CD64: An Attractive Immunotherapeutic Target for M1-type Macrophage Mediated Chronic Inflammatory Diseases.

To date, no curative therapy is available for the treatment of most chronic inflammatory diseases such as atopic dermatitis, rheumatoid arthritis, or autoimmune disorders. Current treatments require a lifetime supply for patients to alleviate clinical symptoms and are unable to stop the course of disease. In contrast, a new series of immunotherapeutic agents targeting the Fc γ receptor I (CD64) have emerged and demonstrated significant clinical potential to actually resolving chronic inflammation driven by M1-type dysregulated macrophages. This subpopulation plays a key role in the initiation and maintenance of a series of chronic diseases. The novel recombinant M1-specific immunotherapeutics offer the prospect of highly effective treatment strategies as they have been shown to selectively eliminate the disease-causing macrophage subpopulations. In this review, we provide a detailed summary of the data generated, together with the advantages and the clinical potential of CD64-based targeted therapies for the treatment of chronic inflammatory diseases.

CD64-directed microtubule associated protein tau kills leukemic blasts ex vivo.

Fc gamma receptor I (FcγRI, CD64) is a well-known target antigen for passive immunotherapy against acute myeloid leukemia and chronic myelomonocytic leukemia. We recently reported the preclinical immunotherapeutic potential of microtubule associated protein tau (MAP) against a variety of cancer types including breast carcinoma and Hodgkin's lymphoma. Here we demonstrate that the CD64-directed human cytolytic fusion protein H22(scFv)-MAP kills ex vivo 15-50% of CD64+ leukemic blasts derived from seven myeloid leukemia patients. Furthermore, in contrast to the nonspecific cytostatic agent paclitaxel, H22(scFv)-MAP showed no cytotoxicity towards healthy CD64+ PBMC-derived cells and macrophages. The targeted delivery of this microtubule stabilizing agent therefore offers a promising new strategy for specific treatment of CD64+ leukemia.

Fully human MAP-fusion protein selectively targets and eliminates proliferating CD64(+) M1 macrophages.

Classical immunotoxins compromise a binding component (for example, a ligand, antibody or fragment thereof) and a cytotoxic component, usually derived from bacteria or plants (for example, Pseudomonas exotoxin A or ricin). Despite successful testing in vitro, the clinical development of immunotoxins has been hampered by immunogenicity and unsatisfactory safety profiles. Therefore, research has focused on fully human pro-apoptotic components suitable for the development of cytolytic fusion proteins (CFP). We recently reported that human microtubule-associated protein tau (MAP) can induce apoptosis when delivered to rapidly proliferating cancer cells. Here, we describe a new fully human CFP called H22(scFv)-MAP, which specifically targets CD64(+) cells. We show that H22(scFv)-MAP can efficiently kill proliferating HL-60 pro-monocytic cells in vitro. In addition, the human CFP specifically eliminates polarized M1 macrophages in a transgenic mouse model of cutaneous chronic inflammation. Because M1 macrophages promote the pathogenesis of many chronic inflammatory diseases, targeting this cell population with H22(scFv)-MAP could help to treat diseases such as atopic dermatitis, rheumatoid arthritis and inflammatory bowel disease.

Targeting CD64 mediates elimination of M1 but not M2 macrophages in vitro and in cutaneous inflammation in mice and patient biopsies.

Macrophages are key players in controlling the immune response that can adapt to microenvironmental signals. This results in distinct polarization states (classical M1 or alternative M2), that play a differential role in immune regulation. In general, the M1 contribute to onset of inflammation, whereas the M2 orchestrate resolution and repair, whereby failure to switch from predominantly M1 to M2 reinforces a pro-inflammatory environment and chronic inflammation. Here, we show selective elimination of M1 macrophages in vitro by a range of CD64-targeted immunotoxins, including H22(scFv)-ETA'. After re-polarization of already polarized macrophages, still only M1 polarization showed sensitivity toward CD64-directed immunotoxins. The selectivity for M1 was found linked to reduced endosomal protease activity in M1 macrophages as demonstrated by inhibition of endosomal proteases. Using the H22(scFv)-ETA' in a transgenic mouse model for chronic cutaneous inflammation, the M1 specificity was confirmed in vivo and a beneficial effect on inflammation demonstrated. Also ex vivo on skin biopsies from atopic dermatitis and diabetes type II patients with chronically-inflamed skin, a clear M1 specific effect was found. This indicates the potential relevance for human application. Our data show that targeting M1 macrophages through CD64 can be instrumental in developing novel intervention strategies for chronic inflammatory conditions.

Recombinant H22(scFv) blocks CD64 and prevents the capture of anti-TNF monoclonal antibody. A potential strategy to enhance anti-TNF therapy.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that plays a critical role in many inflammatory diseases. Soluble TNF can be neutralized by monoclonal antibodies (mAbs), and this is a widely-used therapeutic approach. However, some patients do not respond to anti-TNF therapy due to the increased expression of CD64 on monocytes and macrophages. A recent study has shown that CD64 captures anti-TNF mAbs via their Fcγ domain, which induces the transcription of pro-inflammatory genes. Specific blocking of CD64 could therefore be a promising strategy to improve the response to anti-TNF therapy. We used the CD64-specific antibody fragment H22(scFv) and tested its activity against the human CD64(+) cell line HL-60. When stimulated with interferon gamma (IFN-γ), these cells represent a pro-inflammatory phenotype of the monocyte/macrophage lineage. We found that H22(scFv) binds selectively to and blocks CD64, preventing the capture of anti-TNF mAb. Importantly, H22(scFv) itself does not induce CD64 activation. We also found that transmembrane TNF on HL-60 cells stimulated with IFN-γ also contributes to the capture of anti-TNF mAb, although via their Fab domain. In conclusion, the specific blocking of CD64 by H22(scFv) could be used a possible anti-inflammatory mechanism for potentiating the effect of anti-TNF antibodies.

EpCAM-selective elimination of carcinoma cells by a novel MAP-based cytolytic fusion protein.

In normal epithelia, the epithelial cell adhesion molecule (EpCAM) expression is relatively low and only present at the basolateral cell surface. In contrast, EpCAM is aberrantly overexpressed in various human carcinomas. Therefore, EpCAM is considered to be a highly promising target for antibody-based cancer immunotherapy. Here, we present a new and fully human cytolytic fusion protein (CFP), designated "anti-EpCAM(scFv)-MAP," that is comprised of an EpCAM-specific antibody fragment (scFv) genetically fused to the microtubule-associated protein tau (MAP). Anti-EpCAM(scFv)-MAP shows potent EpCAM-restricted proapoptotic activity toward rapidly proliferating carcinoma cells. In vitro assays confirmed that treatment with anti-EpCAM(scFv)-MAP resulted in the colocalization and stabilization of microtubules, suggesting that this could be the potential mode of action. Dose-finding experiments indicated that anti-EpCAM(scFv)-MAP is well tolerated in mice. Using noninvasive far-red in vivo imaging in a tumor xenograft mouse model, we further demonstrated that anti-EpCAM(scFv)-MAP inhibited tumor growth in vivo. In conclusion, our data suggest that anti-EpCAM(scFv)-MAP may be of therapeutic value for the targeted elimination of EpCAM(+) carcinomas.

Targeted ex vivo reduction of CD64-positive monocytes in chronic myelomonocytic leukemia and acute myelomonocytic leukemia using human granzyme B-based cytolytic fusion proteins.

CMML (chronic myelomonocytic leukemia) belongs to the group of myeloid neoplasms known as myelodysplastic and myeloproliferative diseases. In some patients with a history of CMML, the disease transforms to acute myelomonocytic leukemia (AMML). There are no specific treatment options for patients suffering from CMML except for supportive care and DNA methyltransferase inhibitors in patients with advanced disease. New treatment strategies are urgently required, so we have investigated the use of immunotherapeutic directed cytolytic fusion proteins (CFPs), which are chimeric proteins comprising a selective domain and a toxic component (preferably of human origin to avoid immunogenicity). The human serine protease granzyme B is a prominent candidate for tumor immunotherapy because it is expressed in cytotoxic T lymphocytes and natural killer cells. Here, we report the use of CD64 as a novel target for specific CMML and AMML therapy, and correlate CD64 expression with typical surface markers representing these diseases. We demonstrate that CD64-specific human CFPs kill CMML and AMML cells ex vivo, and that the mutant granzyme B protein R201K is more cytotoxic than the wild-type enzyme in the presence of the granzyme B inhibitor PI9. Besides, the human CFP based on the granzyme B mutant was also able to kill AMML or CMML probes resistant to Pseudomonas exotoxin A.

Human microtubule-associated protein tau mediates targeted killing of CD30(+) lymphoma cells in vitro and inhibits tumour growth in vivo.

Hodgkin lymphoma (HL) and systemic anaplastic large cell lymphoma (sALCL) are rare lymphoproliferative cancer types. Although most HL patients can be cured by chemo- and radio-therapy, 4-50% of patients relapse and have a poor prognosis. The need for improved therapeutic options for patients with relapsed or refractory disease has been addressed by CD30-specific antibody-based immunotherapeutics. However, available CD30-specific monoclonal antibodies (mAbs), antibody drug conjugates (ADCs) or chimeric immunotoxins suffer from the requirement of a functional host immunity, undesirable immune reactions or heterogeneity and instability, respectively. Here, we present a new fusion protein comprised of the CD30-specific antibody single-chain fragment Ki4(scFv) and the human pro-apoptotic effector protein, microtubule-associated protein tau (MAPT). Ki4(scFv)-MAP selectively induced apoptosis in rapidly proliferating L540cy, L428, and Karpas 299 cells in a dose-dependent manner. Tubulin polymerization assays confirmed that Ki4(scFv)-MAP stabilizes microtubules, suggesting a mechanism for its pro-apoptotic action. Dose-finding experiments proved that Ki4(scFv)-MAP is well tolerated in mice compared to the previously reported Ki4(scFv)-ETA'. Ki4(scFv)-MAP significantly inhibited growth of subcutaneous L540cy xenograft tumours in mice. Our data present a novel approach for the treatment of CD30(+) lymphomas, combining the binding specificity of a target-specific antibody fragment with the selective cytotoxicity of MAPT towards proliferating lymphoma cells.

Granzyme M as a novel effector molecule for human cytolytic fusion proteins: CD64-specific cytotoxicity of Gm-H22(scFv) against leukemic cells.

Immunotoxins are promising targeted therapeutic agents comprising an antibody-based ligand that specifically binds to diseased cells, and a pro-apoptotic protein. Toxic components from bacteria or plants can trigger a neutralizing immune response, so that human effector molecules are more suitable. In this context, the protease granzyme B has been successfully tested in cytotoxicity assays against different cancer cells in vitro and in vivo. Our aim here was to introduce granzyme M as an alternative and novel component of human cytolytic fusion proteins. We fused it to the humanized single-chain antibody fragment (scFv) H22 which specifically binds to CD64, an FcγRI receptor overexpressed on activated myeloid cells and leukemic cells. We show that the humanized cytolytic fusion protein Gm-H22(scFv) specifically targets the acute myeloid leukemia cell line HL60 in vitro and is cytotoxic with an IC50 between 1.2 and 6.4 nM. These findings were confirmed ex vivo using leukemic primary cells from patients, which were killed by granzyme M despite the presence of the granzyme B inhibitor serpin B9. In conclusion, granzyme M is a promising new cell-death inducing component for hCFPs because it specifically and efficiently kills target cells when fused to a targeting component.

Macrophage-targeted therapy: CD64-based immunotoxins for treatment of chronic inflammatory diseases.

Diseases caused by chronic inflammation (e.g., arthritis, multiple sclerosis and diabetic ulcers) are multicausal, thus making treatment difficult and inefficient. Due to the age-associated nature of most of these disorders and the demographic transition towards an overall older population, efficient therapeutic intervention strategies will need to be developed in the near future. Over the past decades, elimination of activated macrophages using CD64-targeting immunotoxins has proven to be a promising way of resolving inflammation in animal models. More recent data have shown that the M1-polarized population of activated macrophages in particular is critically involved in the chronic phase. We recapitulate the latest progress in the development of IT. These have advanced from full-length antibodies, chemically coupled to bacterial toxins, into single chain variants of antibodies, genetically fused with fully human enzymes. These improvements have increased the range of possible target diseases, which now include chronic inflammatory diseases. At present there are no therapeutic strategies focusing on macrophages to treat chronic disorders. In this review, we focus on the role of different polarized macrophages and the potential of CD64-based IT to intervene in the process of chronic inflammation.

Labeling of anti-MUC-1 binding single chain Fv fragments to surface modified upconversion nanoparticles for an initial in vivo molecular imaging proof of principle approach.

In vivo optical Imaging is an inexpensive and highly sensitive modality to investigate and follow up diseases like breast cancer. However, fluorescence labels and specific tracers are still works in progress to bring this promising modality into the clinical day-to-day use. In this study an anti-MUC-1 binding single-chain antibody fragment was screened, produced and afterwards labeled with newly designed and surface modified NaYF(4):Yb,Er upconversion nanoparticles as fluorescence reporter constructs. The MUC-1 binding of the conjugate was examined in vitro and in vivo using modified state-of-the-art small animal Imaging equipment. Binding of the newly generated upconversion nanoparticle based probe to MUC-1 positive cells was clearly shown via laser scanning microscopy and in an initial proof of principal small animal optical imaging approach.

Reduction of activated macrophages after ischaemia-reperfusion injury diminishes oxidative stress and ameliorates renal damage.

BACKGROUND: Macrophages are major effectors of the local inflammatory response syndrome (LIRS) and influence the extent of ischaemia/reperfusion injury, thereby impacting organ function. Several subgroups of macrophages exist, representing distinct modes of action. The specific role of the subset expressing Fc gamma receptor (FcγR) 1 in the activated state of macrophages is poorly defined. METHODS: We induced a LIRS via 30 min of ischaemia in uninephrectomized rats, transgenic for the human FcγR1. Six hours after reperfusion, the treatment group was injected with a recombinant immunotoxin (IT) H22(scFv)-ETA' targeted against human FcγR1, which induced apoptosis of target cells. The placebo group received normal saline (NS). Contralateral kidneys served as healthy controls (Ctr). After 24 h of reperfusion, the animals were analysed. RESULTS: Targeted treatment with IT resulted in preserved renal function [NS versus IT treatment and baseline (creatinine: 69.2 ± 2.6, 54.7 ± 3.4 and 27.3 ± 1.0 µmol/L; P < 0.001)]. The number of all infiltrating monocytes were significantly reduced (CD68-positive cells per view field: NS 3.8 ± 0.4, IT 2.5 ± 0.2 and Ctr 1.2 ± 0.4; P < 0.05), renal histology improved and there was a reduced expression of renal fibronectin (NS 4.0 ± 0.4, IT 2.3 ± 0.2 and Ctr 1.1 ± 0.1; P < 0.001). Following IT administration, we also observed less expression of renal monocyte chemoattractant protein-1-positive cells per view field (NS 19.0 ± 1, IT 10.1 ± 0.8 and Ctr 2.0 ± 0.3; P < 0.001) as well as reduced systemic and local oxidative stress [serum malondialdehyde (MDA): NS 340 ± 30, IT 224 ± 36 versus baseline 140 ± 5 nmol/mL; P < 0.01]; renal MDA arbitrary units of fluorescence intensity: NS 3.7 ± 0.2, IT 1.8 ± 0.3 and Ctr 0.4 ± 0.2; P < 0.001. CONCLUSIONS: Reduction of FcγR1-up-regulated monocytic cells leads to preserved renal function and morphology in a rat model of ischaemia-triggered LIRS. Our results show that targeting activated macrophages is a valuable approach for ameliorating ischaemia-induced tissue injury.

In vivo imaging of immunotoxin treatment using Katushka-transfected A-431 cells in a murine xenograft tumour model.

PURPOSE: Preclinical in vivo analyses of treatment responses are an important prerequisite to evaluate new therapeutics. Molecular in vivo imaging in the far red (FR)/near infra red (NIR) is a promising method, as it enables measurements at different time points in individual animals, thereby reducing the number of animals required, while increasing statistical significance. Here, we show the establishment of a method to monitor response to treatment using fluorescent cells, expressing the epidermal growth factor receptor (EGFR), a target already used in therapy. METHODS: We transfected A-431 tumour cells with the far red-emitting protein Katushka (Kat2), resulting in strong fluorescence allowing for the monitoring of tumour growth when implanted in BALB/c nu/nu mice with a CRi Maestro in vivo imager. We targeted A-431 cells with a previously reported immunotoxin (IT), consisting of the anti-EGFR antibody single-chain variable fragment (scFv) 425, fused to Pseudomonas aeruginosa Exotoxin A' (ETA'). In addition, EGFR expression was verified using the 425(scFv) conjugated to a NIR dye BG-747 through a SNAP-tag linker. RESULTS: The results show the feasibility to evaluate response to treatment in vivo by FR imaging, while at the same location detecting EGFR expression. Treatment with 425(scFv)-ETA' resulted in decelerated tumour growth, while not affecting the overall health of the animals. This is in contrast to treatment with Doxorubicin, which, although decreasing the tumour size, resulted in poor health. CONCLUSIONS: We developed a novel method to non-invasively determine treatment responses by in vivo imaging of multiple parameters which showed the efficacy of 425(scFv)-ETA'.

In vivo efficacy of the recombinant anti-CD64 immunotoxin H22(scFv)-ETA' in a human acute myeloid leukemia xenograft tumor model.

Target-specific acute myeloid leukemia (AML) immunotherapy requires selective cell-surface antigens on AML blast cells. CD64 is a promising candidate antigen because it is abundantly expressed on monocytoid differentiated AML subtypes. In previous studies, a chemically linked full-length anti-CD64 immunotoxin based on ricin A showed promising results in several animal models, but further development has been hindered by its substantial, dose-limiting off-target effects. We recently constructed the recombinant immunotoxin H22(scFv)-ETA', comprising a truncated Pseudomonas exotoxin A (PE) and a humanized scFv antibody against CD64. This molecule was shown to kill CD64(+) AML-derived tumor cell lines and primary patient-derived AML cells specifically, both in vitro and ex vivo. Here we describe the in vivo efficiency of H22(scFv)-ETA' in the U937/SCID mouse xenograft model for human AML, by providing immunohistochemical evidence for the elimination of human CD64(+) tumor cells in mouse organs. H22(scFv)-ETA' showed potent antitumor activity against myeloid tumor cells and significantly prolonged the overall survival of AML xenograft animals. In conclusion, H22(scFv)-ETA' is efficacious against AML with monocytoid differentiation in vitro and in animal models in vivo, providing the basis for a novel therapeutic strategy for the treatment of AML patients.

Rapid optical imaging of EGF receptor expression with a single-chain antibody SNAP-tag fusion protein.

PURPOSE: The epidermal growth factor receptor (EGFR) is overexpressed in several types of cancer and its inhibition can effectively inhibit tumour progression. The purpose of this study was to design an EGFR-specific imaging probe that combines efficient tumour targeting with rapid systemic clearance to facilitate non-invasive assessment of EGFR expression. METHODS: Genetic fusion of a single-chain antibody fragment with the SNAP-tag produced a 48-kDa antibody derivative that can be covalently and site-specifically labelled with substrates containing 0 (6)-benzylguanine. The EGFR-specific single-chain variable fragment (scFv) fusion protein 425(scFv)SNAP was labelled with the near infrared (NIR) dye BG-747, and its accumulation, specificity and kinetics were monitored using NIR fluorescence imaging in a subcutaneous pancreatic carcinoma xenograft model. RESULTS: The 425(scFv)SNAP fusion protein accumulates rapidly and specifically at the tumour site. Its small size allows efficient renal clearance and a high tumour to background ratio (TBR) of 33.2 +/- 6.3 (n = 4) 10 h after injection. Binding of the labelled antibody was efficiently competed with a 20-fold excess of unlabelled probe, resulting in an average TBR of 6 +/- 1.35 (n = 4), which is similar to that obtained with a non-tumour-specific probe (5.44 +/- 1.92, n = 4). When compared with a full-length antibody against EGFR (cetuximab), 425(scFv)SNAP-747 showed significantly higher TBRs and complete clearance 72 h post-injection. CONCLUSION: The 425(scFv)SNAP fusion protein combines rapid and specific targeting of EGFR-positive tumours with a versatile and robust labelling technique that facilitates the attachment of fluorophores for use in optical imaging. The same approach could be used to couple a chelating agent for use in nuclear imaging.

Recombinant bispecific single chain antibody fragments induce Fc gamma-receptor-mediated elimination of CD30+ lymphoma cells.

Bispecific molecules (BSMs) facilitate the targeting of immune effector cells to tumor cells. Here we describe the construction and characterization of a recombinant BSM comprising two single chain fragments: H22(scFv), targeting the Fc gamma-receptor (CD64) on monocytes, and Ki4(scFv), targeting CD30 on Hodgkin lymphoma cells. A homologous, chemically-linked BSM has been described previously, but is heterogeneous and difficult to prepare. The recombinant version is easier to prepare and homogeneous, yet retains the antigen specificities and efficiently triggers CD64-related effector functions. The elimination of lymphoma cells was preferentially achieved by phagocytosis, not through the ADCC pathway additionally activated by the chemically-linked molecule.

Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes.

Acute myeloid leukemia (AML) cells of subtypes M4 and M5 show enhanced expression of CD64 (FcgammaRI), the high-affinity receptor for IgG, which is normally expressed at high levels only on activated cells of the myeloid lineage. CD64 is therefore a prime target for the specific delivery of cytotoxic agents. A promising toxin candidate is granzyme B, a human serine protease originating from cytotoxic granules of CD8+ T lymphocytes and natural killer cells. After evaluating the sensitivity of the AML-related cell line U937 toward cytosolic granzyme B, we genetically fused granzyme B to H22, a humanized single-chain antibody fragment (scFv) specific for CD64, to obtain Gb-H22(scFv), a fusion protein lacking the immunogenic properties of nonhuman immunofusions. Gb-H22(scFv) was successfully expressed in human 293T cells, secreted, and purified from cell culture supernatants. The purified protein bound specifically to CD64+ U937 cells. Despite linkage to the binding domain, the proteolytic activity of functional Gb-H22(scFv) was identical to that of free granzyme B. Target cell-specific cytotoxicity was observed with a half-maximal inhibitory concentration (IC50) between 1.7 and 17 nmol/L. In addition, the induction of apoptosis in U937 cells was confirmed by Annexin A5 staining and the detection of activated caspase-3 in the cytosol. Finally, apoptosis was observed in primary CD64+ AML cells, whereas CD64(-) AML cells were unaffected. This is the first report of a completely human granzyme B-based immunotoxin directed against CD64, with activity against an AML-related cell line and primary AML cells.

Depletion of autoreactive B-lymphocytes by a recombinant myelin oligodendrocyte glycoprotein-based immunotoxin.

We report the construction of a fusion protein comprising the extracellular domain of myelin oligodendrocyte glycoprotein (MOG) and a truncated version of Pseudomonas aeruginosa exotoxin A (ETA'). The chimeric immunotoxin targeted MOG-reactive B-lymphocytes by binding selectively to the appropriate receptors, leading to internalization and apoptosis of the target cells. The functionality of the immunotoxin was tested on a MOG-sensitive murine hybridoma cell line and ex vivo on freshly isolated splenocytes from transgenic IgH(MOG) mice. These data demonstrate, for the first time, the specific cytotoxicity of a MOG-containing recombinant immunotoxin expressed in bacteria towards MOG-reactive B-lymphocytes.

CD64-directed immunotoxin inhibits arthritis in a novel CD64 transgenic rat model.

Macrophages are known to play a key role during inflammation in rheumatoid arthritis (RA). Inflammatory macrophages have increased expression of CD64, the high-affinity receptor for IgG. Targeting this receptor through a CD64-directed immunotoxin, composed of an Ab against CD64 and Ricin A, results in effective killing of inflammatory macrophages. In this study, we show elevated levels of CD64 on synovial macrophages in both synovial lining and synovial fluid in RA patients. The CD64-directed immunotoxin efficiently eliminates activated synovial macrophages in vitro, while leaving quiescent, low CD64-expressing macrophages unaffected. To examine whether killing of CD64 macrophages results in therapeutic effects in vivo, we established an adjuvant arthritis (AA) model in newly generated human CD64 (hCD64) transgenic rats. We demonstrate that hCD64 regulation in this transgenic rat model is similar as in humans. After AA induction, treatment with CD64-directed immunotoxin results in significant inhibition of disease activity. There is a direct correlation between immunotoxin treatment and decreased macrophage numbers, followed by diminished inflammation and bone erosion in paws of these hCD64 transgenic rats. These data support synovial macrophages to play a crucial role in joint inflammation in AA in rats and in human RA. Selective elimination of inflammatory macrophages through a CD64-directed immunotoxin may provide a novel approach for treatment of RA.

Recombinant soluble human Fcgamma receptor I with picomolar affinity for immunoglobulin G.

The ectodomain of human FcgammaRI (rsCD64) was expressed in HEK 293T cells and purified by immobilized-metal affinity chromatography. Binding activity to human IgG was verified by ELISA and the isotype-specificity determined by a surface plasmon resonance inhibition assay was found to be the same as for native CD64. The active concentration of the rsCD64 preparation was derived using a solution competition assay and was used for the subsequent kinetic analysis. Binding curves were well described by a simple monovalent interaction model confirming the known stoichiometry of the interaction. Mass-transport limitation was prevented by using sufficiently low surface capacities. For binding to the recombinant mouse/human chimeric antibody cPIPP (IgG1/kappa) a high association rate of k(ass)=1.7 x 10(6) (M s)(-1) and a low dissociation rate of k(diss)=1.8 x 10(-4) s(-1) were observed. The derived dissociation equilibrium constant of K(D)=110 pM was significantly lower than that reported for binding to native FcgammaRI.