies (MAbs), MAb 37 (IgG1) and MAb 65 (IgG2a), which recognize a similar epitope in M2e with similar affinities. The binding of MAb 65 to influenza A virus-infected cells triggered all three activating mouse Fcγ receptors in vitro, whereas MAb 37 activated only FcγRIII. The passive transfer of MAb 37 or MAb 65 in wild-type, Fcer1g-/-, Fcgr3-/-, and Fcgr1-/-Fcgr3-/- BALB/c mice revealed the importance of these receptors for protection against influenza A virus challenge, with a clear requirement of FcγRIII for IgG1 MAb 37 being found. We also report that FcγRIV contributes to protection by M2e-specific IgG2a antibodies.IMPORTANCE There is increased awareness that protection by antibodies directed against viral antigens is also mediated by the Fc domain of these antibodies. These Fc-mediated effector functions are often missed in clinical assays, which are used, for example, to define correlates of protection induced by vaccines. The use of antibodies to prevent and treat infectious diseases is on the rise and has proven to be a promising approach in our battle against newly emerging viral infections. It is now also realized that Fcγ receptors significantly enhance the in vivo protective effect of broadly neutralizing antibodies directed against the conserved parts of the influenza virus hemagglutinin. We show here that two M2e-specific monoclonal antibodies with close to identical antigen-binding specificities and affinities have a very different in vivo protective potential that is controlled by their capacity to interact with activating Fcγ receptors.

Q586B2 is a crucial virulence factor during the early stages of Trypanosoma brucei infection that is conserved amongst trypanosomatids.

Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment.

Development and Characterization of Nanobodies Targeting the Kupffer Cell.

Nanobodies that are derived from single-chain antibodies of camelids have served as powerful tools in diagnostics, therapeutics and investigation of membrane receptors' structure and function. In this study, we developed a series of nanobodies by a phage display screening building from lymphocytes isolated from an alpaca immunized with recombinant mouse Kupffer cell receptor Clec4F, which is involved in pathogen recognition by binding to galactose and N-acetylgalactosamine. Bio-panning selections retrieved 14 different nanobodies against Clec4F with an affinity ranging from 0.2 to 2 nM as determined by SPR. Those nanobodies mainly recognize 4 different epitopes as analyzed via competitive epitope binning. By analysis of the radioactivity in each organ after injection of 99mTc labeled Clec4F nanobodies in naïve mice, we found that these nanobodies are targeting the liver. Furthermore, we performed a structural characterization at atomic resolution of two of the Clec4F nanobodies from different epitope groups, which revealed distinct features within the CDR2 and CDR3 regions. Taken together, we developed a series of nanobodies targeting multiple distinct recognition epitopes of the Kupffer cell-specific receptor Clec4F which may be useful for its structural and functional investigation as well as for use as molecular imaging and therapeutic agents.

Targeting Neuropilin-1 with Nanobodies Reduces Colorectal Carcinoma Development.

Neuropilin-1 (NRP-1) is a co-receptor for semaphorins and vascular endothelial growth factor (VEGF) family members that can be expressed on cancer cells and tumor-infiltrating myeloid, endothelial and lymphoid cells. It has been linked to a tumor-promoting environment upon interaction with semaphorin 3A (Sema3A). Nanobodies (Nbs) targeting NRP-1 were generated for their potential to hamper the NRP-1/Sema3A interaction and their impact on colorectal carcinoma (CRC) development was evaluated in vivo through the generation of anti-NRP-1-producing CRC cells. We observed that tumor growth was significantly delayed and survival prolonged when the anti-NRP-1 Nbs were produced in vivo. We further analyzed the tumor microenvironment and observed that the pro-inflammatory MHC-IIhigh/trophic MHC-IIlow macrophage ratio was increased in tumors that produce anti-NRP-1 Nbs. This finding was corroborated by an increase in the expression of genes associated with MHC-IIhigh macrophages and a decrease in the expression of MHC-IIlow macrophage-associated genes in the macrophage pool sorted from anti-NRP-1 Nb-producing tumors. Moreover, we observed a significantly higher percentage of tumor-associated antigen-specific CD8+ T cells in tumors producing anti-NRP-1 Nbs. These data demonstrate that an intratumoral expression of NRP-1/Sema3A blocking biologicals increases anti-tumor immunity.

Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris.

BACKGROUND: Tumour associated antigens on the surface of tumour cells, such as MUC1, are being used as specific antibody targets for immunotherapy of human malignancies. In order to address the poor penetration of full sized monoclonal antibodies in tumours, intermediate sized antibodies are being developed. The cost-effective and efficient production of these molecules is however crucial for their further success as anti-cancer therapeutics. The methylotropic P. pastoris yeast grows in cheap mineral media and is known for its short process times and the efficient production of recombinant antibody fragments like scFvs, bivalent scFvs and Fabs. RESULTS: Based on the anti-MUC1 PH1 Fab, we have developed bivalent PH1 bibodies and trivalent PH1 tribodies of intermediate molecular mass by adding PH1 scFvs to the C-terminus of the Fab chains using flexible peptide linkers. These recombinant antibody derivatives were efficiently expressed in both mammalian and P. pastoris cells. Stable production in NS0 cells produced 130.5 mg pure bibody and 27 mg pure tribody per litre. This high yield is achieved as a result of the high overall purification efficiency of 77%. Expression and purification of PH1 bibodies and tribodies from Pichia supernatant yielded predominantly correctly heterodimerised products, free of light chain homodimers. The yeast-produced bi- and tribodies retained the same specific activity as their mammalian-produced counterparts. Additionally, the yields of 36.8 mg pure bibody and 12 mg pure tribody per litre supernatant make the production of these molecules in Pichia more efficient than most other previously described trispecific or trivalent molecules produced in E. coli. CONCLUSION: Bi- and tribody molecules are efficiently produced in P. pastoris. Furthermore, the yeast produced molecules retain the same specific affinity for their antigen. These results establish the value of P. pastoris as an efficient alternative expression system for the production of recombinant multivalent Fab-scFv antibody derivatives.

Monoclonal antibody 14C5 targets integrin alphavbeta5.

This study identifies and characterizes the antigen recognized by monoclonal antibody (mAb) 14C5. We compared the expression of antigen 14C5 with the expression of eight integrin subunits (alpha1, alpha2, alpha3, alphav, beta1, beta2, beta3, and beta4) and three integrin heterodimers (alphavbeta3, alphavbeta5, and alpha5beta1) by flow cytometry. Antigen 14C5 showed a similar expression to alphavbeta5 in eight different epithelial cancer cell lines (A549, A2058, C32, Capan-2, Colo16, HT-1080, HT-29, and SKBR-3). Specific binding of P1F6, an anti-alphavbeta5 specific antibody, was blocked by mAb 14C5. After transient expression of alphavbeta5 in 14C5-negative Colo16 cells, mAb 14C5 was able to bind a subpopulation of alphavbeta5-positive cells. We evaluated the tissue distribution of the 14C5 antigen in colon (n = 20) and lung (n = 16) cancer tissues. The colon carcinoma cells stained positive for 14C5 in 50% of tumors analyzed, whereas bronchoalveolar lung carcinoma and typical carcinoid were not positive for the antigen. More common types of non-small cell lung cancer, i.e., squamous (n = 5) and adenocarcinoma (n = 3), stained positive in 2 of 5 squamous carcinomas and in 1 of 3 investigated adenocarcinoma. Colon (95%) and lung (50%) carcinoma tissues showed extensive expression of antigen 14C5 in the stroma surrounding the tumor cells and on the membrane of the adjacent fibroblasts. We show for the first time that mAb 14C5 binds the vascular integrin alphavbeta5, suggesting that mAb 14C5 can be used as a screening agent to select colon and lung cancer patients that are eligible for anti-alphavbeta5-based therapies.

Single-domain antibody fusion proteins can target and shuttle functional proteins into macrophage mannose receptor expressing macrophages.

The tumor microenvironment of numerous prevalent cancer types is abundantly infiltrated with tumor-associated macrophages (TAMs). Macrophage mannose receptor (MMR or CD206) expressing TAMs have been shown to be key promoters of tumor progression and major opponents of successful cancer therapy. Therefore, depleting MMR+ TAMs is an interesting approach to synergize with current antitumor therapies. We studied the potential of single-domain antibodies (sdAbs) specific for MMR to target proteins to MMR+ TAMs. Anti-MMR sdAbs were genetically coupled to a reporter protein, mWasabi (wasabi green, WG), generating sdAb "drug" fusion proteins (SFPs), referred to as WG-SFPs. The resulting WG-SFPs were highly efficient in targeting MMR+ macrophages both in vitro and in vivo. As we showed that second mitochondria-derived activator of caspase (SMAC) mimetics modulate MMR+ macrophages, we further coupled the anti-MMR sdAb to an active form of SMAC, referred to as tSMAC. The resulting tSMAC-SFPs were able to bind and upregulate caspase3/7 activity in MMR+ macrophages in vitro. In conclusion, we report the proof-of-concept of an elegant approach to conjugate anti-MMR sdAbs to proteins, which opens new avenues for targeted manipulation of MMR+ tumor-promoting TAMs.

Evaluating a Single Domain Antibody Targeting Human PD-L1 as a Nuclear Imaging and Therapeutic Agent.

The PD-1:PD-L1 immune checkpoint axis is central in the escape of cancer cells from anticancer immune responses. Monoclonal antibodies (mAbs) specific for PD-L1 have been approved for treatment of various cancer types. Although PD-L1 blockade has proven its merit, there are still several aspects that require further attention to fully capitalize on its potential. One of these is the development of antigen-binding moieties that enable PD-L1 diagnosis and therapy. We generated human PD-L1 binding single domain antibodies (sdAbs) and selected sdAb K2, a sdAb with a high affinity for PD-L1, as a lead compound. SPECT/CT imaging in mice following intravenous injection of Technetium-99m (99mTc)-labeled sdAb K2 revealed high signal-to-noise ratios, strong ability to specifically detect PD-L1 in melanoma and breast tumors, and relatively low kidney retention, which is a unique property for radiolabeled sdAbs. We further showed using surface plasmon resonance that sdAb K2 binds to the same epitope on PD-L1 as the mAb avelumab, and antagonizes PD-1:PD-L1 interactions. Different human cell-based assays corroborated the PD-1:PD-L1 blocking activity, showing enhanced T-cell receptor signaling and tumor cell killing when PD-1POS T cells interacted with PD-L1POS tumor cells. Taken together, we present sdAb K2, which specifically binds to human PD-L1, as a new diagnostic and therapeutic agent in cancer management.

Biodistribution and planar gamma camera imaging of (123)I- and (131)I-labeled F(ab')(2) and Fab fragments of monoclonal antibody 14C5 in nude mice bearing an A549 lung tumor.

UNLABELLED: Detection of antigen 14C5, involved in substrate adhesion and highly expressed on the membrane of many carcinomas, including lung cancer, provides important diagnostic information that can influence patient management. The aim of this study was to evaluate the biodistribution and planar gamma camera imaging characteristics of radioiodinated F(ab')(2) and Fab fragments of monoclonal antibody (mAb) 14C5 in tumor-bearing mice. METHODS: F(ab')(2) and Fab 14C5 fragments were radioiodinated using the Iodo-Gen method. In vitro stability, binding specificity and affinity of (125)I-labeled 14C5 fragments were studied in A549 lung carcinoma cells. Biodistribution, blood clearance and tumor-targeting characteristics of (131)I-labeled 14C5 fragments and intact mAb 14C5 were studied in Swiss nu/nu mice bearing A549 lung carcinoma tumors. Planar gamma imaging illustrated the potential use of these (123)I-labeled 14C5 fragments for radioimmunodetection (RID). RESULTS: Saturation binding experiments showed highest affinity for (125)I-labeled F(ab')(2) fragments (K(d)=0.37+/-0.10 nmol/L) and lowest affinity for (125)I-labeled Fab fragments (K(d)=2.25+/-0.44 nmol/L). Blood clearance studies showed that the alpha half-life (t(1/2)alpha) value for Fab, F(ab')(2) and mAb 14C5 was 14.9, 21 and 118 min, respectively. The beta half-life t(1/2)beta value for Fab, F(ab')(2) and mAb 14C5 was 439, 627 and 4067 min, respectively. (131)I-Fab fragments showed highest tumor uptake 3 h after injection (2.4+/-0.8 %ID/g), (131)I-labeled F(ab')(2) showed highest tumor uptake 6 h after injection (4.7+/-0.7 %ID/g) and for (131)I-labeled mAb highest tumor uptake was observed at 24 h (10.7+/-2.3 %ID/g). In planar gamma imaging, both labeled fragments gave better tumor-to-background contrast than (123)I-mAb 14C5. CONCLUSION: Fab and F(ab')(2) fragments derived from intact mAb 14C5 have significant potential for diagnostic and therapeutic applications and may provide new tools in mAb-based radiopharmaceuticals for targeting non-small cell lung cancer.

Molecular Imaging with Kupffer Cell-Targeting Nanobodies for Diagnosis and Prognosis in Mouse Models of Liver Pathogenesis.

PURPOSE: Kupffer cells (KCs), the liver resident macrophages, are important mediators of tissue homeostasis and pathogen clearance. However, depending on the inflammatory stimuli, KCs have been involved in divergent hepato-protective or hepato-destructive immune responses. The versatility of KCs in response to environmental triggers, in combination with the specific biomarkers they express, make these macrophages attractive in vivo targets for non-invasive monitoring of liver inflammation or pathogenicity. This study aims to determine whether V-set and Ig domain-containing 4 (Vsig4) and C-type lectin domain family (Clec) 4, member F (Clec4F) can be used as imaging biomarkers for non-invasive monitoring of KCs during distinct liver inflammation models. PROCEDURE: Flow cytometry (FACS), immuno-histochemistry (IHC), and single-photon emission computed tomography (SPECT) with Tc-99m labeled anti-Vsig4 or anti-Clec4F nanobodies (Nbs) was performed to evaluate in mice KC dynamics in concanavalin A (ConA)-induced hepatitis and in non-alcoholic steatohepatitis induced via methionine choline deficiency (MCD). RESULTS: In homeostatic mice, Nbs targeting Clec4F were found to accumulate and co-localize with Vsig4-targeting Nbs only in the liver. Upon induction of acute hepatitis using ConA, down-regulation of the in vivo Nb imaging signal was observed, reflecting reduction in KC numbers as confirmed by FACS and IHC. On the other hand, induction of steatohepatitis resulted in higher signals in the liver corresponding to higher density of KCs. The Nb-imaging signals returned to normal levels after resolution of the investigated liver diseases. CONCLUSIONS: Anti-Clec4F and anti-Vsig4 Nbs targeting KCs as molecular imaging biomarkers could allow non-invasive monitoring/staging of liver pathogenesis.

Structural evaluation of a nanobody targeting complement receptor Vsig4 and its cross reactivity.

Vsig4 is a recently identified immune regulatory protein related to the B7 family with dual functionality: a negative regulator of T cell activation and a receptor for the complement components C3b and C3c. Here we present a structural evaluation of a nanobody, Nb119, against the extracellular IgV domain protein of both mouse and human recombinant Vsig4, which have a high degree of sequence identity. Although mouse and human Vsig4 bind to Nb119 with a 250 times difference in dissociation constants, the interaction results in a highly identical assembly with a RMSD of 0.4Å. The molecular determinants for Vsig4 recognition and cross reactivity unveiled by the atomic structure of Nb119 in complex with mVsig4 and hVsig4 afford new insights useful for the further optimization of the nanobody for potential use in humans. Additionally, structural analysis of the Vsig4-Nb119 complexes indicates that Nb119 occupies the interface on Vsig4 recognized by the macroglobulin-like domains MG4 and MG5 of C3b. Thus an affinity-improved Nb119 may have the potential to influence the activation of both T cells and complement.

In vitro and in vivo targeting properties of iodine-123- or iodine-131-labeled monoclonal antibody 14C5 in a non-small cell lung cancer and colon carcinoma model.

PURPOSE: The monoclonal antibody (mAb) 14C5 is a murine IgG1 directed against a yet undefined molecule involved in cell substrate adhesion found on the surface of malignant breast cancer tissue. mAb 14C5 is able to inhibit cell substrate adhesion and invasion of breast cancer cells in vitro. In normal tissues as well as in the stroma surrounding in situ carcinomas of the breast, no expression of the antigen 14C5 occurs. The aim of this study was to investigate the in vitro and in vivo targeting properties of 123I- and 131I-labeled mAb 14C5 as a novel agent for radioimmunodetection and radioimmunotherapy. EXPERIMENTAL DESIGN: Internalization of mAb 14C5 was investigated with 125I-labeled mAb 14C5 and by confocal laser scanning microscopy. Biodistribution studies of 131I-labeled mAb 14C5 and planar gamma imaging were done in nude mice bearing an A549 (non-small cell lung carcinoma) or a LoVo (colon carcinoma) tumor. RESULTS: Internalization studies with both A549 and LoVo cells showed that 125I-labeled mAb 14C5 is slowly internalized with approximately 30% of the initially bound mAb 14C5 internalized after 2 hours at 37 degrees C. Internalization of mAb 14C5 could be visualized with confocal laser scanning microscopy. In vivo, radioisotope uptake peaked at 24 hours for both tumor models (n = 5) with no significant difference in percentage of injected dose/g tissue (A549 10.4 +/- 0.8 and LoVo 9.3 +/- 0.8). Via planar gamma camera imaging, A549 lung tumors as well as LoVo colon tumors could be clearly visualized. CONCLUSIONS: The in vitro and in vivo targeting properties of 123I- and 131I-labeled mAb 14C5 are promising and could provide a new antibody-based agent for radioimmunodetection and radioimmunotherapy of patients bearing antigen 14C5-expressing tumors.

Specificity Evaluation and Disease Monitoring in Arthritis Imaging with Complement Receptor of the Ig superfamily targeting Nanobodies.

Single-photon emission computed tomography combined with micro-CT (SPECT/µCT) imaging using Nanobodies against complement receptor of the Ig superfamily (CRIg), found on tissue macrophages such as synovial macrophages, has promising potential to visualize joint inflammation in experimental arthritis. Here, we further addressed the specificity and assessed the potential for arthritis monitoring. Signals obtained with 99mTc-labelled NbV4m119 Nanobody were compared in joints of wild type (WT) versus CRIg-/- mice with collagen-induced arthritis (CIA) or K/BxN serum transfer-induced arthritis (STIA). In addition, SPECT/µCT imaging was used to investigate arthritis development in STIA and in CIA under dexamethasone treatment. 99mTc-NbV4m119 accumulated in inflamed joints of WT, but not CRIg-/- mice with CIA and STIA. Development and spontaneous recovery of symptoms in STIA was reflected in initially increased and subsequently reduced joint accumulation of 99mTc-NbV4m119. Dexamethasone treatment of CIA mice reduced 99mTc-NbV4m119 accumulation as compared to saline control in most joints except knees. SPECT/µCT imaging with 99mTc-NbV4m119 allows specific assessment of inflammation in different arthritis models and provides complementary information to clinical scoring for quantitatively and non-invasively monitoring the pathological process and the efficacy of arthritis treatment.

Bone marrow-derived monocytes give rise to self-renewing and fully differentiated Kupffer cells.

Self-renewing tissue-resident macrophages are thought to be exclusively derived from embryonic progenitors. However, whether circulating monocytes can also give rise to such macrophages has not been formally investigated. Here we use a new model of diphtheria toxin-mediated depletion of liver-resident Kupffer cells to generate niche availability and show that circulating monocytes engraft in the liver, gradually adopt the transcriptional profile of their depleted counterparts and become long-lived self-renewing cells. Underlining the physiological relevance of our findings, circulating monocytes also contribute to the expanding pool of macrophages in the liver shortly after birth, when macrophage niches become available during normal organ growth. Thus, like embryonic precursors, monocytes can and do give rise to self-renewing tissue-resident macrophages if the niche is available to them.

Monitoring liver macrophages using nanobodies targeting Vsig4: concanavalin A induced acute hepatitis as paradigm.

Kupffer cells (KCs) are liver resident macrophages which are important for tissue homeostasis and have been implicated in immunogenic, tolerogenic and pathogenic immune reactions depending on the insult. These cells and the biomarkers they express thus represent interesting in vivo sensors for monitoring liver inflammation. In the current study, we explored whether KCs can be monitored non-invasively using single-photon-emission computed tomography (SPECT) with (99m)Tc labeled nanobodies (Nbs) targeting selected biomarkers. Nbs targeting V-set and immunoglobulin domain-containing 4 (Vsig4) or macrophage mannose receptor (MMR) accumulated in the liver of untreated mice. The liver targeting of anti-Vsig4 Nbs, but not anti-MMR Nbs, was blunted upon depletion of macrophages, highlighting specificity of anti-Vsig4 Nbs for liver macrophage imaging. Ex vivo flow cytometry and immunohistochemistry analysis confirmed that anti-Vsig4 Nbs specifically targeted KCs but no other cell types in the liver. Upon induction of acute hepatitis using concanavalin A (ConA), down-regulation of the in vivo imaging signal obtained using anti-Vsig4 Nbs reflected reduction in KC numbers and transient modulation of Vsig4 expression on KCs. Overall, these results indicate that Nbs targeting Vsig4 as molecular imaging biomarker enable non-invasive monitoring of KCs during hepatic inflammation.

PET Imaging of Macrophage Mannose Receptor-Expressing Macrophages in Tumor Stroma Using 18F-Radiolabeled Camelid Single-Domain Antibody Fragments.

UNLABELLED: Tumor-associated macrophages constitute a major component of the stroma of solid tumors, encompassing distinct subpopulations with different characteristics and functions. We aimed to identify M2-oriented tumor-supporting macrophages within the tumor microenvironment as indicators of cancer progression and prognosis, using PET imaging. This can be realized by designing (18)F-labeled camelid single-domain antibody fragments (sdAbs) specifically targeting the macrophage mannose receptor (MMR), which has been identified as an important biomarker on this cell population. METHODS: Cross-reactive anti-MMR sdAbs were generated after immunization of an alpaca with the extracellular domains of both human and mouse MMR. The lead binder was chosen on the basis of comparisons of binding affinity and in vivo pharmacokinetics. The PET tracer (18)F-fluorobenzoate (FB)-anti-MMR sdAb was developed using the prosthetic group N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-SFB), and its biodistribution, tumor-targeting potential, and specificity in terms of macrophage and MMR targeting were evaluated in mouse tumor models. RESULTS: Four sdAbs were selected after affinity screening, but only 2 were found to be cross-reactive for human and mouse MMR. The lead anti-MMR 3.49 sdAb, bearing an affinity of 12 and 1.8 nM for mouse and human MMR, respectively, was chosen for its favorable in vivo biodistribution profile and tumor-targeting capacity. (18)F-FB-anti-MMR 3.49 sdAb was synthesized with a 5%-10% radiochemical yield using an automated and optimized protocol. In vivo biodistribution analyses showed fast clearance via the kidneys and retention in MMR-expressing organs and tumor. The kidney retention of the fluorinated sdAb was 20-fold lower than a (99m)Tc-labeled counterpart. Compared with MMR- and C-C chemokine receptor 2-deficient mice, significantly higher uptake was observed in tumors grown in wild-type mice, demonstrating the specificity of the (18)F tracer for MMR and macrophages, respectively. CONCLUSION: Anti-MMR 3.49 was denoted as the lead cross-reactive MMR-targeting sdAb. (18)F radiosynthesis was optimized, providing an optimal probe for PET imaging of the tumor-promoting macrophage subpopulation in the tumor stroma.