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Immune checkpoint inhibitors demonstrate clinical activity in many tumor types, however, only a fraction of patients benefit. Combining CD137 agonists with these inhibitors increases anti-tumor activity preclinically, but attempts to translate these observations to the clinic have been hampered by systemic toxicity. Here we describe a human CD137xPD-L1 bispecific antibody, MCLA-145, identified through functional screening of agonist- and immune checkpoint inhibitor arm combinations. MCLA-145 potently activates T cells at sub-nanomolar concentrations, even under suppressive conditions, and enhances T cell priming, differentiation and memory recall responses. In vivo, MCLA-145 anti-tumor activity is superior to immune checkpoint inhibitor comparators and linked to recruitment and intra-tumor expansion of CD8 + T cells. No graft-versus-host-disease is observed in contrast to other antibodies inhibiting the PD-1 and PD-L1 pathway. Non-human primates treated with 100 mg/kg/week of MCLA-145 show no adverse effects. The conditional activation of CD137 signaling by MCLA-145, triggered by neighboring cells expressing >5000 copies of PD-L1, may provide both safety and potency advantages., The anti-tumour effect of immune checkpoint inhibitors is potentiated by CD137 agonists in preclinical models, but translation of these results to the clinical practice is hampered by toxicity. Authors describe here a human CD137xPD-L1 bispecific antibody with improved anti-cancer activity whilst maintaining low toxicity in non-human primates.

Background Only a fraction of cancer patients benefit from currently available immune checkpoint inhibitors (ICI). Attempts to improve efficacy of ICI by combining with costimulatory receptor agonists such as CD137 (4-1BB) have led to greater anti-tumor activity preclinically but have shown systemic toxicity in the clinic. MCLA-145 is a human CD137xPD-L1 bispecific common light chain antibody (bAb), identified through functional screening of agonist and ICI bAb combinations. Further, MCLA-145 can overcome Treg and macrophage suppression to potently activate T cells in these immune suppressive conditions. In two ICI insensitive xenograft models, MCLA-145 demonstrated good anti-tumor activity and CD8+ T cells were enriched in tumors post treatment (indicative of intratumor expansion and recruitment). No signs of GvHD were observed in mice following treatment with MCLA-145 in contrast to that seen in animals treated with other ICI mAbs. Methods The EC30 from an in vitro T cell transactivation assay based on IFNg was used as an estimate of the MABEL for MCLA-145. A 2 compartment PK model coupled to a target-mediated drug disposition component was generated based on the available cynomolgus monkey PK data. Results Repeated doses of MCLA-145 up to 100 mg/kg/wk in cynomolgus monkeys were well tolerated without major adverse effects, and dose-dependent increases in serum MCLA-145 concentrations were observed. Following allometric scaling, the model was used to predict exposure in humans following MCLA-145 IV given over 2-hours every 2 weeks, including the starting dose for the FIH trial. Conclusions Conditional activation of CD137 signaling by MCLA-145, triggered by a neighboring target cell expressing of PD-L1, may provide both improved efficacy and safety. MCLA-145 is currently undergoing clinical investigation (NCT03922204).

Background MCLA-145 is a CD137 x PD-L1 bispecific antibody that releases PD-L1 mediated T-cell inhibition and activates and expands T cells through agonism of CD137. Immune checkpoint inhibitors (ICI) against PD-(L)1 have demonstrated anti-tumor efficacy in a fraction of patients across a broad range of cancers. CD137 (4-1BB, tumor necrosis factor receptor superfamily 9) is an inducible costimulatory receptor transiently expressed on T cells after TCR engagement. CD137 signaling is triggered by receptor clustering and leads to enhanced cytokine production; T cell proliferation, survival, and effector function; and immunological memory formation. Targeting of PD-L1 and CD137 with MCLA-145 may achieve synergistic activity by simultaneously blocking the inhibitory checkpoint PD-L1 and activating tumor specific T cells through co-stimulation. Methods We performed combinatorial functional screening of bispecific antibodies generated from high affinity inhibitory Fabs binding PD-L1 combined with a large and diverse panel of agonistic CD137 Fabs. Results MCLA-145 was selected based on its in vitro potency in multiple primary human immune cell assays. Further, it displays an ability to reverse T cell suppression mediated by M2 macrophages or Tregs. MCLA-145 binds to a unique epitope in the cysteine rich domain 2 of CD137 that overlaps with the CD137L binding region, and all potent bAbs in the screen were able to bind to this region. MCLA-145 drives activation of CD137 and the degree of CD137 agonistic activity in T cells correlated with the expression level of PD-L1 on neighboring cells. Using proximity ligation assays and confocal microscopy we demonstrated that MCLA-145 clusters CD137 on the surface of T cells resulting in internalization. The binding location of MCLA-145 on CD137 may be optimal for the formation of ‘immunological synapses’ with PD-L1 expressing antigen presenting cells or tumors resulting in the potent activation of tumor specific cytotoxic T cells. Conclusions These experiments demonstrate the dual anti-cancer activity of MCLA-145 in preclinical models: release of T-cell checkpoint inhibition through PD-L1; and activation and expansion of T cells through CD137, therefore overcoming T-cell exhaustion and increasing T-cell presence/activity (infiltration) in tumors. MCLA-145 is currently undergoing clinical development in an ongoing trial (NCT03922204). Ethics Approval Animal experiments were performed according to guidelines for animal care of the local Animal Experiments Committee; Use of human blood cells from healthy volunteers was approved by the blood bank’s Ethical Advisory Council.

Background: The World Health Organization recommends the development of affordable next-generation inactivated poliovirus vaccines (IPV) using attenuated poliovirus Sabin strains. Previously, we introduced a novel PER.C6® cell culture platform, which allows for high yield production of an affordable trivalent Sabin IPV vaccine. Methods: Immunogenicity and safety of this novel PER.C6®-based Sabin-IPV (sIPV) was assessed in rats and non-human primates (NHPs). NHPs received one of four different dose dilutions vaccine according to current human schedule (three prime-immunizations and one boost immunization). For comparison, NHPs received commercially available reference Salk IPV or sIPV. Results: Dose-dependent immunogenicity and good tolerability was observed for the PER.C6®-based sIPV formulations in rats and NHPs. In NHPs, the lowest tested dose that induced anti-Sabin virus-neutralizing antibody titers that were non-inferior to commercial sIPV after three immunizations was 5-7.5-25 D-antigen units for type 1, 2 and 3 respectively. Discussion: PER.C6®-based sIPV induced comparable immunogenicity to commercial Salk IPV and sIPV vaccines in NHPs. Together with the absence of any preclinical safety signals, these data warrant further testing in clinical trials. sIPV produced on the PER.C6® cell platform could be one solution to the need for an affordable and immunogenic IPV to achieve and maintain global polio eradication.

The currently recommended treatment for individuals exposed to rabies virus is the combined administration of rabies vaccine and rabies immune globulin (RIG). This review sets out the criteria used to guide development of a cocktail of human monoclonal antibodies as a replacement for RIG. Using this process as a model, the general requirements for development of safe and efficacious monoclonal antibody alternatives to currently used polyclonal serum products are discussed.

Monoclonal antibodies (MAbs) neutralizing West Nile Virus (WNV) have been shown to protect against infection in animal models and have been identified as a correlate of protection in WNV vaccine studies. In the present study, antibody repertoires from three convalescent WNV-infected patients were cloned into an scFv phage library, and 138 human MAbs binding to WNV were identified. One hundred twenty-one MAbs specifically bound to the viral envelope (E) protein and four MAbs to the premembrane (prM) protein. Enzyme-linked immunosorbent assay-based competitive-binding assays with representative E protein-specific MAbs demonstrated that 24/51 (47%) bound to domain II while only 4/51 (8%) targeted domain III. In vitro neutralizing activity was demonstrated for 12 MAbs, and two of these, CR4374 and CR4353, protected mice from lethal WNV challenge at 50% protective doses of 12.9 and 357 μg/kg of body weight, respectively. Our data analyzing three infected individuals suggest that the human anti-WNV repertoire after natural infection is dominated by nonneutralizing or weakly neutralizing MAbs binding to domain II of the E protein, while domain III-binding MAbs able to potently neutralize WNV in vitro and in vivo are rare.

Anti-rabies virus immunoglobulin combined with rabies vaccine protects humans from lethal rabies infections. For cost and safety reasons, replacement of the human or equine polyclonal immunoglobulin is advocated, and the use of rabies virus-specific monoclonal antibodies (MAbs) is recommended. We produced two previously described potent rabies virus-neutralizing human MAbs, CR57 and CRJB, in human PER.C6 cells. The two MAbs competed for binding to rabies virus glycoprotein. Using CR57 and a set of 15-mer overlapping peptides covering the glycoprotein ectodomain, a neutralization domain was identified between amino acids (aa) 218 and 240. The minimal binding region was identified as KLCGVL (aa 226 to 231), with key residues K-CGV- identified by alanine replacement scanning. The critical binding region of this novel nonconformational rabies virus epitope is highly conserved within rabies viruses of genotype 1. Subsequently, we generated six rabies virus variants escaping neutralization by CR57 and six variants escaping CRJB. The CR57 escape mutants were only partially covered by CRJB, and all CRJB-resistant variants completely escaped neutralization by CR57. Without exception, the CR57-resistant variants showed a mutation at key residues within the defined minimal binding region, while the CRJB escape viruses showed a single mutation distant from the CR57 epitope (N182D) combined with mutations in the CR57 epitope. The competition between CR57 and CRJB, the in vitro escape profile, and the apparent overlap between the recognized epitopes argues against including both CR57 and CRJB in a MAb cocktail aimed at replacing classical immunoglobulin preparations.

Acute myeloid leukemia (AML) has a poor prognosis due to treatment-resistant relapses. A humanized anti-CD33 antibody (Mylotarg) showed a limited response rate in relapsed AML. To discover novel AML antibody targets, we selected a panel of single chain Fv fragments using phage display technology combined with flow cytometry on AML tumor samples. One selected single chain Fv fragment broadly reacted with AML samples and with myeloid cell lineages within peripheral blood. Expression cloning identified the antigen recognized as C-type lectin-like molecule-1 (CLL-1), a previously undescribed transmembrane glycoprotein. CLL-1 expression was analyzed with a human anti-CLL-1 antibody that was generated from the single chain Fv fragment. CLL-1 is restricted to the hematopoietic lineage, in particular to myeloid cells present in peripheral blood and bone marrow. CLL-1 is absent on uncommitted CD34+/CD38− or CD34+/CD33− stem cells and present on subsets of CD34+/CD38+ or CD34+/CD33+ progenitor cells. CLL-1 is not expressed in any other tissue. In contrast, analysis of primary AMLs demonstrated CLL-1 expression in 92% (68 of 74) of the samples. As an AML marker, CLL-1 was able to complement CD33, because 67% (8 of 12) of the CD33− AMLs expressed CLL-1. CLL-1 showed variable expression (10–60%) in CD34+ cells in chronic myelogenous leukemia and myelodysplastic syndrome but was absent in 12 of 13 cases of acute lymphoblastic leukemia. The AML reactivity combined with the restricted expression on normal cells identifies CLL-1 as a novel potential target for AML treatment.

Negative selection eliminates thymocytes bearing autoreactive T cell receptors (TCR) via an apoptotic mechanism. We have cloned an inhibitor of NF-kappa B, I kappa BNS, which is rapidly expressed upon TCR-triggered but not dexamethasone- or gamma irradiation-stimulated thymocyte death. The predicted protein contains seven ankyrin repeats and is homologous to I kappa B family members. In class I and class II MHC-restricted TCR transgenic mice, transcription of I kappa BNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides. I kappa BNS blocks transcription from NF-kappa B reporters, alters NF-kappa B electrophoretic mobility shifts, and interacts with NF-kappa B proteins in thymic nuclear lysates following TCR stimulation. Retroviral transduction of I kappa BNS in fetal thymic organ culture enhances TCR-triggered cell death consistent with its function in selection.

Cleavage of eukaryotic translation initiation factor 4GI (eIF4GI) is required for shutoff of host cell translation during poliovirus (PV) infection of HeLa cells. Reports published by several groups have led to confusion whether this cleavage is mediated by viral 2A protease (2A pro ) or a putative cellular enzyme (termed eIF4Gase) which is activated by 2A pro or other aspects of viral infection. Here we have further investigated eIF4Gase activities in PV-infected cells. Column purification of eIF4GI cleavage activity separated two activities which generated N-terminal cleavage products of different lengths. Both activities were detected using either native eIF4G or radiolabeled recombinant eIF4G as the substrate. Analysis of cleavage products formed by each activity on native and mutant substrates suggests that one activity cleaves eIF4G1 at or very near the 2A pro cleavage site and the other activity cleaves approximately 40 residues upstream of the 2A pro cleavage site. When PV infections in HeLa cells were supplemented with 2 mM guanidine, which indirectly limits expression of 2A pro , two distinct C-terminal cleavage fragments of eIF4GI were detected. These C-terminal cleavage fragments of eIF4GI were purified from infected cells, and a new eIF4GI cleavage site was mapped to a unique site 43 amino acids upstream of the known 2A pro cleavage site. Further, eIF4GI cleavage in vivo could be blocked by addition of zVAD to PV-guanidine infections. zVAD is a broad-spectrum caspase inhibitor which had no effect on 2A pro cleavage activity or PV polyprotein processing. Lastly, similar types of eIF4Gase cleavage activities were also detected in uninfected cells under various conditions, including early apoptosis or during cell cycle transit. The data suggest that the same types of eIF4GI cleavage activities which are generated in PV-infected cells can also be generated in the absence of virus. Taken together, the data support a model in which multiple cellular activities process eIF4GI in PV-infected cells, in addition to 2A pro .

The inhibition of host cell translation by poliovirus, also called host cell shutoff, occurs early in the infectious cycle, typically only 1.5 to 2.5 h postinfection in HeLa cells. This chapter explores information regarding cleavage of new initiation factors and then discusses information relevant to whether these factors, particularly eukaryotic translation initiation factor 4GI (eIF4GI), are cleaved in vivo by viral or cellular proteases or both. Researchers have discovered evidence for several eIF4Gase activities that cleave eIF4GI at different sites to produce distinct types of eIF4G cleavage products. In the chapter, researchers define eIF4Gase activities based on the size of cleavage products generated (which results from use of alternate cleavage sites) and the mode of induction of the activity. Researchers have identified three types of eIF4Gase activities (termed eIF4Gase-α, elF4Gase-β, eIF4Gase-γ) that can be generated without poliovirus (PV) infection; two of these appear similar to activities also present in infected cells. The sites, which generate the three major N-terminal cleavage products seen on our immunoblots, are 149 amino acids (aa) upstream of the 2Apro cleavage site and 106 aa upstream of the putative eIF4Gase-β cleavage site. Researchers have screened many S10 extracts with this procedure and found that this specific cleavage activity, which is often weak, appeared in approximately 40% of the extracts tested.

Induction of apoptosis in a variety of cell types leads to inhibition of protein synthesis. Recently, the cleavage of eukaryotic translation initiation factor 4G (eIF4G) by caspase 3 was described as a possible event contributing to translation inhibition. Here, we report the cleavage of another initiation factor in apoptotic cells, eIF2alpha, that could contribute to regulation of translation during apoptosis. This cleavage event could be completely inhibited by pretreatment of HeLa cells with Z-VAD-fmk. In vitro analysis using purified eIF2 and purified caspases showed cleavage of eIF2alpha by caspase 3, 6, 8, and 10 but not 9. Caspase 3 most efficiently cleaved eIF2alpha and this could be inhibited by addition of Ac-DEVD-CHO in vitro. Comparison of cleavage of phosphorylated versus nonphosphorylated eIF2alpha revealed a modest preference of the caspases for the nonphosphorylated form. When eIF2. 2B complex was used as substrate, only caspase 3 was capable of eIF2alpha cleavage, which was not affected by phosphorylation of the alpha subunit. The eIF2.GDP binary complex was cleaved much less efficiently by caspase 3. Sequence analysis of the cleavage fragment suggested that the cleavage site is located in the C-terminal portion of the protein. Analysis showed that after caspase cleavage, exchange of GDP bound to eIF2 was very rapid and no longer dependent upon eIF2B. Furthermore, in vitro translation experiments indicated that cleavage of eIF2alpha results in functional alteration of the eIF2 complex, which no longer stimulated upstream AUG selection on a mRNA containing a viral internal ribosome entry site and was no longer capable of stimulating overall translation. In conclusion, we describe here the cleavage of a translation initiation factor, eIF2alpha that could contribute to inhibition or alteration of protein synthesis during the late stages of apoptosis.

Human enteroviruses and rhinoviruses rapidly and selectively abolish translation from cellular mRNA upon infection of susceptible cells. Expression of the poliovirus 2A protease (PV 2A pro ) is sufficient to cause host translation shutoff through cleavage of eIF4G (formerly p220, eIF4γ) either directly or indirectly through activation of a cellular factor. Evidence exists for both direct and indirect cleavage mechanisms; however, factors presumed to participate in an indirect mechanism have not yet been purified or defined. Here we show that the dominant eIF4G cleavage activity in lysates from infected HeLa cells was separable from PV 2A pro by size exclusion chromatography. 2A pro separated into two peak fractions which contained activity which cleaved a peptide substrate derived from the poliovirus polyprotein. These peak 2A pro fractions did not cleave eIF4G or an eIF4G-derived peptide, as expected, due to the poor efficiency of direct cleavage reactions. Conversely, fractions which contained peak eIF4G cleavage activity and only trace amounts of 2A pro efficiently cleaved a peptide substrate derived from the previously mapped eIF4G cleavage site and also cleaved a peptide derived from the poliovirus 1D2A region. The dominant eIF4G cleavage activity was highly purified through four chromatography steps and found to be devoid of all traces of 2A pro or its precursors. Quantitation of 2A pro from lysates of infected cells showed that during infections in HeLa cells, 2A pro does not reach molar excess over eIF4G, as previously shown to be required for direct eIF4G cleavage in vitro. Further, infection of HeLa cells in the presence of 2 mM guanidine–HCl, a potent inhibitor of viral RNA replication, suppressed accumulation of 2A pro and its precursor 2ABC below detectable levels but was unable to delay the onset of eIF4G proteolysis in vivo. The eIF4G cleavage activity was still easily detectable in in vitro assays using fractions from guanidine-treated cells. Thus, the data suggest that poliovirus utilizes two catalytic activities to ensure rapid cleavage of eIF4G in vivo. Although it was not directly measurable here, 2A pro likely does cleave a portion of eIF4G in cells. However, the data suggest that a cellular factor which can be activated by small quantities of 2A pro constitutes the bulk of the eIF4G-specific cleavage activity in infected cells and is responsible for the rapid and efficient eIF4G cleavage activity observed in vivo.

Administration of post-exposure prophylaxis (PEP) is recommended for all patients with category III exposure to rabies virus (RABV) and other lyssaviruses.1 A key component of PEP is rabies immune globulins (RIGs), which are obtained from rabies-immunized human or equine donors.5,6 Due to the limited supply of RIGs, monoclonal antibodies (MAbs) are being explored as an alternative in PEP. DNA recombinant technology has been developed to express and purify human MAb cocktails, which are as effective as RIGs,10 but can be produced in much larger quantities at a much smaller cost. This chapter describes the methodology of how to express MAbs in mammalian cell lines, how to purify MAbs via Protein A chromatography, and desalting with both manual and automated methods. Additionally, a variety of methods for how to verify the quality of the end product are also described.

The safety and availability of the human polyclonal sera that is currently utilized for post-exposure treatment (PET) of rabies virus (RABV) infection remain a concern. Recombinant monoclonal antibodies have been postulated as suitable alternatives by WHO. To this extent, CL184, the RABV human antibody combination comprising monoclonal antibodies (mAbs) CR57 and CR4098, has been developed and has delivered promising clinical data to support its use for RABV PET. For this fully human IgG1 cocktail, mAbs CR57 and CR4098 are produced in the PER.C6 human cell line and combined in equal amounts in the final product. During preclinical evaluation, CR57 was shown to bind to antigenic site I whereas CR4098 neutralization was influenced by a mutation of position 336 (N336) located within antigenic site III. Here, alanine scanning was used to analyze the influence of mutations within the potential binding site for CR4098, antigenic site III, in order to evaluate the possibility of mutated rabies viruses escaping neutralization. For this approach, twenty flanking amino acids (10 upstream and 10 downstream) of the RABV glycoprotein (G) asparagine (N336) were exchanged to alanine (or serine, if already alanine) by site-directed mutagenesis. Analysis of G expression revealed four of the twenty mutant Gs to be non-functional, as shown by their lack of cell surface expression, which is a requirement for the production of infectious RABV. Therefore, these mutants were excluded from further study. The remaining sixteen mutants were introduced in an infectious clone of RABV, and recombinant RABVs (rRABVs) were recovered and utilized for in vitro neutralization assays. All of the viruses were effectively neutralized by CR4098 as well as by CR57, indicating that single amino acid exchanges in this region does not affect the broad neutralizing capability of the CL184 mAb combination.

Monoclonal antibodies are successful biologics in treating a variety of diseases, including the prevention or treatment of viral infections. CL184 is a 1:1 combination of two human monoclonal IgG1 antibodies (CR57 and CR4098) against rabies virus, produced in the PER.C6 human cell line. The two antibodies are developed as replacements of human rabies immune globulin (HRIG) and equine rabies immune globulin (ERIG) in postexposure prophylaxis (PEP). The rapid fluorescent focus inhibition test (RFFIT) is a cell-based virus neutralization assay which is usually performed to determine the biological potency of a vaccine and to measure the levels of protection against rabies in humans and animals. In order to confirm the suitability of this assay as a pharmacodynamic assay, we conducted a validation using both HRIG- and CL184-spiked serum samples and sera from vaccinated donors. The validation results met all analytical acceptance criteria and showed that HRIG and CL184 serum concentrations can be compared. Stability experiments showed that serum samples were stable in various suboptimal conditions but that rabies virus should be handled swiftly once thawed. We concluded that the assay is suitable for the measurement of polyclonal and monoclonal rabies neutralizing antibodies in clinical serum samples.

CD3epsilongamma and CD3epsilondelta are noncovalent heterodimers; each consists of Ig-like extracellular domains associated side-to-side via paired terminal beta-strands that are linked to individual subunit membrane proximal stalk segments. CD3epsilon, CD3gamma, and CD3delta stalks contain the RxCxxCxE motif. To investigate the functional importance of a CD3 stalk and terminal beta-strand, we created a CD3gamma double mutant CD3gamma(C82S/C85S) and a CD3gamma beta-strand triple mutant CD3gamma(Q76S/Y78A/Y79A) for use in retroviral transduction of lymphoid progenitors for comparison with CD3gammawt. Although both mutant CD3gamma molecules reduced association with CD3epsilon in CD3epsilongamma heterodimers, CD3gamma(Q76S/Y78A/Y79A) abrogated surface TCR expression whereas CD3gamma(C82S/C85S) did not. Furthermore, CD3gamma(C82S/C85S) rescued thymic development in CD3gamma(-/-) fetal thymic organ culture. However, the numbers of double-positive and single-positive thymocytes after CD3gamma(C82S/C85S) transduction were significantly reduced despite surface pre-TCR and TCR expression comparable to that of CD3gamma(-/-) thymocytes transduced in fetal thymic organ culture with a retrovirus harboring CD3gammawt cDNA. Furthermore, double-negative thymocyte development was perturbed with attenuated double-negative 3/double-negative 4 maturation and altered surface-expressed CD3epsilongamma, as evidenced by the loss of reactivity with CD3gamma N terminus-specific antisera. Single histidine substitution of either CD3gamma stalk cysteine failed to restore CD3epsilongamma association and conformation in transient COS-7 cell transfection studies. Thus, CD3gamma(C82) and CD3gamma(C85) residues likely are either reduced or form a tight intrachain disulfide loop rather than contribute to a metal coordination site in conjunction with CD3epsilon(C80) and CD3epsilon(C83). The implications of these results for CD3epsilongamma and TCR structure and signaling function are discussed.

The World Health Organization estimates human mortality from endemic canine rabies to be 55,000 deaths/year. Limited supply hampers the accessibility of appropriate lifesaving treatment, particularly in areas where rabies is endemic. Anti-rabies antibodies are key to protection against lethal rabies. Currently, only human and equine polyclonal anti-rabies immune globulin (HRIG and ERIG) is available. Replacement of HRIG and ERIG with a safer and more widely available product is recommended. We have recently identified a combination of 2 human monoclonal antibodies (MAbs), CR57 and CR4098, that has high potential. We here describe a head-to-head comparison between an CR57/CR4098 MAb cocktail and HRIG. The MAb cocktail neutralized all viruses from a panel of 26 representative street rabies virus isolates. In combination with vaccine, the MAb cocktail protected Syrian hamsters against lethal rabies when administered 24 h after exposure, comparable with the results obtained with HRIG. Furthermore, the MAb cocktail did not interfere with rabies vaccine differently from HRIG. These results demonstrate that the human MAb cocktail of CR57 and CR4098 is a safe and efficacious alternative to RIG in rabies postexposure prophylaxis.

Antibody phage display technology was used to identify human monoclonal antibodies that neutralize rabies virus (RV). A phage repertoire was constructed using antibody genes harvested from the blood of vaccinated donors. Selections using this repertoire and three different antigen formats of the RV glycoprotein (gp) resulted in the identification of 147 unique antibody fragments specific for the RV gp. Analysis of the DNA sequences of these antibodies demonstrated a large variation in the heavy- and light-chain germ-line gene usage, suggesting that a broad antibody repertoire was selected. The single-chain variable fragment (scFv) antibodies were tested in vitro for RV neutralization, resulting in 39 specificities that neutralize the virus. Of the scFv clones, 21 were converted into full-length human IgG(1) format. Analysis of viral escape variants and binding competition experiments indicated that the majority of the neutralizing antibodies are directed against antigenic site III of the RV gp. The obtained specificities expand the set of human anti-RV antibodies eligible for inclusion in an antibody cocktail aimed for use in rabies post-exposure prophylaxis.

Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease.

The need to replace rabies immune globulin (RIG) as an essential component of rabies postexposure prophylaxis is widely acknowledged. We set out to discover a unique combination of human monoclonal antibodies (MAbs) able to replace RIG. Stringent criteria concerning neutralizing potency, affinity, breadth of neutralization, and coverage of natural rabies virus (RV) isolates and in vitro escape mutants were set for each individual antibody, and the complementarities of the two MAbs were defined at the onset. First, we identified and characterized one human MAb (CR57) with high in vitro and in vivo neutralizing potency and a broad neutralization spectrum. The linear antibody binding site was mapped on the RV glycoprotein as antigenic site I by characterizing CR57 escape mutants. Secondly, we selected using phage display a complementing antibody (CR4098) that recognized a distinct, nonoverlapping epitope (antigenic site III), showed similar neutralizing potency and breadth as CR57, and neutralized CR57 escape mutants. Reciprocally, CR57 neutralized RV variants escaping CR4098. Analysis of glycoprotein sequences of natural RV isolates revealed that the majority of strains contain both intact epitopes, and the few remaining strains contain at least one of the two. In vitro exposure of RV to the combination of CR57 and CR4098 yielded no escape mutants. In conclusion, a novel combination of human MAbs was discovered suitable to replace RIG.

Acute myeloid leukemia (AML) has a poor prognosis due to treatment-resistant relapses. A humanized anti-CD33 antibody (Mylotarg) showed a limited response rate in relapsed AML. To discover novel AML antibody targets, we selected a panel of single chain Fv fragments using phage display technology combined with flow cytometry on AML tumor samples. One selected single chain Fv fragment broadly reacted with AML samples and with myeloid cell lineages within peripheral blood. Expression cloning identified the antigen recognized as C-type lectin-like molecule-1 (CLL-1), a previously undescribed transmembrane glycoprotein. CLL-1 expression was analyzed with a human anti-CLL-1 antibody that was generated from the single chain Fv fragment. CLL-1 is restricted to the hematopoietic lineage, in particular to myeloid cells present in peripheral blood and bone marrow. CLL-1 is absent on uncommitted CD34(+)/CD38(-) or CD34(+)/CD33(-) stem cells and present on subsets of CD34(+)/CD38(+) or CD34(+)/CD33(+) progenitor cells. CLL-1 is not expressed in any other tissue. In contrast, analysis of primary AMLs demonstrated CLL-1 expression in 92% (68 of 74) of the samples. As an AML marker, CLL-1 was able to complement CD33, because 67% (8 of 12) of the CD33(-) AMLs expressed CLL-1. CLL-1 showed variable expression (10-60%) in CD34(+) cells in chronic myelogenous leukemia and myelodysplastic syndrome but was absent in 12 of 13 cases of acute lymphoblastic leukemia. The AML reactivity combined with the restricted expression on normal cells identifies CLL-1 as a novel potential target for AML treatment.

Eukaryotic translation initiation factor 4G (eIF4G), which has two homologs known as eIF4GI and eIF4GII, functions in a complex (eIF4F) which binds to the 5' cap structure of cellular mRNAs and facilitates binding of capped mRNA to 40S ribosomal subunits. Disruption of this complex in enterovirus-infected cells through eIF4G cleavage is known to block this step of translation initiation, thus leading to a drastic inhibition of cap-dependent translation. Here, we show that like eIF4GI, the newly identified homolog eIF4GII is cleaved during apoptosis in HeLa cells and can serve as a substrate for caspase 3. Proteolysis of both eIF4GI and eIF4GII occurs with similar kinetics and coincides with the profound translation inhibition observed in cisplatin-treated HeLa cells. Both eIF4GI and eIF4GII can be cleaved by caspase 3 with similar efficiency in vitro, however, eIF4GII is processed into additional fragments which destroy its core central domain and likely contributes to the shutoff of translation observed in apoptosis. Cell Death and Differentiation (2000) 7, 1234 - 1243.

Although much is known about the multiple mechanisms which induce apoptosis, comparatively little is understood concerning the execution phase of apoptosis and the mechanism(s) of cell killing. Several reports have demonstrated that cellular translation is shut off during apoptosis; however, details of the mechanism of translation inhibition are lacking. Translation initiation factor 4G (eIF4G) is a crucial protein required for binding cellular mRNA to ribosomes and is known to be cleaved as the central part of the mechanism of host translation shutoff exerted by several animal viruses. Treatment of HeLa cells with the apoptosis inducers cisplatin and etoposide resulted in cleavage of eIF4G, and the extent of its cleavage correlated with the onset and extent of observed inhibition of cellular translation. The eIF4G-specific cleavage activity could be measured in cell lysates in vitro and was inhibited by the caspase inhibitor Ac-DEVD-CHO at nanomolar concentrations. A combination of in vivo and in vitro inhibitor studies suggest the involvement of one or more caspases in the activation and execution of eIF4G cleavage. Furthermore recombinant human caspase 3 was expressed in bacteria, and when incubated with HeLa cell lysates, was shown to produce the same eIF4G cleavage products as those observed in apoptotic cells. In addition, purified caspase 3 caused cleavage of purified eIF4G, demonstrating that eIF4G could serve as a substrate for caspase 3. Taken together, these data suggest that cellular translation is specifically inhibited during apoptosis by a mechanism involving cleavage of eIF4G, an event dependent on caspase activity.