Portable transdermal electrical stimulation (TES) applicators for modifying a subject's cognitive state by applying stimulation to the subject's skin. One or more electrode may be on the subject's mastoid, and/or on or near the back of the subject's neck. The portable applicators are configured and adapted to be lightweight and may be wearable, and to deliver a high-intensity TES able to evoke or enhance a predetermined cognitive effect to stimulate either the trigeminal, facial and/or cervical plexus. These TES applicators may include a pair of electrodes and a TES control module comprising a processor, a timer and a waveform generator.

The brain is composed of neurons and other cell types in connected networks that process sensory input, generate motor commands, and control all other behavioral and cognitive functions. Neurons communicate primarily through electrochemical pulses that transmit signals between connected cells within and between brain areas. Noninvasive neuromodulation technologies that affect neuronal activity can modulate the pattern of neural activity and may cause altered behavior, cognitive states, perception, and motor output without requiring an invasive procedure.
Non-invasive neuromodulation includes the broad category of "transdermal electrical stimulation," which generally refers to electrical stimulation of the nervous system (brain, cranial nerves, peripheral nerves, etc.) through a subject's skin. Specific examples of transdermal electric stimulation (hereinafter "TES") may include transcranial stimulation, for example, through scalp electrodes and have been used to affect brain function in humans in the form of transcranial alternating current stimulation (hereinafter "tACS"), transcranial direct current stimulation (hereinafter "tDCS"), cranial electrotherapy stimulation (hereinafter "CES"), and transcranial random noise stimulation (hereinafter "tRNS"). Systems and methods for TES have been disclosed (see for example, Capel U.S. Pat. No. 4,646,744; Haimovich et al. U.S. Pat. No. 5,540,736; Besio et al. U.S. Pat. No. 8,190,248; Hagedorn and Thompson U.S. Pat. No. 8,239,030; Bikson et al. U.S. Patent Application Publication No. 2011/0144716; and Lebedev et al. U.S. Patent Application Publication No. 2009/0177243). tDCS systems with numerous electrodes and a high level of configurability have been disclosed (see for example Bikson et al. U.S. Patent Application Publication Nos. 2012/0209346, 2012/0265261, and 2012/0245653), as have portable TES systems for auto-stimulation (Brocke U.S. Pat. No. 8,554,324).
In general, TES has been shown to improve motor control and motor learning, improve memory consolidation during slow-wave sleep, regulate decision-making and risk assessment, affect sensory perception, and cause movements. TES has been used therapeutically in various clinical applications, including treatment of pain, depression, epilepsy, and tinnitus. In at least some cases of TES (e.g., tDCS) therapeutic use, more data concerning the efficacy of tDCS in treatment is needed.
Despite research done on TES neuromodulation, existing systems and methods for TES are lacking in at least some cases in their capacity to safely and robustly affect cognitive function and induce cognitive states in human subjects. The development of new TES methods, TES stimulation protocols, TES systems, and TES electrode configurations that induce substantial changes in cognitive function and/or cognitive state comfortably would be advantageous. Existing systems and methods can cause skin irritation or pain and are lacking with regard to the reliability and amount of change in cognitive state that can be achieved.
Electrotherapy for muscles and other peripheral nervous system applications (e.g. TENS and transdermal drug delivery) have used strategies to reduce pain, irritation, and tissue damage, including (1) higher frequencies of alternating current stimulation and (2) a beat frequency generally between 1 Hz and 200 Hz created from a difference frequency of two channels (anode-cathode pairs) of electrodes. Reduced side-effects (e.g. pain and irritation) are approximately linear across a wide range from ˜1 kHz to 100 kHz. Skin impedance is frequency dependent, with lower impedances at higher electrical stimulation frequencies. For interferential stimulation, a beat frequency of between 1 and 200 Hz is an advantageous frequency to avoid activating pain and muscle fibers that are perceived as irritating or painful. Power density also affects skin resistivity, with lower resistivity occurring at higher power densities. However, systems and methods for TES are lacking in terms of mitigation of pain, irritation, and tissue damage.
Typical transcranial alternating current stimulation protocols are also typically below 150 Hz (see Paulus 2011), consistent with frequencies of brain rhythms or below 640 Hz as used in tRNS protocols. Recently, Chaieb et al. used 1 kHz, 2 kHz, and 5 kHz tACS to induce neuromodulation (Chaieb L, Antal A, Paulus W. "Transcranial alternating current stimulation in the low kHz range increases motor cortex excitability." Restor Neurol Neurosci. 2011; 29(3):167-75, incorporated fully herein by reference). International Publication No. WO 2012/089588 by inventors Paulus and Warschewske describes systems and methods of tACS at frequencies between 1 Hz and 50 kHz, including interferential tACS from two anode-cathode electrode pairs and pulsed tACS. However, existing tACS systems for neuromodulation are less than ideal for inducing cognitive effects robustly and comfortably.
One advantage of transcranial alternating current stimulation relative to transcranial direct current stimulation is reduced pain and irritation. However, existing tACS systems for neuromodulation are less than ideal in at least some instances, because alternating currents affect nervous system function (i.e., brain function) differently than direct currents. One advantage of pulsed transcranial direct current stimulation relative to unpulsed transcranial direct current stimulation is reduced pain and irritation. Pulsed transcranial direct current stimulation has been previously reported for peripheral use in patients but has not been used for targeting the brain transcranially. The Idrostar Iontophoresis Machine (STD Pharmaceutical Products Ltd, Hereford, England) delivers pulsed direct current stimulation (7 kHz, about 42% duty cycle) to address hyperhidrosis (excess sweating). Alternative transcranial electrical stimulation protocols that achieve desired effects on the nervous system with manageable amounts of pain and/or irritation would be advantageous.
It would generally be advantageous to provide devices and methods that allow transdermal electrical stimulation in a manner that overcomes the problems with pain and efficacy discussed above. In particular, it would be beneficial to provide TES devices and methods for modulating (e.g., inducing, enhancing, reversing, or otherwise increasing or changing) a cognitive effect and/or mental state. For example, TES stimulation protocols and electrode configurations that induce a relaxing, calming, anxiolytic, dissociated, high mental clarity, or worry-free state of mind in a subject would be advantageous for improving the subject's experiences and state of mind, as well as addressing insomnia and mitigating negative responses to stress. Similarly TES stimulation protocols and apparatuses that increase a subject's motivation, subjective (and/or physiological) energy level, or focus would be advantageous for improving a subject's productivity and providing beneficial states of mind.
Systems and methods for inducing these states via transdermal electrical stimulation targeting peripheral nerves at any (or multiple) locations would be a beneficial improvement by permitting targeting more broadly (i.e. for wearable systems on users with varying anatomy of bones (which may restrict conformity of a wearable system) and hair (which limits low-impedance, uniform contact to skin for stimulation). The anatomy of various peripheral nerves, including cranial and cervical spinal nerves (among others) are well-known and generally conserved across individuals. Moreover, electrophysiological or other mapping may be used to more accurately identify the location of branches of a targeted peripheral nerve.
Personalizing or optimizing TES for a subject (electrode position and waveform parameters) would be beneficial given inherent variability between individuals. Existing systems are less than ideal, because they lack physiological measurements from sensor systems of a TES apparatus to provide feedback to a TES controller and/or TES user.
In some instances, being able to place electrodes and/or a wearable neurostimulator module of a TES system on a part of the body other than the head or neck may be advantageous for comfortably inducing a cognitive effect in a less obtrusive way (relative to having a neurostimulator on the temple area).
Sympathetic nervous system activity can only be directly assessed through neurophysiological recordings from sympathetic nerve fibers or from plasma measurements of norepinephrine spillover. Of the two, recording of muscle sympathetic nerve activity has higher temporal resolution and is both easier technically and provides real-time data. Accordingly, direct microneurographic recordings of muscle sympathetic nerve activity (MSNA) are considered the gold standard for assaying sympathetic outflow or tone. Previous TES systems are lacking in part because they have not incorporated methods for MSNA to assess effects on sympathetic nervous system activity.
Many TES systems described to date use fixed waveform parameters, limiting the adaptability and effectiveness of TES systems. TES systems and methods for using them that apply more general principles of TES waveform design would be beneficial for improved consistency, effectiveness, and comfort of TES for inducing a change in cognitive state. The systems and methods described herein address this deficiency.
Described herein are methods and apparatuses (including devices and systems) and methods that may address the problems and opportunities discussed above.

Apparatuses and methods for transdermal electrical stimulation of nerves to modify or induce a cognitive state


A global navigation system includes a first navigation receiver located in a rover and a second navigation receiver located in a base station. Single differences of measurements of satellite signals received at the two receivers are calculated and compared to single differences derived from an observation model. Anomalous measurements are detected and removed prior to performing computations for determining the output position of the rover and resolving integer ambiguities. Detection criteria are based on the residuals between the calculated and the derived single differences. For resolving integer ambiguities, computations based on Cholesky information Kalman filters and Householder transformations are advantageously applied. Changes in the state of the satellite constellation from one epoch to another are included in the computations.

The present invention relates generally to global navigation satellite systems, and more particularly to detection and correction of anomalous measurements and ambiguity estimation in a navigation receiver.
Global navigation satellite systems (GNSSs) can determine locations with high accuracy. Currently deployed global navigation satellite systems are the United States Global Positioning System (GPS) and the Russian GLONASS. Other global navigation satellite systems, such as the European GALILEO system, are under development. In a GNSS, a navigation receiver receives and processes radio signals transmitted by satellites located within a line-of-sight distance of the navigation receiver. The satellite signals comprise carrier signals modulated by pseudo-random binary codes. The navigation receiver measures the time delays of the received signals relative to a local reference clock or oscillator. Code measurements enable the navigation receiver to determine the pseudo-ranges between the navigation receiver and the satellites. The pseudo-ranges differ from the actual ranges (distances) between the navigation receiver and the satellites due to various error sources and due to variations in the time scales of the satellites and the navigation receiver. If signals are received from a sufficiently large number of satellites, then the measured pseudo-ranges can be processed to determine the code coordinates and coordinate time scales at the navigation receiver. This operational mode is referred to as a stand-alone mode, since the measurements are determined by a single navigation receiver. A stand-alone system typically provides meter-level accuracy.
To improve the accuracy, precision, stability, and reliability of measurements, differential navigation (DN) systems have been developed. In a DN system, the position of a user is determined relative to a base station (also referred to as a base) whose coordinates are precisely known. The base contains a navigation receiver that receives satellite signals. The user, whose position is to be determined, can be stationary or mobile and is often referred to as a rover. The rover also contains a navigation receiver that receives satellite signals. Signal measurements processed at the base are transmitted to the rover via a communications link. The communications link, for example, can be provided over a cable or optical fiber. To accommodate a mobile rover, the communications link is often a wireless link.
The rover processes the measurements received from the base, along with measurements taken with its own navigation receiver, to improve the accuracy of determining its position. Accuracy is improved in the differential navigation mode because errors incurred by the navigation receiver at the rover and by the navigation receiver at the base are highly correlated. Since the coordinates of the base are accurately known, measurements from the base can be used to compensate for the errors at the rover. A differential global positioning system (DGPS) computes locations based on pseudo-ranges only.
The location determination accuracy of a differential navigation system can be further improved by supplementing the code pseudo-range measurements with measurements of the phases of the satellite carrier signals. If the carrier phases of the signals transmitted by the same satellite are measured by both the navigation receiver at the base and the navigation receiver at the rover, processing the two sets of carrier phase measurements can yield a location determination accuracy to within several percent of the carrier's wavelength. A differential navigation system that computes locations based on real-time carrier signals, in addition to the code pseudo-ranges, is often referred to as a real-time kinematic (RTK) system. Processing carrier phase measurements to determine coordinates includes the step of ambiguity resolution; that is, determining the integer number of cycles in the carrier signal received by a navigation receiver from an individual satellite.
In many instances, a navigation receiver (in particular, the navigation receiver at the rover) operates in a complex environment in which various external influences cause measurement errors. For example, external signals can interfere with the satellite signals, and structures and terrain can result in multipath errors. Errors can be classified into two broad categories: normal errors and abnormal errors. Normal errors are normally-distributed white noise errors that can be compensated for during calculation of location coordinates. Abnormal errors are large systematic errors that can prevent the system from calculating an accurate location. In some instances, abnormal errors are caused by spikes of intrinsic noise. More often, they result from environmental conditions. For example, strong reflected signals that interfere with the direct satellite signal can cause an abnormal error. Similarly, extreme radio interference can also result in abnormal errors.
Partial or complete shading of the navigation receiver can result in errors due to radio wave diffraction. If the shading is partial and minor, the measurement error can be minimal. If a satellite is completely shaded (that is, blocked), however, only the multipath signal remains. As a result, tracking in the channel is interrupted, and the measured phase is lost, resulting in an abnormal error. Dynamic effects on the navigation receiver (for example, specific motions of the rover) can also cause abnormal errors. Impulse accelerations impact both the receiving antenna and the quartz crystal of the local reference oscillator, resulting in drift of the intermediate carrier frequency and measured phase.
One specific type of abnormal error is a phase-lock loop (PLL) cycle slip, which is a cycle slip in the PLL circuits that track the satellite carrier signal. After a cycle slip occurs, the PLL circuit transitions to a new point of steady balance, after which it continues tracking the satellite carrier signal. If a cycle slip occurs during signal tracking, an abnormal error equal to several integer number of semi-cycles (half-cycles) is introduced into the carrier phase measurements. If a cycle slip occurs after signal lock, an abnormal error equal to several integer number of cycles is introduced into the carrier phase measurements.
Calculating coordinates from received satellite signals entails the calculation of complex mathematical algorithms. These algorithms are computationally intense, often utilizing high processor and memory capacity. What are needed are methods and apparatus for detection and correction, or elimination, of abnormal measurements prior to execution of complex algorithms.

Detection and correction of anomalous measurements and ambiguity resolution in a global navigation satellite system receiver


There is provided an isolated immunoglobulin comprising two heavy polypeptide chains sufficient for the formation of a complete antigen binding site or several antigen binding sites, wherein the immunoglobulin is further devoid of light polypeptide chains.

The invention relates to new isolated immunoglobulins which are devoid of light polypeptide chains. These immunoglobulins do not consist in the degradation products of immunoglobulins composed of both heavy polypeptide and light polypeptide chains but to the contrary, the invention defines a new member of the family of the immunoglobulins, especially a new type of molecules capable of being involved in the immune recognition. Such immunoglobulins can be used for several purposes, especially for diagnosis or therapeutical purposes including protection against pathological agents or regulation of the expression or activity of proteins.
Up to now the structure proposed for immunoglobulins consists of a four-chain model referring to the presence of two identical light polypeptide chains (light chains) and two identical heavy polypeptide chains (heavy chains) linked together by disulfide bonds to form a y- or T-shaped macromolecules. These chains are composed of a constant region and a variable region, the constant region being subdivided in several domains. The two heavy polypeptide chains are usually linked by disulphide bounds in a so-called xe2x80x9chinge regionxe2x80x9d situated between the first and second domains of the constant region.
Among the proteins forming the class of the immunoglobulins, most of them are antibodies and accordingly present an antigen binding site or several antigen binding sites.
According to the four-chain model, the antigen binding site of an antibody is located in the variable domains of each of the heavy and light chains, and requires the association of the heavy and the light chains variable domains.
For the definition of these four-chain model immunoglobulins, reference is made to Roitt. I et al (Immunology-second-Edition Gower Medical Publishing USA, 1989). Reference is especially made to the part concerning the definition of the four-chain immunoglobulins, their polypeptidic and genetic structures, the definition of their variable and constant regions and the obtention of the fragments produced by enzymatic degradation according to well known techniques.
The inventors have surprisingly established that different molecules can be isolated from animals which naturally produce them, which molecules have functional properties of immunoglobulins these functions being in some cases related to structural elements which are distinct from those involved in the function of four-chain immunoglobulins due for instance to the absence of light chains.
The invention relates to two-chain model immunoglobulins which neither correspond to fragments obtained for instance by the degradation in particular the enzymatic degradation of a natural four-chain model immunoglobulin, nor correspond to the expression in host cells, of DNA coding for the constant or the variable region of a natural four-chain model immunoglobulin or a part of these regions, nor correspond to antibodies produced in lymphopaties for example in mice, rats or human.
E. S. Ward et al (1) have described some experiments performed on variable domains of heavy polypeptide chains (VH) or/and light polypeptide chains (VK/FV) to test the ability of these variable domains, to bind specific antigens. For this purpose, a library of VH genes was prepared from the spleen genomic DNA of mice previously immunized with these specific antigens.
Ward et al have described in their publication that VH domains are relatively sticky, presumably due to the exposed hydrophobic surface normally capped by the VK or Vxcex domains. They consequently envisage that it should be possible to design VH domains having improved properties and further that VH domains with binding activities could serve as the building blocks for making variable fragments (Fv fragments) or complete antibodies.
The invention does not start from the idea that the different fragments (light and heavy chains) and the different domains of these fragments of four-chain model immunoglobulin can be modified to define new or improved antigen binding sites or a four-chain model immunoglobulin.
The inventors have determined that immunoglobulins can have a different structure than the known four-chain model and that such different immunoglobulins offer new means for the preparation of diagnosis reagents, therapeutical agents or any other reagent for use in research or industrial purposes.
Thus the invention provides new immunoglobulins which are capable of showing functional properties of four-chain model immunoglobulins although their structure appears to be more appropriate in many circumstances for their use, their preparation and in some cases for their modification. Moreover these molecules can be considered as lead structures for the modification of other immunoglobulins. The advantages which are provided by these immunoglobulins comprise the possibility to prepare them with an increased facility.
The invention accordingly relates to immunoglobulins characterized in that they comprise two heavy polypeptide chains sufficient for the formation of a complete antigen binding site or several antigen binding sites, these immunoglobulins being further devoid of light polypeptide chains. In a particular embodiment of the invention, these immunoglobulins are further characterized by the fact that they are the product of the expression in a prokaryotic or in a eukaryotic host cell, of a DNA or of a cDNA having the sequence of an immunoglobulin devoid of light chains as obtainable from lymphocytes or other cells of Camelids.
The immunoglobulins of the invention can be obtained for example from the sequences which are described in FIG. 7.
The immunoglobulins of the invention, which are devoid of light chains are such that the variable domains of their heavy chains have properties differing from those of the four-chain immunoglobulin VH. The variable domain of a heavy-chain immunoglobulin of the invention has no normal interaction sites with the VL or with the CH1 domain which do not exist in the heavy chain immunoglobulins. it is hence a novel fragment in many of its properties such as solubility and position of the binding site. For clarity reasons we will call it VHH in this text to distinguish it from the classical VH of four-chain immunoglobulins.
By xe2x80x9ca complete antigen binding sitexe2x80x9d it is meant according to the invention, a site which will alone allow the recognition and complete binding of an antigen. This could be verified by any known method regarding the testing of the binding affinity.
These immunoglobulins which can be prepared by the technique of recombinant DNA, or isolated from animals, will be sometimes called xe2x80x9cheavy-chain immunoglobulinsxe2x80x9d in the following pages. In a preferred embodiment of the invention, these immunoglobulins are in a pure form.
In a first embodiment, the immunoglobulins of the invention are obtainable in prokaryotic cells, especially in E. coli cells by a process comprising the steps of:
a) cloning in a Bluecript vector of a DNA or cDNA sequence coding for the VHH domain of an immunoglobulin devoid of light chain obtainable for instance from lymphocytes of Camelids,
b) recovering the cloned fragment after amplification using a 5xe2x80x2 primer containing an Xho site and a 3xe2x80x2 primer containing the Spe site having the following sequence TC TTA ACT AGT GAG GAG ACG GTG ACC TG SEQ ID NO: 51,
c) cloning the-recovered fragment in phase in the immuno PBS vector after digestion of the vector with Xho and Spe restriction enzymes,
d) transforming host cells, especially E. coli by transfection with the recombinant immuno PBS vector of step c,
e) recovering the expression product of the VHH coding sequence, for instance by using antibodies raised against the dromadary VHH domain.
In another embodiment the immunoglobulins are hetero-specific immunoglobulins obtainable by a process comprising the steps of:
obtaining a first DNA or cDNA sequence coding for a VHH domain or part thereof having a determined specificity against a given antigen and comprised between Xho and Spe sites,
obtaining a second DNA or cDNA sequence coding for a VHH domain or part thereof, having a determined specificity different from the specificity of the first DNA or cDNA sequence and comprised between the Spe and EcoRI sites,
digesting an immuno PBS vector with EcoRI and XhoI restriction enzymes,
ligating the obtained DNA or cDNA sequences coding for VHH domains, so that the DNA or cDNA sequences are serially cloned in the vector,
transforming a host cell, especially E. coli cell by transfection, and recovering the obtained immunoglobulins.
In another embodiment, the immunoglobulins are obtainable by a process comprising the steps of:
obtaining a DNA or cDNA sequence coding for a VHH domain or part thereof, having a determined specific antigen binding site,
amplifying the obtained DNA or cDNA, using a 5xe2x80x2 primer containing an initiation codon and a HindIII site, and a 3xe2x80x2 primer containing a termination codon having a XhoI site,
recombining the amplified DNA or cDNA into the HindIII (position 2650) and XhoI (position 4067) sites of a plasmid pMM984,
transfecting permissive cells especially NB-E cells with the recombinant plasmid,
recovering the obtained products.
Successful expression can be verified with antibodies directed against a region of a VHH domain, especially by an ELISA assay.
According to another particular embodiment of this process, the immunoglobulins are cloned in a parvovirus.
In another example these immunoglobulins are obtainable by a process comprising the further cloning of a second DNA or cDNA sequence having another determined antigen binding site, in the pMM984 plasmid.
Such an Immunoglobulin can be further characterized in that it is obtainable by a process wherein the vector is Yep 52 and the transformed recombinant cell is a yeast especially S. cerevisiae.
A particular immunoglobulin is characterized in that it has a catalytic activity, especially in that it is directed against an antigen mimicking an activated state of a given substrate. These catalytic antibodies can be modified at the level of their biding site, by random or directed mutagenesis in order to increase oe modify their catalytic function. Reference may be made to the publication of Lerner et al (TIBS November 1987. 427-430) for the general technique for the preparation of such catalytic immunoglobulins.
According to a preferred embodiment, the immunoglobulins of the invention are characterized in that their variable regions contain in position 45, an amino-acid which is different from leucine, proline or glutamine residue.
Moreover the heavy-chain immunoglobulins are not products characteristic of lymphocytes of animals nor from lymphocytes of a human patient suffering from lymphopathies. Such immunoglobulins produced in lymphopathies are monoclonal in origin and result from pathogenic mutations at the genomic level. They have apparently no antigen binding site.
The two heavy polypeptide chains of these immunoglobulins can be linked by a hinge region according to the definition of Roitt et al.
In a particular embodiment of the invention, immunoglobulins corresponding to the above-defined molecules are capable of acting as antibodies.
The antigen binding site(s) of the immunoglobulins of the invention are located in the variable region of the heavy chain.
In a particular group of these immunoglobulins each heavy polypeptide chain contains one antigen binding site on its variable region, and these sites correspond to the same amino-acid sequence.
In a further embodiment of the invention the immunoglobulins are characterized in that their heavy polypeptide chains contain a variable region (VHH) and a constant region (CH) according to the definition of Roitt et al, but are devoid of the first domain of their constant region. This first domain of the constant region is called CH1.
These immunoglobulins having no CH1 domain are such that the variable region of their chains is directly linked to the hinge region at the C-terminal part of the variable region.
The immunoglobulins of the type described here-above can comprise type G immunoglobulins and especially immunoglobulins which are defined as immunoglobulins of class 2 (IgG2) or immunoglobulins of class 3 (IgG3).
The absence of the light chain and of the first constant domain lead to a modification of the nomenclature of the immunoglobulin fragments obtained by enzymatic digestion, according to Roitt et al.
The terms Fc and pFc on the one hand, Fcxe2x80x2 and pFcxe2x80x2 on the other hand corresponding respectively to the papain and pepsin digestion fragments are maintained.
The terms Fab F(ab)2 F(abxe2x80x2)2 Fabc, Fd and Fv are no longer applicable in their original sense as these fragments have either a light chain, the variable part of the light chain or the CH1 domain.
The fragments obtained by papain digestion and composed of the VHH domain and the hinge region will be called FVHHh or F(VHHh)2 depending upon whether or not they remain linked by the disulphide bonds.
In another embodiment of the invention, immunoglobulins replying to the hereabove given definitions can be originating from animals especially from animals of the camelid family. The inventors have found out that the heavy-chain immunoglobulins which are present in camelids are not associated with a pathological situation which would induce the production of abnormal antibodies with respect to the four-chain immunoglobulins. On the basis of a comparative study of old world camelids (Camelus bactrianus and Camelus dromaderius) and new world camelids (for example Lama Paccos, Lama Glama, and Lama Vicuqna) the inventors have shown that the immunoglobulins of the invention, which are devoid of light polypeptide chains are found in all species. Nevertheless differences may be apparent in molecular weight of these immunoglobulins depending on the animals. Especially the molecular weight of a heavy chain contained in these immunoglobulins can be from approximately 43 kd to approximately 47 kd, in particular 45 kd.
Advantageously the heavy-chain immunoglobulins of the invention are secreted in blood of camelids.
Immunoglobulins according to this particular embodiment of the invention are obtainable by purification from serum of camelids and a process for the purification is described in details in the examples. In the case where the immunoglobulins are obtained from Camelids, the invention relates to immunoglobulins which are not in their natural biological environment.
According to the invention immunoglobulin IgG2 as obtainable by purification from the serum of camelids can be characterized in that:
it is not adsorbed by chromatography on Protein G Sepharose column,
it is adsorbed by chromatography on Protein A Sepharose column,
it has a molecular weight of around 100 kd after elution with a pH 4.5 buffer (0.15 M NaCl, 0.58% acetic acid adjusted to pH 4.5 by NaOH),
it consists of heavy xcex32 polypeptide chains of a molecular weight of around 46 kd preferably 45 after reduction.
According to a further embodiment of the invention another group of immunoglobulins corresponding to IgG3, as obtainable by purification from the serum of Camelids is characterized in that the immunoglobulin:
is adsorbed by chromatography on a Protein A Sepharose column,
has a molecular weight of around 100 kd after elution with a pH 3.5 buffer (0.15 M NaCl, 0.58% acetic acid),
is adsorbed by chromatography on a Protein G Sepharose column and eluted with pH 3.5 buffer (0.15 M NaCl, 0.58% acetic acid).
consists of heavy xcex33 polypeptide chains of a molecular weight of around 45 Kd in particular between 43 and 47 kd after reduction.
The immunoglobulins of the invention which are devoid of light chains, nevertheless comprise on their heavy chains a constant region and a variable region. The constant region comprises different domains.
The variable region of immunoglobulins of the invention comprises frameworks (FW) and complementarity determining regions (CDR), especially 4 frameworks and 3 complementarity regions. It is distinguished from the four-chain immunoglobulins especially by the fact that this variable region can itself contain an antigen binding site or several, without contribution of the variable region of a light chain which is absent.
The amino-acid sequences of frameworks 1 and 4 comprise among others respectively amino-acid sequences which can be selected from the following:
for the framework 1 domain
GGSVQTGGSLRLSCEISGLTFD SEQ ID NO: 1
GGSVQTGGSLRLSCAVSGFSFS SEQ ID NO: 2
GGSEQGGGSLRLSCAISGYTYG SEQ ID NO: 3
GGSVQPGGSLTLSCTVSGATYS SEQ ID NO: 4
CGSVQAGGSLRLSCTGSGFPYS SEQ ID NO: 5
GGSVQAGGSLRLSCVAGFGTS SEQ ID NO: 6
GGSVQAGGSLRLSCVSFSPSS SEQ ID NO: 7
for the framework 4 domain
WGQGTQVTVSS SEQ ID NO: 8
WGQGTLVTVSS SEQ ID NO: 9
WGQGAQVTVSS SEQ ID NO: 10
WGQGTQVTASS SEQ ID NO: 11
RGQGTQVTVSL SEQ ID NO: 12
for the CDR3 domain
As stated above, the immunoglobulins of the invention are preferably devoid of the totality of their CH1 domain.
Such immunoglobulins comprise CH2 and CH3 domains in the C-terminal region with respect to the hinge region.
According to a particular embodiment of the invention the constant region of the immunoglobulins comprises CH2 and CH3 domains comprising an amino-acid sequence selected from the following:
for the CH2 domain:
APELLGGPTVFIFPPKPKDVLSITLTP SEQ ID NO: 31
APELPGGPSVFVFPTKPKDVLSISGRP SEQ ID NO: 32
APELPGGPSVFVFPPKPKDVLSISGRP SEQ ID NO: 33
APELLGGPSVFIFPPKPKDVLSISGRP SEQ ID NO: 34
for the CH3 domain:
GQTREPQVYTLA SEQ ID NO: 35
GQTREPQVYTLAPXRLEL SEQ ID NO: 36
GQPREPQVYTLPPSRDEL SEQ ID NO: 109
GQPREPQVYTLPPSREEM SEQ ID NO: 110
GQPREPQVYTLPPSQEEM SEQ ID NO: 111
Interestingly the inventors have shown that the hinge region of the immunoglobulins of the invention can present variable lengths. When these immunoglobulins act as antibodies, the length of the hinge region will participate to the determination of the distance separating the antigen binding sites.
Preferably an immunoglobulin according to the invention is characterized in that its hinge region comprises from 0 to 50 amino-acids.
Particular sequences of hinge region of the immunoglobulins of the invention are the following.
GTNEVCKCPKCP SEQ ID NO: 37
or,
EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP SEQ ID NO: 38
The short hinge region corresponds to an IgG3 molecule and the long hinge sequence corresponds to an IgG2 molecule.
Isolated VHH derived from heavy chain immunoglobulins or VHH libraries corresponding to the heavy chain immunoglobulins can be distinguished from VHH cloning of four-chain model immunoglobulins on the basis of sequence features characterizing heavy chain immunoglobulins.
The camel heavyxe2x80x94chain immunoglobulin VHH region shows a number of differences with the VHH regions derived from 4-chain immunoglobulins from all species examined. At the levels of the residues involved in the VHH/VL interactions, an important difference is noted at the level of position 45 (FW) which is practically always leucine in the 4-chain immunoglobulins (98%), the other amino acids at this position being proline (1%) or glutamine (1%).
In the camel heavy-chain immunoglobulin, in the sequences examined at present, leucine at position 45 is only found once. It could originate from a four-chain immunoglobulin. In the other cases, it is replaced by arginine, cysteine or glutamic acid residue. The presence of charged amino acids at this position should contribute to making the VHH more soluble.
The replacement by camelid specific residues such as those of position 45 appears to be interesting for the construction of engineered VHH regions derived from the VHH repertoire of 4-chain immunoglobulins.
A second feature specific of the camelid VHH domain is the frequent presence of a cysteine in the CDR3 region associated with a cysteine in the CDR1 position 31 or 33 or FW2 region at position 45. The possibility of establishing a disulphide bond between the CDR3 region and the rest of the variable domain would contribute to the stability and positioning of the binding site.
With the exception of a single pathogenic myeloma protein (DAW) such a disulphide bond has never been encountered in immunoglobulin V regions derived from 4 chain immunoglobulins.
The heavy-chain immunoglobulins of the invention have further the particular advantage of being not sticky. Accordingly these immunoglobulins being present in the serum, aggregate much less than isolated heavy chains of a four-chain immunoglobulins. The immunoglobulins of the invention are soluble to a concentration above 0.5 mg/ml, preferably above 1 mg/ml and more advantageously above 2 mg/ml.
These immunoglobulins further bear an extensive antigen binding repertoire and undergo affinity and specificity maturation in vivo. Accordingly they allow the isolation and the preparation of antibodies having defined specificity, regarding determined antigens.
Another interesting property of the immunoglobulins of the invention is that they can be modified and especially humanized. Especially it is possible to replace all or part of the constant region of these immunoglobulins by all or part of a constant region of a human antibody. For example the CH2 and/or CH3 domains of the immunoglobulin could be replaced by the CH2 and/or CH3 domains of the IgG xcex33 human immunoglobulin.
In such humanized antibodies it is also possible to replace a part of the variable sequence, namely one or more of the framework residues which do not intervene in the binding site by human framework residues, or by a part of a human antibody.
Conversely features (especially peptide fragments) of heavy-chain immunoglobulin VHH regions, could be introduced into the VH or VL regions derived from four-chain immunoglobulins with for instance the aim of achieving greater solubility of the immunoglobulins.
The invention further relates to a fragment of an immunoglobulin which has been described hereabove and especially to a fragment selected from the following group:
a fragment corresponding to one heavy polypeptide chain of an immunoglobulin devoid of light chains,
fragments obtained by enzymatic digestion of the immunoglobulins of the invention, especially those obtained by partial digestion with papain leading to the Fc fragment (constant fragment) and leading to FVHHh fragment (containing the antigen binding sites of the heavy chains) or its dimer F(VHHh)2, or a fragment obtained by further digestion with papain of the Fc fragment, leading to the pFc fragment corresponding to the C-terminal part of the Fc fragment,
homologous fragments obtained with other proteolytic enzymes,
a fragment of at least 10 preferably 20 amino acids of the variable region of the immunoglobulin, or the complete variable region, especially a fragment corresponding to the isolated VHH domains or to the VHH dimers linked to the hinge disulphide,
a fragment corresponding to the hinge region of the immunoglobulin,or to at least 6 amino acids of this hinge region,
a fragment of the hinge region comprising a repeated sequence of Pro-X,
a fragment corresponding to at least 10 preferably 20 amino acids of the constant region or to the complete constant region of the immunoglobulin.
The invention also relates to a fragment comprising a repeated sequence, Pro-X which repeated sequence contains at least 3 repeats of Pro-X, X being any amino-acid and preferably Gln (glutamine), Lys (lysine) or Glu (acide glutamique); a particular repeated fragment is composed of a 12-fold repeat of the sequence Pro-X.
Such a fragment can be advantageously used as a link between different types of molecules.
The amino-acids of the Pro-X sequence are chosen among any natural or non natural amino-acids.
The fragments can be obtained by enzymatic degradation of the immunoglobulins. They can also be obtained by expression in cells or organisms, of nucleotide sequence coding for the immunoglobulins, or they can be chemically synthesized.
The invention also relates to anti-idiotypes antibodies belonging to the heavy chain immunoglobulin classes. Such anti-idiotypes can be produced against human or animal idiotypes. A property of these anti-idiotypes is that they can be used as idiotypic vaccines, in particular for vaccination against glycoproteins or glycolipids and where the carbohydrate determines the epitope.
The invention also relates to anti-idiotypes capable of recognizing idiotypes of heavy-chain immunoglobulins.
Such anti-idiotype antibodies can be either syngeneic antibodies or allogenic or xenogeneic antibodies.
The invention also concerns nucleotide sequences coding for all or part of a protein which amino-acid sequence comprises a peptide sequence selected from the following:
GGSVQTHGSLRLSCEISGLTFD SEQ ID NO: 1
GGSVQTGGSLRLSCAVSGFSFS SEQ ID NO: 2
GCSEQGGGSLRLSCAISGYTYG SEQ ID NO: 3
GGSVQPGGSLTLSCTVSGATYS SEQ ID NO: 4
GGSVQAGGSLRLSCTGSGFPYS SEQ ID NO: 5
GGSVQAGGSLRLSCVAGFGTS SEQ ID NO: 6
CGSVQAGGSLRLSCVSFSPSS SEQ ID NO: 7
WGQGTQVTVSS SEQ ID NO: 8
WGQGTLVTVSS SEQ ID NO: 9
WGQGAQVTVSS SEQ ID NO: 10
WGQGTQVTASS SEQ ID NO: 11
RGQGTQVTVSL SEQ ID NO: 12
APELLGGPSVFVFPPKPKDVLSISGXPK SEQ ID NO: 39
APELPGGPSVFVFPTKPKDVLSISGRPK SEQ ID NO: 40
APELPGGPSVFVFPPKPKDVLSISGRPK SEQ ID NO: 41
APELLGGPSVFIFPPKPKDVLSISGRPK SEQ ID NO: 42
GQTREPQVYTLAPXRLEL SEQ ID NO: 36
GQPREPQVYTLPPSRDEL SEQ ID NO: 109
GQPREPQVYTLPPSREEM SEQ ID NO: 110
GQPREPQVYTLPPSQEEM SEQ ID NO: 111
VTVSSGTNEVCKCPKCPAPELPGGPSVFVFP SEQ ID NO: 43
or,
VTVSSEPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCPAPELLGGPSVFIFP SEQ ID NO: 44
GTNEVCKCPKCP SEQ ID NO: 37
APELPGGPSVFVFP SEQ ID NO: 45
EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP SEQ ID NO: 38
APELLGGPSVFIFP SEQ ID NO: 46
Such nucleotide sequences can be deduced from the amino-acid sequences taking into account the deneneracy of the genetic code. They can be synthesized or isolated from cells producing immunoglobulins of the invention.
A procedure for the obtention of such DNA sequences is described in the examples.
The invention also contemplates RNA, especially mRNA sequences corresponding to these DNA sequences, and also corresponding cDNA sequences.
The nucleotide sequences of the invention can further be used for the preparation of primers appropriate for the detection in cells or screening of DNA or cDNA libraries to isolate nucleotide sequences coding for immunoglobulins of the invention.
Such nucleotide sequences can be used for the preparation of recombinant vectors and the expression of these sequences contained in the vectors by host cells especially prokaryotic cells like bacteria or also eukaryotic cells and for example CHO cells, insect cells, simian cells like Vero cells, or any other mammalian cells. Especially the fact that the immunoglobulins of he invention are devoid of light chains permits to secrete them in eukaryotic cells since there is no need to have recourse to the step consisting in the formation of the BIP protein which is required in the four-chain immunoglobulins.
The inadequacies of the known methods for producing monoclonal antibodies or immunoglobulins by recombinant DNA technology comes from the necessity in the vast majority of cases to clone simultaneously the VH and VL domains corresponding to the specific binding site of 4 chain immunoglobulins. The animals and especially camelids which produce heavy-chain immunoglobulins according to the invention, and possibly other vertebrate species are capable of producing heavy-chain immunoglobulins of which the binding site is located exclusively in the VHH domain. Unlike the few heavy-chain immunoglobulins produced in other species by chain separation or by direct cloning, the camelid heavy-chain immunoglobulins have undergone extensive maturation in vivo. Moreover their V region has naturally evolved to function in absence of the VL. They are therefore ideal for producing monoclonal antibodies by recombinant DNA technology. As the obtention of specific antigen binding clones does not depend on a stochastic process necessitating a very large number of recombinant cells, this allows also a much more extensive examination of the repertoire.
This can be done at the level of the non rearranged VHH repertoire using DNA derived from an arbitrarily chosen tissue or cell type or at the level of the rearranged VHH repertoire, using DNA obtained from B lymphocytes. More interesting however is to transcribe the mRNA from antibody producing cells and to clone the cDNA with or without prior amplification into an adequate vector. This will result in the obtention of antibodies which have already undergone affinity maturation.
The examination of a large repertoire should prove to be particularly useful in the search for antibodies with catalytic activities.
The invention thus provides libraries which can be generated in a way which includes part of the hinge sequence, the identification is simple as the hinge is directly attached to the VHH domain.
These libraries can be obtained by cloning cDNA from lymphoid cells with or without prior PCR amplification. The PCR primers are located in the promoter, leader or framework sequences of the VHH for the 5xe2x80x2 primer and in the hinge, CH2, CH3, 3xe2x80x2 untranslated region or polyA tail for the 3xe2x80x2 primer. A size selection of amplified material allows the construction of a library limited to heavy chain immunoglobulins.
In a particular example, the following 3xe2x80x2 primer in which a KpnI site has been constructed and which corresponds to amino-acids 313 to 319 (CGC CAT CAA GGT AAC AGT TGA) SEQ ID NO: 47 is used in conjunction with mouse VHH primers described by Sestry et al and containing a Xho site
These primers yield a library of camelid heavy chain immunoglobulins comprising the VHH region (related to mouse or human subgroup III), the hinge and a section of CH2.
In another example, the cDNA is polyadenylated at its 5xe2x80x2 end and the mouse specific VHH primers are replaced by a poly T primer with an inbuilt XhoI site, at the level of nucleotide 12.
CTCGAGT12.
The same 3xe2x80x2 primer with a KpnI site is used.
This method generates a library containing all subgroups of immunoglobulins.
Part of the interest in cloning a region encompassing the hinge-CH2 link is that in both xcex32 and xcex33, a Sac site is present immediately after the hinge. This site allows the grafting of the sequence coding for the VHH and the hinge onto the Fc region of other immunoglobulins, in particular the human IgG1 and IgG3 which have the same amino acid sequence at this site (Glu246 Leu247).
As an example, the invention contemplates a cDNA library composed of nucleotide sequences coding for a heavy-chain immunoglobulin, such as obtained by performing the following steps:
a) treating a sample containing lymphoid cells, especially periferal, lymphocytes, spleen cells, lymph nodes or another lyphoid tissue from a healthy animal, especially selected among the Camelids, in order to separate the lymphoid cells,
b) separating polyadenylated RNA from the other nucleic acids and components of the cells,
c) reacting the obtained RNA with a reverse transcriptase in order to obtain the corresponding cDNA,
d) contacting the cDNA of step c) with 5xe2x80x2 primers corresponding to mouse VH domain of four-chain immunoglobulins, which primer contains a determined restriction site, for example an XhoI site and with 3xe2x80x2 primers corresponding to the N-terminal part of a CH2 domain containing a KpnI site,
e) amplifying the DNA,
f) cloning the amplified sequence in a vector, especially in a bluescript vector,
g) recovering the clones hybridizing with a probe corresponding to the sequence coding for a constant domain from an isolated heavy-chain immunoglobulin.
This cloning gives rise to clones containing DNA sequences including the sequence coding for the hinge. It thus permits the characterization of the subclass of the immunoglobulin and the SacI site useful for grafting the FVHHh to the Fc region.
The recovery of the sequences coding for the heavy-chain immunoglobulins can also be achieved by the selection of clones containing DNA sequences having a size compatible with the lack of the CH1 domain.
It is possible according to another embodiment of the invention, to add the following steps between steps c) and d) of the above process:
in the presence of a DNA polymerase and of deoxyribonucleotide triphosphates, contacting said cDNA with oligonucleotide degenerated primers, which sequences are capable of coding for the hinge region and N-terminal VHH domain of an immunoglobulin, the primers being capable of hybridizing with the cDNA and capable of initiating the extension of a DNA sequence complementary to the cDNA used as template,
recovering the amplified DNA.
The clones can be expressed in several types of expression vectors. As an example using a commercially available vector Immuno PBS (Huse et al: Science (1989) 246, 1275), clones produced in Bluescript(copyright) according to the above described procedure, are recovered by PCR using the same XhoI containing 5xe2x80x2 primer and a new 3xe2x80x2 primer, corresponding to residues 113-103 in the framework of the immunoglobulins, in which an Spe site has been constructed: TC TTA ACT AGT GAG GAG ACG GTG ACC TG SEQ ID NO: 51. This procedure allows the cloning of the VHH in the Xho/Spe site of the Immuno PBS vector. However, the 3xe2x80x2 end of the gene is not in phase with the identification xe2x80x9ctagxe2x80x9d and the stop codon of the vector. To achieve this, the construct is cut with Spe and the 4 base overhangs are filled in, using the Klenow fragment after which the vector is religated. A further refinement consists in replacing the marker (xe2x80x9ctagxe2x80x9d) with a poly histidine so that metal purification of the cloned VHH can be performed. To achieve this a Spe/EcoRI double stranded oligo-nucleotide coding for 6 histidines and a termination codon is first constructed by synthesis of both strands followed by heating and annealing:
CTA GTG CAC CAC CAT CAC CAT CAC TAA TAG SEQ ID NO: 52
AC GTG GTG GTA GTG GTA GTG ATT ATC TTA A SEQ ID NO: 53
The vector containing the insert is then digested with SpeI and EcoRI to remove the resident xe2x80x9ctagxe2x80x9d sequence which can be replaced by the poly-His/termination sequence. The produced VHH can equally be detected by using antibodies raised against the dromedary VHH regions. Under laboratory conditions, VHH regions are produced in the Immuno PBS vector in mg amounts per liter.
The invention also relates to a DNA library composed of nucleotide sequences coding for a heavy-chain immunoglobulin, such as obtained from cells with rearranged immunoglobulin genes.
In a preferred embodiment of the invention, the library is prepared from cells from an animal previously immunized against a determined antigen. This allows the selection of antibodies having a preselected specificity for the antigen used for immunization.
In another embodiment of the invention, the amplification of the cDNA is not performed prior to the cloning of the cDNA.
The heavy-chain of the four-chain immunoglobulins remains sequestered in the cell by a chaperon protein (BIP) until it has combined with a light chain. The binding site for the chaperon protein is the CH1 domain. As this domain is absent from the heavy chain immunoglobulins, their secretion is independent of the presence of the BIP protein or of the light chain. Moreover the inventors have shown that the obtained immunoglobulins are not sticky and accordingly will not abnormally aggregate.
The invention also relates to a process for the preparation of a monoclonal antibody directed against a determined antigen, the antigen binding site of the antibody consisting of heavy polypeptide chains and which antibody is further devoid of light polypeptide chains, which process comprises:
immortalizing lymphocytes, obtained for example from the peripheral blood of Camelids previously immunized with a determined antigen, with an immortal cell and preferably with myeloma cells, in order to form a hybridoma,
culturing the immortalized cells (hybridoma) formed and recovering the cells producing the antibodies having the desired specificity.
The preparation of antibodies can also be performed without a previous immunization of Camelids.
According to another process for the preparation of antibodies, the recourse to the technique of the hybridoma cell is not required.
According to such process, antibodies are prepared in vitro and they can be obtained by a process comprising the steps of:
cloning into vectors, especially into phages and more particularly filamentous bacteriophages, DNA or cDNA sequences obtained from lymphocytes especially PBLs of Camelids previously immunized with determined antigens,
transforming prokaryotic cells with the above vectors in conditions allowing the production of the antibodies,
selecting the antibodies for their heavy-chain structure and further by subjecting them to antigen-affinity selection,
recovering the antibodies having the desired specificity,
In another embodiment of the invention the cloning is performed in vectors, especially into plasmids coding for bacterial membrane proteins. Procaryotic cells are then transformed with the above vectors in conditions allowing the expression of antibodies in their membrane.
The positive cells are further selected by antigen affinity selection.
The heavy chain antibodies which do not contain the CH1 domain present a distinct advantage in this respect. Indeed, the CH1 domain binds to BIP type chaperone proteins present within eukaryotic vectors and the heavy chains are not transported out of the endocytoplasmic reticulum unless light chains are present. This means that in eukaryotic cells, efficient cloning of 4-chain immunoglobulins in non mammalian cells such as yeast cells can depend on the properties of the resident BIP type chaperone and can hence be very difficult to achieve. In this respect the heavy chain antibodies of the invention which lack the CH1 domain present a distinctive advantage.
In a preferred embodiment of the invention the cloning can be performed in yeast either for the production of antibodies or for the modification of the metabolism of the yeast. As example, Yep 52 vector can be used. This vector has the origin of replication (ORI) 2xcexc of the yeast together with a selection marker Leu 2.
The cloned gene is under the control of gall promoter and accordingly is inducible by galactose. Moreover, the expression can be repressed by glucose which allows the obtention of very high concentration of cells before the induction.
The cloning between BamHI and SalI sites using the same strategy of production of genes by PCR as the one described above, allows the cloning of camelid immunoglobulin genes in E. coli. As example of metabolic modulation which can be obtained by antibodies and proposed for the yeast, one can site the cloning of antibodies directed against cyclins, that is proteins involved in the regulation of the cellular cycle of the yeast (TIBS 16 430 J. D. Mc Kinney, N. Heintz 1991). Another example is the introduction by genetic engineering of an antibody directed against CD28, which antibody would be inducible (for instance by gall), within the genome of the yeast. The CD28 is involved at the level of the initiation of cell division, and therefore the expression of antibodies against this molecule would allow an efficient control of multiplication of the cells and the optimization of methods for the production in bioreactors or by means of immobilized cells.
In yet another embodiment of the invention, the cloning vector is a plasmid or a eukaryotic virus vector and the cells to be transformed are eukaryotic cells, especially yeast cells, mammalian cells for example CHO cells or simian cells such as Vero cells, insect cells, plant cells, or protozoan cells.
For more details concerning the procedure to be applied in such a case, reference is made to the publication of Marks et al, J. Mol. Biol. 1991, 222:581-597.
Furthermore, starting from the immunoglobulins of the invention, or from fragments thereof, new immunoglobulins or derivatives can be prepared.
Accordingly immunoglobulins replying to the above given definitions can be prepared against determined antigens. Especially the invention provides monoclonal or polyclonal antibodies devoid of light polypeptide chains or antisera containing such antibodies and directed against determined antigens and for example against antigens of pathological agents such as bacteria, viruses or parasites. As example of antigens or antigenic determinants against which antibodies could be prepared, one can cite the envelope glycoproteins of viruses or peptides thereof, such as the external envelope glycoprotein of a HIV virus, the surface antigen of the hepatitis B virus.
Immunoglobulins of the invention can also be directed against a protein, hapten, carbohydrate or nucleic acid.
Particular antibodies according to the invention are directed against the galactosylxcex1-1-3-galactose epitope.
The immunoglobulins of the invention allow further the preparation of combined products such as the combination of the heavy-chain immunoglobulin or a fragment thereof with a toxin, an enzyme, a drug, a hormone.
As example one can prepare the combination of a heavy-chain immunoglobulin bearing an antigen binding site recognizing a myeloma immunoglobulin epitope with the abrin or mistletoe lectin toxin. Such a construct would have its uses in patient specific therapy.
Another advantageous combination is that one can prepare between a heavy-chain immunoglobulins recognizing an insect gut antigen with a toxin specific for insects such as the toxins of the different serotypes of Bacillus thuringiensis or Bacillus sphaericus. Such a construct cloned into plants can be used to increase the specificity or the host range of existing bacterial toxins.
The invention also proposes antibodies having different specificities on each heavy polypeptide chains. These multifunctional, especially bifunctional antibodies could be prepared by combining two heavy chains of immunoglobulins of the invention or one heavy chain of an immunoglobulin of the invention with a fragment of a four-chain model immunoglobulin.
The invention also provides hetero-specific antibodies which can be used for the targetting of drugs or any biological substance like hormones. In particular they can be used to selectively target hormones or cytokines to a limited category of cells. Examples are a combination of a murine or human antibody raised against interleukin 2 (IL2) and a heavy-chain antibody raised against CD4 cells. This could be used to reactivate CD4 cells which have lost their IL2 receptor.
The heavy-chain immunoglobulins of the invention can also be used for the preparation of hetero-specific antibodies. These can be achieved either according to the above described method by reduction of the bridges between the different chains and reoxydation, according to the usual techniques, of two antibodies having different specificities, but it can also be achieved by serial cloning of two antibodies for instance in the Immuno pBS vector.
In such a case, a first gene corresponding to the VHH domain comprised between Xho site and a Spe site is prepared as described above. A second gene is then prepared through an analogous way by using as 5xe2x80x2 extremity a primer containing a Spe site, and as 3xe2x80x2 extremity a primer containing a termination codon and an EcoRI site. The vector is then digested with EcoRI and XhoI and further both VHH genes are digested respectively by Xho/Spe and by Spe/EcoRI.
After ligation, both immunoglobulin genes are serially cloned. The spacing between both genes can be increased by the introduction of addition codons within the 5xe2x80x2 SpeI primer.
In a particular embodiment of the invention, the hinge region of IgG2 immunoglobulins according to the invention is semi-rigid and is thus appropriate for coupling proteins. In such an application proteins or peptides can be linked to various substances, especially to ligands through the hinge region used as spacer. Advantageously the fragment comprises at least 6 amino acids.
According to the invention it is interesting to use a sequence comprising a repeated sequence Pro-X, X being any amino-acid and preferably Gln, Lys or Glu, especially a fragment composed of at least a 3-fold repeat and preferably of a 12-fold repeat, for coupling proteins to ligand, or for assembling different protein domains.
The hinge region or a fragment thereof can also be used for coupling proteins to ligands or for assembling different protein domains.
Usual techniques for the coupling are appropriate and especially reference may be made to the technique of protein engineering by assembling cloned sequences.
The antibodies according to this invention could be used as reagents for the diagnosis in vitro or by imaging techniques. The immunoglobulins of the invention could be labelled with radio-isotopes, chemical or enzymatic markers or chemiluminescent markers.
As example and especially in the case of detection or observation with the immunoglobulins by imaging techniques, a label like technetium, especially technitium 99 is advantageous. This label can be used for direct labelling by a coupling procedure with the immunoglobulins or fragments thereof or for indirect labelling after a step of preparation of a complex with the technitium.
Other interesting radioactive labels are for instance indium and especially indium 111, or iodine, especially I131, I125 and I123.
For the description of these techniques reference is made to the FR patent application published under number 2649488.
In these applications the small size of the VHH fragment is a definitive advantage for penetration into tissue.
The invention also concerns monoclonal antibodies reacting with anti-idiotypes of the above-described antibodies.
The invention also concerns cells or organisms in which heavy-chain immunoglobulins have been cloned. Such cells or organisms can be used for the purpose of producing heavy-chain immunoglobulins having a desired preselected specificity, or corresponding to a particular repertoire. They can also be produced for the purpose of modifying the metabolism of the cell which expresses them. In the case of modification of the metabolism of cells transformed with the sequences coding for heavy-chain immunoglobulins, these produced heavy-chain immunoglobulins are used like antisense DNA. Antisense DNA is usually involved in blocking the expression of certain genes such as for instance the variable surface antigen of trypanosomes or other pathogens. Likewise, the production or the activity of certain proteins or enzymes could be inhibited by expressing antibodies against this protein or enzyme within the same cell.
The invention also relates to a modified 4-chain immunoglobulin or fragments thereof, the VH regions of which has been partialy replaced by specific sequences or amino acids of heavy chain immunoglobulins, especially by sequences of the VHH domain. A particular modified VH domain of a four-chain immunoglobulin, is characterized in that the leucine, proline or glutamine in position 45 of the VH regions has been replaced by other amino acids and preferably by arginine, glutamic acid or cysteine.
A further modified VH or VL domain of a four-chain immunoglobulin, is characterized by linking of CDR loops together or to FW regions by the introduction of paired cysteines, the CDR region being selected between the CDR1 and the CDR3, the FW region being the FW2 region, and especially in which one of the cysteines introduced is in position 31, 33 of the CDR1 or 45 of FW2; and the other in CDR3.
Especially the introduction of paired cysteines is such that the CDR3 loop is linked to the FW2 or CDR1 domain and more especially the cysteine of the CDR3 of the VH is linked to a cysteine in position 31, 33 of the CDR1 or in position 45 of FW2.
In another embodiment of the invention, plant cells can be modified by the heavy-chain immunoglobulins according to the invention, in order that they acquire new properties or increased properties.
The heavy-chain immunoglobulins of the invention can be used for gene therapy of cancer for instance by using antibodies directed against proteins present on the tumor cells.
In such a case, the expression of one or two VHH genes can be obtained by using vectors derived from parvo or adeno viruses. The parvo viruses are characterized by the fact that they are devoid of pathogenicity or almost not pathogenic for normal human cells and by the fact that they are capable of easily multiplying in cancer cells (Russel S. J. 1990, Immunol. Today II. 196-200).
The heavy-chain immunoglobulins are for instance cloned within HindIII/XbaI sites of the infectious plasmid of the murine MVM virus (pMM984). (Merchlinsky et al, 1983, J. Virol. 47, 227-232) and then placed under the control of the MVM38 promoter.
The gene of the VHH domain is amplified by PCR by using a 5xe2x80x2 primer containing an initiation codon and a HindIII site, the 3xe2x80x2 primer containing a termination codon and a XbaI site.
This construct is then inserted between positions 2650 (HindIII) and 4067 (XbaI) within the plasmid.
The efficiency of the cloning can be checked by transfection. The vector containing the antibody is then introduced in permissive cells (NB-E) by transfection.
The cells are recovered after two days and the presence of VHH regions is determined with an ELISA assay by using rabbit antiserum reacting with the VHH part.
The invention further allows the preparation of catalytic antibodies through different ways. The production of antibodies directed against components mimicking activated states of substrates (as example vanadate as component mimicking the activated state of phosphate in order to produce their phosphoesterase activities, phosphonate as compound mimicking the peptidic binding in order to produce proteases) permits to obtain antibodies having a catalytic function. Another way to obtain such antibodies consists in performing a random mutagenesis in clones of antibodies for example by PCR, in introducing abnormal bases during the amplification of clones. These amplified fragments obtained by PCR are then introduced within an appropriate vector for cloning. Their expression at the surface of the bacteria permits the detection by the substrate of clones having the enzymatic activity. These two approaches can of course be combined. Finally, on the basis of the data available on the structure, for example the data obtained by XRay crystallography or NMR, the modifications can be directed. These modifications can be performed by usual techniques of genetic engineering or by complete synthesis. One advantage of the VHH of the heavy chain immunoglobulins of the invention is the fact that they are sufficiently soluble.
The heavy chain immunoglobulins of the invention can further be produced in plant cells, especially in transgenics plants. As example the heavy chain immunoglobulins can be produced in plants using the pMon530 plasmid (Roger et al. Meth Enzym 153 1566 1987) constitutive plant expression vector as has been described for classical four chain antibodies (Hiat et al. Nature 342 76-78, 1989) once again using the appropriate PCR primers as described above, to generate a DNA fragment in the right phase.


Techniques include receiving a pressure input from a user of a user device at a pressure-sensitive display of the device and determining that the display is covered (e.g., obscured from the user's view) using the device. The techniques further include, in response to receiving the pressure input and determining that the display is covered, receiving a search query from the user at the device via voice input, transmitting the query to a search system via a network using the device, and receiving search results from the system at the device subsequent to transmitting the query. The techniques also include performing one or more actions associated with the search results using the user device (e.g., displaying the results to the user at the display, providing a text-to-speech or playback output for the results to the user, and/or forwarding the results to another computing device for display and/or text-to-speech or playback output).

In recent years, the use of computers, tablets, smartphones, smart watches, and other stationary and mobile computing devices has grown significantly. Additionally, the inclusion of various touchscreen technologies configured to receive user inputs at display screens of these and other computing devices has also increased. Today, many consumer and industrial computing devices and appliances are capable of receiving user inputs at display screens via any of a variety of different touchscreen technologies, including resistive, capacitive, surface-acoustic-wave (SAW), optical, acoustic, as well as any other touchscreen technologies or types.

This patent is subject to a terminal disclaimer.

Performing searches using computing devices equipped with pressure-sensitive displays


A system and methods for the automated display of a borderless sun glare block area and sunshield in a windshield, which can be made of electrochromatic glass, in a motor vehicle driven at any time and at any location on earth is presented. The first method, which accurately calculates the precise position of the sun glare block area, is based on the apparent solar position, the direction of travel relative to the true North, the slope of the road, the windshield tilt angle, and the dynamic position of the driver's eyes. It uses sets of different formulas depending on the position of the sun glare on the windshield and the slope of the road. The second method calculates the changing opacities of the borderless sun glare block area, whose opacity decreases from its center to its edge and matches that of the sunshield, based on the ambient light intensity.

1. Technical Field of the Invention
This invention relates generally to sun glare on a vehicular windshield, and more particularly to providing a method for the calculation of the sun glare position and a system for displaying a darkened area in the windshield to reduce the intensity of the sun glare or to block the sun glare from the driver's eyes.
2. Description of the Related Art
Sun glare is one of the most overlooked dangers of driving. It causes fatal accidents and injuries, creates highway havoc, and drastically slows traffic. The situation is worst during the height of morning and evening commutes for east- and west-bound traffic, respectively. When the sun directly perches on the horizon, a sudden, direct flash of sunlight lasting a mere decisecond can easily blind the driver. Considering the significant potential impacts of sun glare on drivers, many systems and methods of reducing or blocking sun glare have been presented in the past two decades. The most relevant arts prior to this invention are described in the following paragraphs.
Many visor extensions are available on the market and/or have been patented. Some are manual and others are automatic (U.S. Pat. No. 5,271,653, December 1993, Shirley; U.S. Pat. No. 6,286,887 B1, September 2001, Hashmi; U.S. Patent Application 2002/0130530, September 2002, Zenisek; U.S. Pat. No. 7,163,252 B1, January 2007, Neault; U.S. Pat. No. 4,923,238, May 1990, Morgulis, Tilman, Shemes). However, these visor extensions provide protection only if the sun glare is in the top region of the windshield and they may obstruct the driver's view. When the area of intense sun glare lies in the lower region of the windshield, the driver has to raise one hand to shield the sun's piercing light and keep only one hand on the steering wheel, causing a dangerous situation.
A number of inventions consist of manual laterally movable sun visors. Although these visors can move in both the vertical and horizontal directions, they still have the shortcomings of a visor because they are inconvenient and may divert the driver's attention away from the road. A movable visor that is automatically positioned laterally across the windshield and extended downward is documented in U.S. Pat. No. 4,874,938. The positioning is based on the azimuth and elevation of the sun and the angle of the sun's direction relative to the driver's eyes. Angles are detected by a complicated detection system, and the positioning requires a sophisticated motor and cable system. Although the positioning is automatic and may be accurate, the invention has the shortcomings of a visor and the system requires significant hardware additions and modifications. Other forms of sun glare shields are also available, such as an extendable sliding shield of polarized material having a black glass coating (U.S. Pat. No. 5,513,892, May 1996, Thomas). However, these also require extensive hardware addition and modification and obstruct the driver's view.
Another group of inventions employs electrochromatic elements whose transparency can be varied according to the amount of electric voltage or current applied. Some inventions involve coverings over the entire area of the windshield while others restrict the change in opacity in only the upper region, certain fixed sections, or shading bands of the windshield. Several more advanced inventions incorporate changes in opacity of the windshield from the top edge throughout the entire windshield in a propagating manner, such as in U.S. Patent Application 2007/0133078 A1. However, none of the aforementioned systems provide a method that is automatic or darkens only a specific area in the windshield needed to block the sun glare. Another related invention is an anti-dazzle device, installed between the driver and the windshield, with optical sensors that will darken the electro-sensitive screen as documented in U.S. Pat. No. 5,258,607. Although it is automatic with an optical sensor, the device is cumbersome. All the inventions noted in this paragraph unnecessarily darken areas of the windshield that are not in the path of the sun glare to the driver's eyes, which will inadvertently obstruct the driver's view.
An electrochromatic visor that darkens based on the intensity of sunlight detected, consisting of a sophisticated light-detecting apparatus for monitoring sunlight incident upon the face of the driver, is documented in U.S. Pat. No. 6,811,201 B2. The system can darken a specific required area in the visor. However, this invention has the limitation of a visor; a visor fulfills its purpose only if the sun glare is viewed in the top region of the windshield. It also requires a sophisticated digital camera and sensor system. A window pane with a visor-like segment and a glare sensor measuring the eye position of the driver is documented in DE 102005007427A1. The transparency of the segment can be adjusted based on the output signal of the glare sensor. Similar to the visor, the visor segment only serves its purpose when the sun glare area is in the top region of the windshield, and the invention requires a sophisticated glare sensor system.
An electrochromic windshield that can have a specific darkened section controlled by the driver is documented in U.S. Pat. No. 4,892,394. However, the only mode of control is manual through a touch pad that allows up-down and left-right relocation of the section. Since the system is not automatic, it will divert the driver's attention away from the road. Another invention for blocking sun glare incorporates a liquid crystal film as documented in JP 2003159942A2. The light permeability of its specific area can be varied according to signals from a light sensor system providing a visual angle that relatively matches that of the driver. The system allows an automatic darkening of a specific area. However, it requires a sophisticated and expensive sensor system.
One other invention is to provide filtering, such as the use of a liquid crystal display (LCD) screen with camera and sensors to detect the existence and position of a light source. These components, along with the visual detection of the driver, can be used to determine which portions of the LCD screen should be activated to block the light source from the driver's eyes. However, this solution has the disadvantages of being expensive, in that extra sensor and camera hardware is needed to detect not only the driver's eye position, but also the light source position.
A system that selectively attenuates the light passing through an area on an electro-optical element, coated on the windshield, is documented in U.S. Pat. No. 7,134,707 B2. The system consists of a navigation system that determines the vehicle's location and direction of travel, a memory that contains a general location of the driver's eyes within the vehicle, an ephemeris system for providing the position of the sun, and a controller for the calculations. This invention will apply when the light source is in areas not covered by a visor and will perform automatic attenuation of the light passing through the area between the light source and the driver's eyes. Yet, there are a few limitations and complications with this invention. First, the patent does not disclose or describe the details—only the conceptual idea—on how the information from the navigation and ephemeris systems and data on the general location of the driver's eyes are translated into pixels on the electro-optical element. Second, only a generalized location of the driver's eyes is considered in the system. There is no real-time tracking of the driver's eye positions. As indicated in the patent, the driver may have to move his or her head if the light intensity attenuated areas are not in the line of sight between the glare and the driver's eyes. This would be an unnecessary strain on the driver. Third, the system does not consider the slope (inclination or declination) of the road. One of the most dangerous situations for an accident is when there is an abrupt change in the slope of the road and the sun glare will be blinding. Fourth, the patent's method provides only an estimation of the area for opacity to be attenuated because it does not consider the windshield tilt angle, which can vary substantially among different makes and models of motor vehicles.
Accordingly, the present invention offers a new system and method that overcomes the aforementioned problems and drawbacks. Contrary to prior inventions, the present system automatically blocks sun glare on the windshield for motor vehicle drivers under all typical situations and circumstances. This invention details (describes) the method of calculating the sun glare position and changing the opacity of a specific area in the windshield, or the "sun glare block area." The invention also details the method of calculating and changing the opacity, to a lesser extent than that of the sun glare block area, of the entire area of the windshield, with the windshield acting as the "sunshield."
3. References
U.S. Patent or Patent Application Documents
4,874,938October 1989Chuang4,892,394January 1990Bidabad5,258,607November 1993Agostini, Agostini, Noli5,271,653December 1993Shirley5,513,892May 1996Thomas6,056,424May 2000DiNunzio6,286,887 B1September 2001Hashmi2002/0130530September 2002Zenisek6,811,201 B2November 2004Naik7,134,707 B2November 2006Isaac7,163,252 B1January 2007Neault2007/0133078 A1June 2007Fanton, Claude, Dubrenat, Schuett
Foreign Patent Documents
JP 2003159942 A2June 2003Motoi, ShunichiDE 102005007427 A1August 2006Sinnhuber, Wohllebe, Jan, Walter

System and methods for an automated sun glare block area and sunshield in a vehicular windshield


There is provided a medical device having a reminder function, the medical device comprising a reminder generator for issuing a reminder to a user to perform a predetermined action with the medical device; and a controller for selecting the reminder from a plurality of available reminders and for controlling the reminder generator to issue the selected reminder in accordance with a reminding schedule; wherein the controller is configured to maintain a measure of the effectiveness of each of said available reminders and to select the reminder from the plurality of available reminders based on said measures.

In the healthcare sector, many devices and systems are being developed that allow a patient to carry out monitoring of various conditions in their home or work environment or while on the move, without requiring the direct supervision of a healthcare professional.
In these systems, the patient can be provided with a monitoring device, such as a blood sugar meter, a blood INR meter, a heart rate monitor, a blood pressure meter, and so on, that the patient uses to take regular or frequent measurements of some physiological parameter(s). The monitoring device can then provide the results of the measurement to a computer terminal in the patient's home, and the computer terminal can forward the results to a remote station (such as a healthcare professional's computer terminal), where the results can be collated and reviewed by a healthcare professional. The healthcare professional can contact the patient if the results are unsatisfactory or if the patient needs to change their treatment regimen.
However, this type of system is only effective if the patient uses the monitoring device at the required intervals.
A similar problem exists with home medicament dispensing devices that regularly or frequently dispense a medicament to a patient. The medication therapies can only be effective if the patient takes their medicament at the appropriate time.
Therefore, there is a need for a medical device having a reminder function that motivates the patient or user to use the medical device at the appropriate time.

Medical device having a reminder function


A clip for retaining a trim cover assembly against a foam pad of a vehicle seat including a base having a hook end and a lock end. The base also includes a flange extending out therefrom. The hook end defines a hook, which secures the trim cover assembly to the clip. The lock end includes a lock and a lock hook. The foam pad includes a pair of inner foam pad walls that define a trench, through which the clip initially passes before contacting the foam pad. The clip is inserted into the foam pad so that the lock end passes completely through the foam pad to an underlying “B” surface. The lock hook is then rotated relative to the base until the lock hook engages the flange. The clip is firmly anchored within the foam pad by the lock end, which has a width greater than the trench.

1. Field of the Invention
The invention relates to vehicle seat assemblies. More particularly, this invention is directed to a clip for retaining a trim cover assembly against a foam pad of a vehicle seat and a method for using the same.
2. Description of Related Art
Trim cover assemblies extend over vehicle seats to provide passenger comfort as well as aesthetic appeal. Passenger comfort is ensured by a secure attachment of the trim cover assembly to a foam pad layered over a vehicle seat frame. Several attachment methods and devices are known for effecting such attachment of the trim cover assembly to the foam pad. These attachment methods and devices include hog ring clips, mold-in hook fasteners, and injection-molded clips.
Hog ring clip methods require first embedding a wire into a foam pad. Next, a C-shaped metal piece is wrapped around the wire embedded in the foam pad and is threaded into an opening between a cord sewn on the inside of a trim cover. Finally, the C-shaped connector is compressed by a closure device to entrap the cord within the foam pad. Accuracy of this method depends in large part on the assembler operating the closure device.
Mold-in hook fasteners are generally suitable for attaching straight-line trim cover assemblies. However, for attaching trim cover assemblies with non-linear edges, die-cutting materials must be used, adding further costs to the attachment process. Additionally, mold-in hook fasteners provide for undesired variation in attachment stability due to the fact that proper attachment is not guaranteed each time an assembler attaches the fastener.
Injection-molded clips require considerable investment due to the intricate cavity design for the tooling. And once the tooling is available, the tools are difficult to modify due to the multi-cavity tool construction. More specifically, many or all of the tool parts require modification for each individual change. In addition, injection-molded clips require additional structural elements, such as hog ring clips, for anchoring in the foam pad.
There is a need for a cost-effective, accurate, and easy to assemble device for retaining trim cover assemblies to foam pads of a vehicle seat frame.

Extruded flexible self-locking trim cover assembly retention clip and method for using same


A streamlined housing contains an omnidirectional circular light source, an airflow channel with a flow restriction located just below the light source, and a motor driven fan with the fan blade located just below the restricted flow section of the airflow channel. The light source may be selectively directed by a movable reflecting screen. The fan motor and light source may be powered by a 12-volt D.C. source. The airflow channel is shaped to funnel airflow around the light source through the flow restricted area at high velocity, throwing the insects into a base container with deadly force. The base container has a large exhaust port and forms a part of the airflow channel. The airflow channel also acts to funnel and disperse the light from the light source so that the ground below the light source, as well as all directions above the level of the light source, are illuminated.

1. Field of the Invention
The present invention relates generally to improvements in outdoor lights for camping or home use and, more particularly, pertains to a new and improved lighting structure which also silently eradicates the insects that are attracted by the light.
2. Description of Related Art
Lighting systems that are used privately at camp sites or at home, and traditionally are driven by a low voltage power source, make no provision for eradicating the flying insects that are attracted to them. Although flying insect eradicating devices which utilize a light source are in use, these devices are built for the purpose of eradicating insects, and not for the purpose of providing light to the surrounding area. In other words, the light source in the prior art flying insect eradicating devices has no useful purpose other than to attract the insects to the eradicating device.
It is the object and general purpose of the present invention to provide an outdoor lantern for use in back yards or camp sites, providing the light required for the area surrounding the placement of the lantern, while at the same time silently eradicating all insects that are attracted to the lantern.

Combination lantern and flying insect eradicator


A semiconductor apparatus includes a first device cell and a second device cell. The first device cell includes a first active region including a first set of device fins, an insulator layer disposed over the first set of device fins, a first gate fin over the first set of fins, and a first edge fin disposed over a first edge of the first active region. The second device cell is adjacent the first device cell and includes a second active region including a second set of device fins, the insulator layer disposed over the second set of device fins, a second gate fin over the second set of device fins, and a second edge fin disposed over a second edge of the second active region. The first edge fin and the second edge fin are connected to a power rail, a ground rail, or to each other to define a capacitor between the first device cell and the second device cell.

With increasing consumer and business demand for electronics and interconnectivity, there is greater demand for increased processing power. Such increased processing power can be derived from increased processor clock speeds, increased device complexity, and improved device reliability.
One technology supporting these improvements is FinFET technology in which a gate is placed on two or three sides of a channel or wrapped around the channel. Using FinFETs, switching times can approach 10 ps or less, which corresponds to a primary frequency of 100 Ghz. To support such switching times, local energy storage is utilized to provide the energy for switching. This energy storage is generally implemented using local decoupling capacitors connected between power and ground. However, conventional capacitor devices are quite large, utilizing 5 or more poly grid spaces to implement, thus decreasing the space available for other devices or increasing the physical size, which causes larger parasitic losses and thus, greater energy consumption.
The use of the same reference symbols in different drawings indicates similar or identical items.

Decoupling capacitors using regularity finFET structures and methods for making same