Salivary Gland Mucosa-Associated Lymphoid Tissue-Type Lymphoma From Sjögren's Syndrome Patients in the Majority Express Rheumatoid Factors Affinity-Selected for IgG.

OBJECTIVE: Patients with SjÓ§gren's syndrome (SS) have an increased risk of developing malignant B cell lymphomas, particularly mucosa-associated lymphoid tissue (MALT)-type lymphomas. We have previously shown that a predominant proportion of patients with SS-associated salivary gland MALT lymphoma express somatically hypermutated IgM with strong amino acid sequence homology with stereotypic rheumatoid factors (RFs). The present study was undertaken in a larger cohort of patients with SS-associated MALT lymphoma to more firmly assess the frequency of RF reactivity and the significance of somatic IGV-region mutations for RF reactivity. METHODS: B cell antigen receptors (BCRs) of 16 patients with SS-associated salivary gland MALT lymphoma were analyzed. Soluble recombinant IgM was produced of 12 MALT lymphoma samples, including 1 MALT lymphoma sample that expressed an IgM antibody fitting in a novel IGHV3-30-encoded stereotypic IGHV subset. For 4 of the 12 IgM antibodies from MALT lymphoma samples, the somatically mutated IGHV and IGKV gene sequences were reverted to germline configurations. Their RF activity and binding affinity were determined by enzyme-linked immunosorbent assay and surface plasmon resonance, respectively. RESULTS: Nine (75%) of the 12 IgM antibodies identified in patients with SS-associated salivary gland MALT lymphoma displayed strong monoreactive RF activity. Reversion of the IGHV and IGKV mutations to germline configuration resulted in RF affinities for IgG that were significantly lower for 3 of the 4 somatically mutated IgM antibodies. In stereotypic IGHV3-7/IGKV3-15-encoded RFs, a recurrent replacement mutation in the IGKV3-15-third complementarity-determining region was found to play a pivotal role in the affinity for IgG-Fc. CONCLUSION: A majority of patients with SS-associated salivary gland MALT lymphoma express somatically mutated BCRs that are selected for monoreactive, high-affinity binding of IgG-Fc. These data underscore the notion that soluble IgG, most likely in immune complexes in inflamed tissues, is the principal autoantigen in the pathogenesis of a variety of B cell lymphomas, particularly SS-associated MALT lymphomas.

Highly Efficient Mono-Functionalization of Knob-in-Hole Antibodies with Strain-Promoted Click Chemistry.

Knob-in-hole antibodies can be utilized to introduce a single tag for chemo-enzymatic functionalization. By either introducing a single C-terminal sortase tag (sortase-tag expressed protein ligation) or tyrosine tag (G4Y), mono-functionalization of the monoclonal antibody trastuzumab was achieved rapidly and in high yields. This method was applied to selectively and efficiently introduce a single fluorescent tag, cytokine or single-chain variable fragment, as well as produce clean homo dimers of trastuzumab.

A Chemo-enzymatically Linked Bispecific Antibody Retargets T Cells to a Sialylated Epitope on CD43 in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a high-risk disease with a poor prognosis, particularly in elderly patients. Because current AML treatment relies primarily on untargeted therapies with severe side effects that limit patient eligibility, identification of novel therapeutic AML targets is highly desired. We recently described AT1413, an antibody produced by donor B cells of a patient with AML cured after allogeneic hematopoietic stem cell transplantation. AT1413 binds CD43s, a unique sialylated epitope on CD43, which is weakly expressed on normal myeloid cells and overexpressed on AML cells. Because of its selectivity for AML cells, we considered CD43s as a target for a bispecific T-cell-engaging antibody (bTCE) and generated a bTCE by coupling AT1413 to two T-cell-targeting fragments using chemo-enzymatic linkage. In vitro, AT1413 bTCE efficiently induced T-cell-mediated cytotoxicity toward different AML cell lines and patient-derived AML blasts, whereas endothelial cells with low binding capacity for AT1413 remained unaffected. In the presence of AML cells, AT1413 bTCE induced upregulation of T-cell activation markers, cytokine release, and T-cell proliferation. AT1413 bTCE was also effective in vivo. Mice either coinjected with human peripheral blood mononuclear cells or engrafted with human hematopoietic stem cells [human immune system (HIS) mice] were inoculated with an AML cell line or patient-derived primary AML blasts. AT1413 bTCE treatment strongly inhibited tumor growth and, in HIS mice, had minimal effects on normal human hematopoietic cells. Taken together, our results indicate that CD43s is a promising target for T-cell-engaging antibodies and that AT1413 holds therapeutic potential in a bTCE-format. SIGNIFICANCE: These findings offer preclinical evidence for the therapeutic potential of a bTCE antibody that targets a sialylated epitope on CD43 in AML.

Preparation of bispecific antibody-protein adducts by site-specific chemo-enzymatic conjugation.

Historically, bispecific antibodies have been constructed through the genetic fusion of additional binding domains to the constant domains of the antibody heavy- or light chains. We present an alternative method for the introduction of additional functional domains to an antibody: site-specific chemo-enzymatic conjugation. This method relies on the combination of site-specific transpeptidases and bioorthogonal chemistry. Transpeptidases are used to site-specifically introduce chemical handles, which can then be used to couple new functional groups by means of a bioorthogonal chemical reaction. We demonstrate site-specific chemo-enzymatic linkage using the transpeptidase sortase (hereafter: sortase) and either a strain-promoted alkyne-azide cycloaddition (SPAAC) or an inverse-electron demand Diels-Alder reaction. Other transpeptidases and bioorthogonal reactions suitable for this purpose exist. Site-specific chemo-enzymatic linkage is a modular method. After introduction of a chemical handle in the antibody, any functional group of interest may then be attached. The modularity of this conjugation method allows for a 'plug-and-play' approach to prepare new antibody conjugates, thus bypassing the need for (potentially) laborious genetic fusions. Moreover, as sortase is used to specifically modify the exact C-termini of the antibody chains, the final product will be fused in a C-to-C orientation, which is impossible to achieve by genetic manipulations alone. Here we demonstrate the utility of site-specific chemo-enzymatic conjugation to prepare antibody heterodimers, bispecific T-cell engager antibodies, and immunocytokines, discussing purification methods and describing possible pitfalls.

AML-specific cytotoxic antibodies in patients with durable graft-versus-leukemia responses.

Most patients with acute myeloid leukemia (AML) can only be cured when allogeneic hematopoietic stem-cell transplantation induces a graft-versus-leukemia immune response (GVL). Although the role of T cells and natural killer cells in tumor immunology has been established, less is known about the contribution of B cells. From B cells of high-risk patients with AML with potent and lasting GVL responses, we isolated monoclonal antibodies directed against antigens expressed on the cell surface of AML cells but not on normal hematopoietic and nonhematopoietic cells. A number of these donor-derived antibodies recognized the U5 snRNP200 complex, a component of the spliceosome that in normal cells is found in the cell. In AML however, the U5 snRNP200 complex is exposed on the cell membrane of leukemic blasts. U5 snRNP200 complex-specific antibodies induced death of AML cells in an Fc receptor-dependent way in the absence of cytotoxic leukocytes or complement. In an AML mouse model, treatment with U5 snRNP200 complex-specific antibodies led to significant tumor growth inhibition. Thus, donor-derived U5 snRNP200 complex-recognizing AML-specific antibodies may contribute to antitumor responses.

Inducible, Site-Specific Protein Labeling by Tyrosine Oxidation-Strain-Promoted (4 + 2) Cycloaddition.

Genetically encoded tyrosine (Y-tag) can be utilized as a latent anchor for inducible and site-selective conjugation. Upon oxidation of tyrosine with mushroom tyrosinase, strain-promoted cycloaddition (SPOCQ) of the resulting 1,2-quinone with various bicyclo[6.1.0]nonyne (BCN) derivatives led to efficient conjugation. The method was applied for fluorophore labeling of laminarinase A and for the site-specific preparation of an antibody-drug conjugate.

Patient-derived antibody recognizes a unique CD43 epitope expressed on all AML and has antileukemia activity in mice.

Immunotherapy has proven beneficial in many hematologic and nonhematologic malignancies, but immunotherapy for acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) is hampered by the lack of tumor-specific targets. We took advantage of the tumor-immunotherapeutic effect of allogeneic hematopoietic stem cell transplantation and searched the B-cell repertoire of a patient with a lasting and potent graft-versus-AML response for the presence of AML-specific antibodies. We identified an antibody, AT1413, that was of donor origin and that specifically recognizes a novel sialylated epitope on CD43 (CD43s). Strikingly, CD43s is expressed on all World Health Organization 2008 types of AML and MDS. AT1413 induced antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity of AML cells in vitro. Of note, AT1413 was highly efficacious against AML cells in a humanized mouse model without affecting nonmalignant human myeloid cells, suggesting AT1413 has potential as a therapeutic antibody.

Hepatitis C virus Broadly Neutralizing Monoclonal Antibodies Isolated 25 Years after Spontaneous Clearance.

Hepatitis C virus (HCV) is world-wide a major cause of liver related morbidity and mortality. No vaccine is available to prevent HCV infection. To design an effective vaccine, understanding immunity against HCV is necessary. The memory B cell repertoire was characterized from an intravenous drug user who spontaneously cleared HCV infection 25 years ago. CD27+IgG+ memory B cells were immortalized using BCL6 and Bcl-xL. These immortalized B cells were used to study antibody-mediated immunity against the HCV E1E2 glycoproteins. Five E1E2 broadly reactive antibodies were isolated: 3 antibodies showed potent neutralization of genotype 1 to 4 using HCV pseudotyped particles, whereas the other 2 antibodies neutralized genotype 1, 2 and 3 or 1 and 2 only. All antibodies recognized non-linear epitopes on E2. Finally, except for antibody AT12-011, which recognized an epitope consisting of antigenic domain C /AR2 and AR5, all other four antibodies recognized epitope II and domain B. These data show that a subject, who spontaneously cleared HCV infection 25 years ago, still has circulating memory B cells that are able to secrete broadly neutralizing antibodies. Presence of such memory B cells strengthens the argument for undertaking the development of an HCV vaccine.

The Production of a Stable Infliximab Powder: The Evaluation of Spray and Freeze-Drying for Production.

In prospect of developing an oral dosage form of Infliximab, for treatment of Crohn's disease and rheumatoid arthritis, freeze-drying (vial vs Lyoguard trays) and spray-drying were investigated as production method for stable powders. Dextran and inulin were used in combination with sucrose as stabilizing excipients. The drying processes did not affect Infliximab in these formulations, i.e. both the physical integrity and biological activity (TNF binding) were retained. Accelerated stability studies (1 month at 60°C) showed that the TNF binding ability of Infliximab was conserved in the freeze-dried formulations, whereas the liquid counterpart lost all TNF binding. After thermal treatment, the dried formulations showed some chemical modification of the IgG in the dextran-sucrose formulation, probably due to Maillard reaction products. This study indicates that, with the appropriate formulation, both spray-drying and freeze-drying may be useful for (bulk) powder production of Infliximab.

Effective Inhibition of Bone Morphogenetic Protein Function by Highly Specific Llama-Derived Antibodies.

Bone morphogenetic proteins (BMP) have important but distinct roles in tissue homeostasis and disease, including carcinogenesis and tumor progression. A large number of BMP inhibitors are available to study BMP function; however, as most of these antagonists are promiscuous, evaluating specific effects of individual BMPs is not feasible. Because the oncogenic role of the different BMPs varies for each neoplasm, highly selective BMP inhibitors are required. Here, we describe the generation of three types of llama-derived heavy chain variable domains (VHH) that selectively bind to either BMP4, to BMP2 and 4, or to BMP2, 4, 5, and 6. These generated VHHs have high affinity to their targets and are able to inhibit BMP signaling. Epitope binning and docking modeling have shed light into the basis for their BMP specificity. As opposed to the wide structural reach of natural inhibitors, these small molecules target the grooves and pockets of BMPs involved in receptor binding. In organoid experiments, specific inhibition of BMP4 does not affect the activation of normal stem cells. Furthermore, in vitro inhibition of cancer-derived BMP4 noncanonical signals results in an increase of chemosensitivity in a colorectal cancer cell line. Therefore, because of their high specificity and low off-target effects, these VHHs could represent a therapeutic alternative for BMP4(+) malignancies.

Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies.

UNLABELLED: Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCE: Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell.

Bispecific antibody generated with sortase and click chemistry has broad antiinfluenza virus activity.

Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners.

Genetic manipulation of B cells for the isolation of rare therapeutic antibodies from the human repertoire.

Antibody based therapies are increasingly applied to prevent and treat human disease. While the majority of antibodies currently on the market are chimeric or humanized antibodies from rodents, the focus has now shifted to the isolation and development of fully human antibodies. By retroviral transduction of B cell lymphoma-6 (BCL-6), which prevents terminal differentiation of B cells and, the anti-apoptotic gene B-cell lymphoma-extra large (Bcl-xL) into primary human B cells we efficiently immortalize antibody-producing B cells allowing the isolation of therapeutic antibodies. Selection of antigen-specific B cell clones was greatly facilitated because the transduced B cells retain surface immunoglobulin expression and secrete immunoglobulin into the culture supernatant. Surface immunoglobulin expression can be utilized to stain and isolate antigen specific B cell clones with labeled antigen. Immunoglobulins secreted in culture supernatant can directly be tested in functional assays to identify unique B cell clones. Here we describe the key features of our Bcl-6/Bcl-xL culture platform (AIMSelect).

A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi.

B cell chronic lymphocytic leukemia (CLL), the most common leukemia in adults, is a clonal expansion of CD5(+)CD19(+) B lymphocytes. Two types of CLLs are being distinguished as carrying either unmutated or somatically mutated immunoglobulins (Igs), which are associated with unfavorable and favorable prognoses, respectively. More than 30% of CLLs can be grouped based on their expression of stereotypic B cell receptors (BCRs), strongly suggesting that distinctive antigens are involved in the development of CLL. Unmutated CLLs, carrying Ig heavy chain variable (IGHV) genes in germline configuration, express low-affinity, poly-, and self-reactive BCRs. However, the antigenic specificity of CLLs with mutated IGHV-genes (M-CLL) remained elusive. In this study, we describe a new subset of M-CLL, expressing stereotypic BCRs highly specific for ß-(1,6)-glucan, a major antigenic determinant of yeasts and filamentous fungi. ß-(1,6)-glucan binding depended on both the stereotypic Ig heavy and light chains, as well as on a distinct amino acid in the IGHV-CDR3. Reversion of IGHV mutations to germline configuration reduced the affinity for ß-(1,6)-glucan, indicating that these BCRs are indeed affinity-selected for their cognate antigen. Moreover, CLL cells expressing these stereotypic receptors proliferate in response to ß-(1,6)-glucan. This study establishes a class of common pathogens as functional ligands for a subset of somatically mutated human B cell lymphomas.

Notch controls the magnitude of T helper cell responses by promoting cellular longevity.

Generation of effective immune responses requires expansion of rare antigen-specific CD4(+) T cells. The magnitude of the responding population is ultimately determined by proliferation and survival. Both processes are tightly controlled to limit responses to innocuous antigens. Sustained expansion occurs only when innate immune sensors are activated by microbial stimuli or by adjuvants, which has important implications for vaccination. The molecular identity of the signals controlling sustained T-cell responses is not fully clear. Here, we describe a prominent role for the Notch pathway in this process. Coactivation of Notch allows accumulation of far greater numbers of activated CD4(+) T cells than stimulation via T-cell receptor and classic costimulation alone. Notch does not overtly affect cell cycle entry or progression of CD4(+) T cells. Instead, Notch protects activated CD4(+) T cells against apoptosis after an initial phase of clonal expansion. Notch induces a broad antiapoptotic gene expression program that protects against intrinsic, as well as extrinsic, apoptosis pathways. Both Notch1 and Notch2 receptors and the canonical effector RBPJ (recombination signal binding protein for immunoglobulin kappa J region) are involved in this process. Correspondingly, CD4(+) T-cell responses to immunization with protein antigen are strongly reduced in mice lacking these components of the Notch pathway. Our findings, therefore, show that Notch controls the magnitude of CD4(+) T-cell responses by promoting cellular longevity.

Role of the insertion domain and the zinc-finger motif of Escherichia coli UvrA in damage recognition and ATP hydrolysis.

UvrA is the initial DNA damage-sensing protein in bacterial nucleotide excision repair. Each protomer of the UvrA dimer contains two ATPase domains, that belong to the family of ATP-binding cassette domains. Three structural domains are inserted in these ATPase domains: the insertion domain (ID) and UvrB binding domain (in ATP domain I) and the zinc-finger motif (in ATP domain II). In this paper we analyze the function of the ID and the zinc finger motif in damage specific binding of Escherichia coli UvrA. We show that the ID is not essential for damage discrimination, but it does stabilize UvrA on the DNA, most likely by forming a clamp around the DNA helix. We present evidence that two conserved arginine residues in the ID contact the phosphate backbone of the DNA, leading to strand separation after the ATPase-driven movement of the ID's. Remarkably, deletion of the ID generated a phenotype in which UV-survival strongly depends on the presence of photolyase, indicating that UvrA and photolyase form a ternary complex on a CPD-lesion. The zinc-finger motif is shown to be important for the transfer of the damage recognition signal to the ATPase of UvrA. In the absence of this domain the coupling between DNA binding and ATP hydrolysis is completely lost. Mutation of the phenylalanine residue in the tip of the zinc-finger domain resulted in a protein in which the ATPase was already triggered when binding to an undamaged site. As the zinc-finger motif is connected to the DNA binding regions on the surface of UvrA, this strongly suggests that damage-specific binding to these regions results in a rearrangement of the zinc-finger motif, which in its turn activates the ATPase. We present a model how damage recognition is transmitted to activate ATP hydrolysis in ATP binding domain I of the protein.

Role of the two ATPase domains of Escherichia coli UvrA in binding non-bulky DNA lesions and interaction with UvrB.

The UvrA protein is the initial DNA damage-sensing protein in bacterial nucleotide excision repair and detects a wide variety of structurally unrelated lesions. After initial recognition of DNA damage, UvrA loads the UvrB protein onto the DNA. This protein then verifies the presence of a lesion, after which UvrA is released from the DNA. UvrA contains two ATPase domains, both belonging to the ABC ATPase superfamily. We have determined the activities of two mutants, in which a single domain was deactivated. Inactivation of either one ATPase domain in Escherichia coli UvrA results in a complete loss of ATPase activity, indicating that both domains function in a cooperative way. We could show that this ATPase activity is not required for the recognition of bulky lesions by UvrA, but it does promote the specific binding to the less distorting cyclobutane-pyrimidine dimer (CPD). The two ATPase mutants also show a difference in UvrB-loading, depending on the length of the DNA substrate. The ATPase domain I mutant was capable of loading UvrB on a lesion in a 50 bp fragment, but this loading was reduced on a longer substrate. For the ATPase domain II mutant the opposite was found: UvrB could not be loaded on a 50 bp substrate, but this loading was rescued when the length of the fragment was increased. This differential loading of UvrB by the two ATPase mutants could be related to different interactions between the UvrA and UvrB subunits.

Single-molecule analysis reveals two separate DNA-binding domains in the Escherichia coli UvrA dimer.

The UvrA protein is the initial damage-recognizing factor in bacterial nucleotide excision repair. Each monomer of the UvrA dimer contains two ATPase sites. Using single-molecule analysis we show that dimerization of UvrA in the presence of ATP is significantly higher than with ADP or nonhydrolyzable ATPgammaS, suggesting that the active UvrA dimer contains a mixture of ADP and ATP. We also show that the UvrA dimer has a high preference of binding the end of a linear DNA fragment, independent on the presence or type of cofactor. Apparently ATP binding or hydrolysis is not needed to discriminate between DNA ends and internal sites. A significant number of complexes could be detected where one UvrA dimer bridges two DNA ends implying the presence of two separate DNA-binding domains, most likely present in each monomer. On DNA containing a site-specific lesion the damage-specific binding is much higher than DNA-end binding, but only in the absence of cofactor or with ATP. With ATPgammaS no discrimination between a DNA end and a DNA damage could be observed. We present a model where damage recognition of UvrA depends on the ability of both UvrA monomers to interact with the DNA flanking the lesion.