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It is always a thrill to catch sight of a leopard in the wild. Two leopards, well, that's sheer bush ecstasy! These felines are elusive loners and very independant, so finding them is quite difficult and tracking them, well, hang on to your hat. Their markings and stealth movements allow them to disappear in front of your very eyes. But if you have an experienced guide at the wheel, you might be in for quite a treat! And so it was for me last October while staying at Leadwood Lodge in the Sabi Sands, one of my favourite of & Beyond's property's.
I was guided by Mack, a very experienced and photography-savvy ranger and our talented tracker, who's name I wish I could remember. Two things that make an enormous difference when you are in the bush searching for wildlife and unique photo opportunities–a knoweldgeable ranger/tracker team and a reliable, well-tricked out Land Rover. The & Beyond folks tick both boxes.
Early on our second morning we came across this mother with her adolescent cub. As soon as we spotted them, they were on the move! Through thickets and tall grass, down raveens and around termite mounds, they slipped in and out of sight. Unbelieveably, Mack and our tracker stayed with them, which involved some serious off road manoeuvers. Thankfully our truck easily ploughed through almost anything!
The cats moved through tall grass, the occassional tip of a tail or back of an ear bobbing up here and there, welcome landmarks in bad weather. Finally they emmerged from the thickets into the open and once again we were able to track more closely as they followed the bend in the road in front of us.
But not for long! Into the grass and down the banks they went, forcing us to once again to take our search off-road, bouncing along over tree stumps and thick bushes–with one happy photographer hanging on to a roll bar in one hand and a Canon 5D Mlll in the other!
The guys were quick to respond and years of tracking these fabulous animals has provided keen insights into anticipating which way they might be heading. Mack gunned the engine (as much as you can power forward in 3 feet of underbush!) and moved the truck through the fields, pulling a quick u-turn and magically emmerging back on the road–with the leopards walking towards us!!The kept moving forward, straight at the truck, disappearing through my minimum focus range. They barely gave us a look as they slipped by the open-sided vehicle. I resisted the urge to try to scratch some kitty ears as they floated by.
Finally, they wandered into a cool clearing and lay down to tumble together and enjoy some serious grooming. Leopard watching just does not get better than this!
This post was previously published last July.
Click here for Why I Love Africa
Check out the Big Cats Gallery and Lions at Night


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News Section: Local news
Port Manatee Says Goodbye to Outstanding Employee
PALMETTO – Frank Holden got lots of hugs at Thursday's Port Authority meeting as he said goodbye to PA commissioners, staff and Port Director Carlos Buqueras. Holden is retiring from his job of 14 years as senior director of operations and special projects.
Holder has had a stellar career; 32 years as a police officer, serving 18 as chief of police for two separate Montgomery County, Pennsylvania municipalities.
At Port Manatee he held many positions; starting as a temporary employee warehouseman on to warehouse manager. He soon moved on to compliance officer, then director of seaport security, and then senior director of seaport security.
Holders previous career in law enforcement made him a natural for all of his security position with the last being, senior director of operations and special projects.
As commissioners and port staff reflected on what they all described as outstanding service, Holder humbly accepted a duplicate of the port's bell.
Frank and his wife Judy are looking forward to spending a lot more time together.
Frank, you will be missed by all.
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subset: commoncrawl


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Original recipe yields 20 servings
Based on a 2,000 calorie diet
Ready In
I was amazed at how easy they were to make but also how really yummy they were too! This recipe is great anytime but especially when you don't have the time to make dough from scratch!
i have made these for years. they stay soft for days, if they last that long. you can use cream cheese and jam instead of butter and cinnamon in the middle for a different taste. the whipping ...
These were easy and delicious! Much cheaper than the store bought version as well. Cinnamon rolls are a Christmas tradition at our house and these will replace the store bought version from now ...


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CoolSafe 95/55-80 models are designed for pilot scale, vial or bulk freeze drying pro...
CoolSafe PRO CoolSafe PRO Superior 100-9 XS & 95-15 XL fully Automatic research freez...
CoolSafe PRO the optimal freeze dryers with full display, control and measurement of ...
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ScanCool Snow-Bird…Ultra Freezer The under-bench/ personal, -90°C Ultra-Freezer, t...
LaboGene is a Danish Company that specializes in the design, development, manufacture and sales of laboratory and industrial equipments in the fields of Clean Air & Laminar Flow, Vacuum & Cooling, Centrifugation and Environmental control.
The ScanCell-Caron range of CO2 Incubators, Plant Growth & Environmental Chambers from LaboGene brings a new dimension to cell culture and environmental sciences by offering innovative design, complete sample security together with the integration of advanced monitoring technologies. ScanCell-Caron represents the best in Incubator design and Cell Culture technology for your laboratory and research protocols.
LaboGene introduces ScanConstruct the marquee that symbolises the best in design & build solutions for your specific clean-air requirements. ScanConstruct, with over 30 years experience in bespoke modular constructions, offers the best in clean air and contamination control services.
LaboGeneTM introduces ScanLaf, the name that epitomizes the ultimate in the design, innovation and energy saving technologies, in the manufacture of Clean Air and Biological Safety cabinets that are available for today's laboratories.
ScanSpeed a range of centrifuges of distinction from LaboGene from simple separation to sequencing, discover the ideal centrifuge and accessories for your every day needs and your research programs from the ScanSpeed range. A revolution in centrifuge design and quality is the hallmark of ScanSpeed Centrifuges.
ScanVac, the name that epitomizes the best in Freeze Drying & Vacuum Concentration equipments for the laboratory, research and pilot plant protocols. All models are complimented by a complete and diverse range of innovative accessories and options.
ScanCool the brand that describes a range of Ultra Low Temperature Freezers for sample storage offering a choice of Chest or Upright models from 100 to 600 litres capacity. Environmentally friendly and energy saving design concepts are standard features of all units in the ScanCool range.
The ChillSafe range of cooling & cryogenic baths and circulators with capacities of 8 to 18 litres and temperatures from -30 ºC to -90 ºC completes the ScanCool offering.
Having been involved in the design and manufacture of world renowned cabinets and work stations for IVF applications for over 25 years, ORIGIO ScanLab Equipment a/s today lays claim to being the market leader in this specialised discipline that is the "Science of life." ORIGIO ScanLab Equipment a/s is a joint venture of ORIGIO a/s and LaboGene ApS.


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Hanley school to stage horse racing event to boost cyclist's funds
Posted: May 24, 2014|
A SCHOOL worker raising money with a cycle ride to Italy is not resting on his laurels after a successful community day.
Angelo Antinoro, of Barlaston, held the event at Forest Park Primary School in Hanley on Wednesday to raise funds for the 1,500-mile bike ride from Stoke-on-Trent to Sicily in July.
Now a horse racing event will be held at the school on June 13.
Mr Antinoro said: "The day was brilliant, raising an estimated £1,200, and hopefully more people will donate now."
Related content
The 47-year-old is doing the ride for bereavement charity A Child of Mine, in memory of his late sister, Maria Liew, who passed away in 2009.
To support Angelo, text NORO47 and the amount you want to donate to 70070.


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Kitchen tip
Whenever making deviled eggs I put my mixture in a ziploc bag and cut a corner. It makes it so much easier to fill the cut eggs and it looks pretty too.=)
I also do this with frosting when decorating cookies with the kids.
I do the same thing. When I want to be really fancy, I put them in a pastry bag with my largest decorating tip and make pretty designs.
I recently read a tip somewhere to make the whole thing right in the bag. Use a heavy duty freezer bag appropriate to the amount you're making. Seal it and squish the yolks. Add your other ingredients and then just kneed until it's mixed. Snip off the tip and pipe. It saves you from having to wash the bowl.
Great tip!! I love deviled eggs and will definately try this!
Great idea. Thanks.
I love them too but mine taste horrible. Share your recipes ladies!:)
With Easter coming up this is a great idea
Great Idea, no mess and throw it in the bin when you're done, no mess Perfect!!!
Sounds like a great tip... even for me! The best tip in the kitchen my family has is keep me out of the kitchen! I'll give this idea to dd she'll love it!
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Gear Review
Solid little filter
A friend had the little Hyperflow MSR filter and it stopped working after 1 day. Made me glad I always have my Katadyn Hiker with me in the backcountry. It's not the fastest, or lightest, but it just works year after year.
View More Details on Katadyn Hiker Water Microfilter


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An illuminated controller having a membrane potentiometer for use as a light dimmer or other voltage controlling device. The controller has a backplate that acts as a sturdy base, and a cover, having sidewalls and a top wall with a central opening. The cover is mounted on the backplate and a layered structure is interposed between the cover and backplate. The layered structure includes a membrane potentiometer for varying voltage, and an electroluminescent panel, for generating light to illuminate the controller.
1. A controller that is illuminated, for receiving a first voltage and outputing a second voltage, comprising:
a) a backplate for providing a sturdy base for the controller;
b) a cover, having sidewalls and a top wall which has a central opening defining a cavity therein, for mounting on the backplate;
c) a membrane potentiometer, interposed between the cover and the backplate, for receiving a first voltage, outputing a second voltage that is a percentage of the first voltage; and
d) an electroluminescent panel, interposed between the cover and the membrane potentiometer, for generating a light to illuminate the controller and being exposed through the central opening, the top wall extending over and retaining the membrane potentiometer and the electroluminescent panel to the backplate.
2. The controller of claim 1, further including an electrically insulative sheet, located above the membrane potentiometer and electroluminescent panel but beneath the cover.
3. The controller of claim 2, wherein the electrically insulative sheet is transparent.
4. The controller of claim 2, wherein the electrically insulative sheet has graphic overlays to aid the user in controlling the controller.
5. The controller of claim 1, wherein the membrane potentiometer and electroluminescent panel are formed between common flexible films.
6. The controller of claim 1, further comprising a stiffener board for pressing the membrane potentiometer thereagainst.
7. The controller of claim 1, wherein the cover is transparent and light from the electroluminescent panel is transmitted therethrough.
8. The controller of claim 7, wherein the electroluminescent panel extends beyond the membrane potentiometer and the stiffener board.
9. The controller of claim 1, wherein light generation is restricted to a periphery of the electroluminescent panel.
10. The controller of claim 1, wherein a plurality of electrical terminations for both the membrane potentiometer and electroluminescent panel are mounted on common film layers and the electrical terminations extend through a slot in the backplate.
11. A controller that is illuminated for receiving a first voltage and outputing a second voltage, comprising:
a) a backplate for providing a sturdy base for the controller;
b) a light transmissive cover, having sidewalls and a top wall with a central opening, for mounting on the backplate;
c) a membrane potentiometer, interposed between the membrane potentiometer and the backplate, for receiving a first voltage, outputing a second voltage that is a percentage of the first voltage; and
d) an illumination means, interposed between the cover and the backplate for illuminating at least a portion of the cover by transmitting light through the side and top walls.
12. The controller of claim 11, wherein the illumination means comprises an electroluminescent panel for generating light.
13. The controller of claim 12, wherein the cover is light transmissive for transmitting light to illuminate the controller.
14. The controller of claim 12, wherein the cover comprises light piping for illuminating selected portions of the controller.
15. The controller of claim 12, wherein the cover comprises fiber optics for illuminating selected portions of the controller.
16. The controller of claim 11, wherein a plurality of electrical connectors attached to both the membrane potentiometer and electroluminescent panel extend through a slot in the backplate.
17. The controller of claim 11, wherein the membrane potentiometer prevents light from being directly transmitted through the central opening.
1. Field of the Invention
This invention relates to an illuminated controller, and more specifically to an illuminated membrane potentiometer for use as a light dimmer or other voltage controlling device.
2. Description of the Related Art
Potentiometers are well known for controlling voltage selection in numerous types of applications in both home and industry. For example, these devices may be used in the control panels of such things as aircraft and aerospace applications, computers, arcade games, kitchen appliances, or lighting systems to name a few.
In the past, prior art potentiometers for providing voltage selection have typically been operated using a mechanical wiper that is typically controlled by a knob or slider on a control panel. The wiper contacts a resistive element, which provides for voltage selection over a continuous range of voltages. The constant mechanical contact between the wiper and the resistive element tends to accelerate the wear and eventual failure of this type of potentiometer.
A more recently devised potentiometer uses a membrane comprising a continuous length of electrically resistive material, a corresponding continuous length of electrically conductive material, which serves as a wiper, and an electrically insulating spacer which serves to simultaneously support and separate the resistive and conductive layers. Either one or both of the resistive and conductive materials are designed as flexible members so that they can be pressed together at any selected location along their length to bring them into electrical contact.
If a voltage is applied across the resistive material, a voltage gradient is established along the length of the material. Thus, touching either the top or bottom surface of the flexible member will cause the resistive and conductive materials to contact each other, producing a voltage output on the conductive material or wiper. The advantage of this device is that it minimizes wiper contact and reduces wear and yet provides a continuous range of output voltage levels.
A potentiometer membrane as described can be used to make a reliable and economical dimmer and/or switch for use with a light fixture. However, since light switches and dimmers and other potentiometer controls must sometimes be found and used in poorly lit rooms, it is desirable to provide an illumination source on the control. Accordingly, it is known in the prior art to place an L.E.D on a potentiometer control (U.S. Pat. No. 4,962,364 to Okuya) or to use a photo-conductive element routed to the control knob surface (U.S. Pat. No. 4,523,174 to Oyama). However, these devices are only illuminated by a small point source of light. It is desirable to have a larger surface of the potentiometer illuminated so that it will be easier to see and softer on the eyes.
3. Related Art
Examples of patents that are related to the present invention are as follows, and each patent is herein incorporated by reference for the supporting teachings:
U.S. Pat. No. 5,241,308 is a force sensitive touch panel.
U.S. Pat. No. 5,228,562 is a membrane switch and fabrication method.
U.S. Pat. No. 5,962,364 is an illuminating type linear resistor for volume control.
U.S. Pat. No. 4,939,501 is a sliding foot controller.
U.S. Pat. No. 4,725,817 is a pressure responsive panel.
U.S. Pat. No. 4,542,366 is an illuminated variable resistor.
U.S. Pat. No. 4,523,174 is an illumination type rotary variable resistor.
U.S. Pat. No. 4,494,105 is a touch controlled circuit apparatus for voltage selection.
U.S. Pat. No. 4,017,848 is a transparent keyboard switch and array.
U.S. Pat. No. 4,005,381 is a slide potentiometer.
U.S. Pat. No. 3,968,467 is a touch controlled voltage-divider device.
U.S. Pat. No. Re. 33,089 is a position sensing and indicating device.
The foregoing patents reflect the state of the art of which the applicant is aware and are tendered with the view toward discharging applicant's acknowledged duty of candor in disclosing information which may be pertinent in the examination of this application. It is respectfully stipulated, however, that none of these patents teach or render obvious, singly or when considered in combination, applicant's claimed invention.
It is a feature of the invention to provide an illuminated controller having a membrane potentiometer for use as a light dimmer or other voltage controlling device.
An additional feature of the invention is to provide a device that has a backplate, to act as a sturdy base, and a cover that has sidewalls and a top with a central opening. The cover is mounted on the backplate and a layered structure is interposed between the cover and backplate. The layered structure includes a membrane potentiometer for varying voltage, and an electroluminescent panel, for generating light to illuminate the controller.
A further feature of the invention is to provide a device with a transparent cover so that light from the electroluminescent panel can be transmitted therethrough.
Yet another feature of the invention is to provide an illuminated controller wherein electrical terminations for both the membrane potentiometer and electroluminescent panel are mounted on common film layers.
The invention resides not in any one of these features per se, but rather in the particular combination of all of them herein disclosed and claimed and it is distinguished from the prior art in this particular combination of all of its structures for the functions specified. Other features of the present invention will become more clear from the following detailed description of the invention, taken in conjunction with the accompanying drawings and claims, or may be learned by the practice of the invention.
There has thus been outlined, rather broadly, the more important features of the invention in order that the detailed description thereof that follows may be better understood, and in order that the present contribution to the art may be better appreciated. There are, of course, additional features of the invention that will be described hereinafter and which will form the subject matter of the claims appended hereto. Those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.
FIG. 1 is an exploded perspective view of a first embodiment of a luminescent controller.
FIG. 2 is a cross section of the luminescent controller of FIG. 1 taken transverse to the longitudinal direction.
FIG. 3 is an exploded perspective view of a second embodiment of a the layered structure of the luminescent controller.
FIG. 4 is a cross section of the layered structure of FIG. 3 taken transverse to the longitudinal direction.
FIG. 5 is an exploded perspective view of a third embodiment of a layered structure for a luminescent controller having an electroluminescent panel that lights up a cover.
FIG. 6 is a cross section of the layered structure of FIG. 5 taken transverse to the longitudinal direction.
It is noted that the drawings of the invention are not to scale. The drawings are merely schematic representations, not intended to portray specific parameters of the invention. The drawings are intended to depict only typical embodiments of the invention, and therefore should not be considered as limiting the scope of the invention. The invention will be described with additional specificity and detail through the use of the accompanying drawings. In the drawings, like numbering represent like elements between the drawings.
The present invention provides a luminescent controller for use as a light dimmer or similar application. Regarding FIG. I and FIG. 2, there is a luminescent controller 10 having a backplate 62 and a cover 60. Interposed between backplate 62 and cover 60 is a layered structure 12. Layered structure 12 includes a stiffener board 20, a membrane potentiometer 30, electroluminescent panel 40, and a transparent sheet with graphic overlays 50. Controller 10 is held together by locking tabs 64 which releasably locks into holes 66.
Membrane potentiometer 30 and electroluminescent panel 40 have electrical connectors 32 and 42, respectively. Connectors 42 are designed to fit on either side of aligned connectors 32 when layered structure 12 is assembled. Furthermore, connectors 32 and 42 are designed to be inserted through holes 22 and slot 68. The connectors can then be connected to a circuit board (not shown) having dimmer control electronics (not shown) which are well known in the art. Membrane potentiometer 30 includes a two flexible film layers 31, conductive layer 32 and a resistive layer 34, which are separated by dielectric 36, thereby forming air space 38.
The light 64 generated by electroluminescent panel 40 is transmitted through transparent layer 50 to illuminate controller 10. The details of panel 40, which are well known in the art, are depicted in FIG. 4.
In FIGS. 3 and 4, another embodiment of the invention is shown. This embodiment is similar to the embodiment shown in FIG. 1, except that in layered structure 12a, the electroluminescent panel is incorporated into membrane potentiometer 30a. Membrane potentiometer 30a includes a transparent flexible film layer 31a, which can be made from a polyester film such as Mylar. Film layer 31a is coated with a transparent conductive coating 39 such as Indium Tin Oxide (ITO). On top of the ITO, a layer of electroluminescent material 33 such as phosphor is applied. Over layer 33, an electrically insulative material layer 35, like Barium Titanium Oxide, is deposited. The final layer of membrane 30a consists of a screen deposited conductor layer 37. The lighting of membrane 30a is produced when an alternating voltage is applied to ITO layer 39 and conductive layer 37. The voltage creates a capacitive effect thereby exiting and illuminating electroluminescent material layer 33.
The remaining portion of membrane potentiometer 30a is similar to that in FIG. 2. Connectors 32a are a combination of both connectors 32 and 42 shown in FIG. 1. Additionally, connectors 32a are mounted on film layers 31 and 31 a, which form the outer layers of and are common to both membrane potentiometer 30a and the electroluminescent panel contained therein.
A third embodiment, illustrated in FIGS. 5 and 6, is designed so that layered structure 12b includes an electroluminescent panel 40a is located beneath stiffener board 20. In this embodiment, the electroluminescent material 43 is confined to a periphery 47 of transparent film 44 leaving an air space 45 in the central portion of panel 40a. The electroluminescent structure of panel 40a is similar that of FIG. 4, other than being confined to a periphery. Furthermore, as can be seen in FIG. 6, the luminescent periphery 47 of electroluminescent panel 40a extends beyond stiffener board 20 and under a lip 61 of cover 60. In this embodiment, light 64 does not shine directly through transparent sheet 50 as stiffener board 20 and membrane potentiometer 30 do not transmit light. Instead, light 64 is conducted through cover 60 and exits the top and side surfaces thereof to illuminate controller 10.
This design offers an advantage over the prior art in that an illuminated controller is provided that provides a soothing easy to spot light over a surface area of the controller as opposed to a single point source of light. Furthermore, the invention provides a reliable and cost effective design to accomplish the foregoing. Another unique aspect of the invention is that it is operated by pressing through the electroluminescent panel incorporated in the controller.
One of ordinary skill in the art of designing and using controllers of the type disclosed will realize a device of this type can be used to adjust the voltage of a multitude of mechanisms such as a light fixture or volume of a stereo. The controller receives a first voltage and outputs a second voltage, which is a fraction or percentage of the first voltage. Typically, the voltage is adjusted by a user pressing a finger against transparent sheet 50. This causes conductive layer 32 to touch resistive layer 34 at the point pressed. Accordingly, the electronics (not shown) will detect the change of resistance in the circuit and adjust the voltage to the device to be controlled. The graphics on transparent sheet 50, which are illuminated by electroluminescent panel 30, show the user where to press to adjust the voltage level of the device to the desired setting. Furthermore, transparent sheet 50 provides an additional layer of electrical insulation to protect the user from electrical shock if a lower layer were to tear.
Although the illustrated embodiments display specific designs of the shape of controller 10, one skilled in the art will realize that the preferred embodiment would work with most any shape. For example, the controller could be round, oval or triangular without departing from the disclosed structure. It is also contemplated that the transparent sheet 50 be provided without graphics as the cover could be used to impart control information to the user via molded in designs, stickers or painted designs.
Connectors 32 and 42 can also be varied from the pins shown in the preferred embodiments. Any type of electrical connection known in the art could be used without departing from the scope of the invention. Also, the backplate 62 and cover 60 could be varied in innumerable ways. For example, screws could be used to secure the cover instead of the tabs depicted in FIG. 1. Backplate 62 and cover 60 could even be molded as a single unit. Another option would be to make a portion of cover 60 opaque in the third embodiment so that a desired pattern or design is illuminated on it.
Additionally, it is contemplated that in the first and second embodiment, backplate 62 could also serve as the stiffener board so that conductive layer 32 will come in contact with resistive layer 34 when pressure is applied to transparent layer 50.
Another alternative is to add an actuator such as a slide adjustment that would be moved by a user. The slide would be designed such that it will press conductive layer 32 and resistive layer 34 together at the point where the slide is positioned.
In the third preferred embodiment, the electroluminescent material 43 is only placed around the periphery of panel 40a to save material; however, the entire panel could just as easily be made luminescent. Likewise, the electroluminescent material in the first and second embodiments could be limited to the periphery of panel 40 or 30a, or in the alternative the electroluminescent material could be deposited in any design desired to be illuminated. Another variation of the third embodiment would be to direct light through the cover using fiber optics or light-piping.
While the invention has been taught with specific reference to these embodiments, someone skilled in the art will recognize that changes can be made in form and detail without departing from the spirit and the scope of the invention. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.
Patent Citations
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ABCbodybuilding - View Single Post - Commanding My Body
Commanding My Body
View Single Post
is enjoying bodybuilding
Super Moderator
Join Date: Sep 2007
Location: North East U.S.A.
Posts: 6,774
Friday - Bench
* Upperback work in between some sets
* Bench felt AWESOME today. 280 was dominated for 2 reps (as much as you can dominate 93% of a touch n' go max). All reps on 231 were explosive, it may not have looked explosive to an observer based on bar speed, but I was really focused. Had great tightness, nice arch, squeezing the bar, everything was clicking.
Bench Press
220, 230, 240, 250, 260, 270, 280x2
231x8, 7, 6
165x4, 4, 3, 3, 2, 2, 1, 1 - 10 second rest
1 more round with 165
Ring Dips
BWx7, 6, 6, 8
ss w/
Barbell Row (pulling to chest to hit upperback more than lats)
117.5x15, 3 sets
Bench (Chains)
117.5 (+50) x 5, 3 sets
* Changed thing up slightly. Instead of higher rep finisher, I went explosive on Bench Press with Chains. Also threw in an extra set of dips as they nail the chest nicely. Felt great.
Saturday - Messed around at a hotel gym
My oldest son competed in a statewide mathematics event
Sunday - Legs
Leg Curl Warmup
50x20, 3 sets
Squat Chains (+50)
220, 240, 260, (add belt) 280, 300x2, 307.5x2
261x8, 8, 6
201x4, 4, 3, 3, 2, 2, 1, 1 - 10 second rest between mini sets
Squat (no chains) - hold performed with legs at about 90 degrees, below mid range ROM
146x5, 5 second hold, 4 reps, 4 second hold, 3 reps, 3 second hold, 2 reps, 2 second hold, 1 rep, 1 second hold - all done continuously, no rest
1 more round with 146
Leg Curl
60x12, 2 sets
Somersault Squats
BWx15, 3 sets
Monday AM - Arms
1. DB Hammer Curl
28.5x12, 4 sets
ss w/
Tricep Rope Pressdown
41x15, 2 sets
41x14, drop set to 31x6
2. Preacher Curl
52.5x10, 3 sets
ss w/
Landmine Shoulder Press with Band, then 1.5 Pushups (repeat top half ROM), and some DB Floor Press
* I don't really care for the landmine shoulder press, so I ditched them after 1 set in favor of 2 other compound movements today. I think I will settle back on DB Floor Press for next workout.
3. Barbell Curl - slow descent
40x8, 3 sets
ss w/
Ring OH Tricep Ext - ditched these in favor of more barbell OH extensions
BWx10, 1 set
4. Incline OH Barbell Tricep Ext
51x15, 15, 15, 12
ss w/
Incline Curl
22x8, 6
5. Band Curl
20 reps x 2 sets
ss w/
Band Tricep Extensions
15 reps x 2 sets
James 1:16-17 ESV
Do not be deceived, my beloved brothers. Every good gift and every perfect gift is from above, coming down from the Father of lights
With God's help...
Mens sana in corpore sano
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DURHAM, N.H. – The University of New Hampshire men's and women's track and field teams open the 2013-14 indoor season when they host the University of Maine Friday, Dec. 6 at Paul Sweet Oval.
Fresh off an America East Cross Country title, the women's team looks forward to a successful indoor campaign. The women placed fifth in the America East during the 2012-13 indoor season. UNH notched a victory over Maine in the squad's home meet (Dec. 8, 2012) and took fourth out of 11 teams at the URI Invitational (Feb. 2).
Last year the men's squad claimed fifth place at the America East Championship and earned three top-three finishes. The Wildcat men earned first place in a dual meet with Holy Cross (Jan. 19) and took second place at the Boston Indoor Games (Jan. 25) and the URI Invitational (Feb. 2).
The women's squad returns 18 seniors, 16 juniors, and 23 sophomores, while welcoming in 23 freshmen. The men's team consists of 15 seniors, 14 juniors, 13 sophomores, and 16 freshmen.
The UNH women's team has also announced its four senior captains for the upcoming season. Distance runner Elise Beattie (Woodstock, Ill.), sprinter Caroline Bonsaint (Hampton, N.H.), sprinter Allison Brehm (Pembroke, N.H.), and thrower Rosemary Reed (West Greenwich, R.I.) were named captains by coaches Casey Carroll and Robert Hoppler.
This is the second year in a row that UNH will host a dual meet versus Maine. Last year the women took a 109-80 victory over the Black Bears, while the men fell to Maine 102-89.
For the latest news on UNH men's and women's track and field, tune in to www.unhwildcats.com.


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Described is a new class of polypeptide cell modulators characterized by being composed of two covalently linked cell modulators in a linear polypeptide sequence. Such dual function polypeptides have new and particularly useful activities when the component polypeptide cell modulators are interferons, lymphokines or cytotoxins which act through different and specific cell receptors to initiate complementary biological activities.
1. A composition of the formula
wherein R1 is gamma interferon or a biologically active modified gamma interferon which has one of the following activities:
(a) the ability to protect Vero cells against the cytopathic effect of encephalomyocarditis virus infection; or
(b) antiproliferative activity against HEp-2 cells;
(c) an immunomodulating activity of native gamma interferon;
R2 is human lymphotoxin or a biologically active modified human lymphotoxin which retains a biological activity of native human lymphotoxin; and L is a peptide linker segment of from about 1 to about 500 amino acid residues wherein the L retains or potentiates the biological activity of R1 and/or R2.
2. A composition according to claim 1 wherein L is methionine.
3. A composition according to claim 1 wherein L is mouse IgG 2b "hinge" with each of the four cysteines replaced by serine.
4. A composition according to claim 1 wherein R1 is gamma interferon.
5. A composition according to claim 1 wherein R2 is human lymphotoxin.
6. A composition according to claim 1 wherein R1 is gamma interferon, R2 is human lymphotoxin, and L is a peptide linker segment of from about 1 to about 500 amino acid residues.
7. A composition according to claim 6 wherein L is methionine.
8. A composition according to claim 6 wherein L is mouse IgG 2b "hinge" with each of the four cysteines replaced by serine.
9. The protein identified in FIG. 12 and FIG. 13 as IFNX 603 and having the amino acid sequence shown in FIG. 12 and FIG. 13.
10. The protein identified in FIG. 14 and FIG. 15 as IFNX 604 and having the amino acid sequence shown in FIG. 14 and FIG. 15.
11. A pharmaceutical composition comprising a therapeutically effective amount of a composition of the formula
wherein R1 is gamma interferon or a biologically active modified gamma interferon which has one of the following activities:
(a) the ability to protect Vero cells against the cytopathic effect of encephalomyocarditis virus infection; or
(b) antiproliferative activity against HEp-2 cells;
(c) an immunomodulating activity of native gamma interferon;
R2 is human lymphotoxin or a biologically active modified human lymphotoxin which retains a biological activity of native human lymphotoxin; and L is a peptide linker segment of from about 1 to about 500 amino acid residues wherein the L retains or potentiates the biological activity of R1 and/or R2 admixed with a pharmaceutically acceptable carrier.
12. A pharmaceutical composition comprising a therapeutically effective amount of the composition of claim 2 admixed with a pharmaceutically acceptable carrier.
13. A pharmaceutical composition comprising a therapeutically effective amount of the composition of claim 3 admixed with a pharmaceutically acceptable carrier.
14. A pharamaceutical composition comprising a therapeutically effective amount of the composition of claim 9 admixed with a pharmaceutically acceptable carrier.
15. A pharmaceutical composition comprising a therapeutically effective amount of the composition of claim 10 admixed with a pharmaceutically acceptable carrier.
16. A method of treating viral infections in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 1.
17. A method of regulating cell growth in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 1.
18. A method of regulating the immune system in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 1.
19. A method of treating viral infections in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 2.
20. A method of regulating cell growth in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 2.
21. A method of regulating the immune system in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 2.
22. A method of treating viral infections in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 3.
23. A method of regulating cell growth in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 3.
24. A method of regulating the immune system in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 3.
25. A method of treating viral infections in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 9.
26. A method of regulating cell growth in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 9.
27. A method of regulating the immune system in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 9.
28. A method of treating viral infections in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 10.
29. A method of regulating cell growth in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 10.
30. A method of regulating the immune system in an animal in need of such treatment comprising the administration of an effective therapeutic amount of the composition of claim 10.
This is a continuation of application Ser. No. 06/803,748, filed Dec. 2, 1985, now U.S. Pat. No. 4,935,233.
1. Field of the Invention
This invention relates to covalently linked polypeptide cell modulators, each of which acts through a different and specific cell receptor to initiate complementary biological activities. Polypeptide cell modulators include lymphokines, monokines, interferons, polypeptide hormones or cytotoxins as well as modifications and active segments of such peptides. Also described are DNA sequences, plasmids and hosts capable of expressing the linked polypeptide cell modulators.
2. Description of Prior Art
One class of polypeptide cell modulators can be defined whose members exert an antiproliferative effect almost specifically on tumour cells and possess immunomodulatory activity, but lack antiviral activity. Among the members of this class are human lymphotoxin and tumour necrosis factor (Gray, P. W. et. al. Nature 312, 721, 1984; Pennica D. et. al. Nature 312, 724, 1984).
Human lymphotoxin (hLT) is a cytotoxin induced in lymphocytes by a specific antigen or by bacteria or parasites and has a cytotoxic or cytostatic action on a variety of tumour cells in vivo or in vitro. hLT has been implicated to play a role in cell-mediated immunity and its potent anti-tumour effect suggests it may be of value therapeutically (Ruddle, N. H. et. al. Lymphokine Res. 2, 23, 1983).
Another class of lymphokine can be defined whose members induce an antiviral state in responsive cells, and also have antiproliferative and immunomodulating activity. Among the members of this class are leukocyte interferon (IFN-alpha), fibroblast interferon (IFN-beta) and immune interferon (IFN-gamma).
It has been reported that mixtures of type I interferons (IFN-beta or IFN-alpha) and type II interferons (IFN-gamma) are highly synergistic in exerting an antiviral or antiproliferative effect. (Fleishmann, W. R. et. al. Infect. Immun. 26, 248, 1979; Czarniecki, C. W. et. al. J. Virol. 49, 490, 1984).
In mixtures, much lower concentrations of type I and type II interferons can achieve a particular level of response. Several authors have also described IFN-gamma/hLT and IFN-alpha/hLT synergy or related synergies (Lee, S. H. et. al. J. Immunol. 133, 1083, 1984; Stone-wolff, D. S. et. al. J. Exp. Med. 159, 828, 1984; Williams, T. W. Lymphokine Res. 3, 113, 1984), European Patent Application (EPO 107 498), (EPO 128009).
However, in these instances, there was no disclosure of covalent linkage of the two classes of molecules that were synergistic.
Additional patent publications have described the primary amino acid sequences of human IFN-gamma (GB 2 107 718 A), the IFN-gamma (IFN X918) described herein (PCT 83/04053), IFN-alphas (U.S. Pat. No. 4,414,150--Aug. 11, 1983 and IFN-beta (e.g. GB 0689 70B; GB 2098996A). A modified IFN-beta (IFN X430) described herein is identical to human fibroblast IFN-beta except that amino acids 36 to 48 inclusive are replaced with amino acids 34 to 46 inclusive from human IFN-alpha 1 (European Patent Application 85105914.7 and (Taniguchi, T. et. al. Nature 285, 547, 1980).
This invention encompasses mixed function proteins formed from covalently linked polypeptide cell modulators, each of which acts through a different and specific cell receptor to initiate complementary biological activities. Novel compounds of this invention are represented by the formula
where R1 is a polypeptide cell modulator with one activity, R2 is a polypeptide cell modulator with a different but complementary activity. By complementary activity is meant activity which enhances or changes the response to another cell modulator. The polypeptide cell modulators are either directly bonded to one another or are each bound to a polypeptide linker segment. Thus L represents a chemical bond or a polypeptide linker segment to which both R1 and R2 are bound, most commonly L is a linear peptide to which R1 and R2 are bound by amide bonds linking the carboxy terminus of R1 to the amino terminus of L and the carboxy terminus of L to the amino terminus of R2. The linking group is generally a polypeptide of between 1 and 500 amino acids in length.
The term polypeptide cell modulator encompasses a large variety of peptides which elicit a biological response by binding to a specific binding site on a cell. It is known that mixtures of polypeptide cell modulators such as beta and gamma interferon exhibit a synergistic effect. In this invention the polypeptide cell modulators are bound together to produce the same synergistic effect as a mixture of the polypeptide cell modulators or a further enhanced effect or a different effect with the advantage of a single dosage form.
Compounds of this invention are preferably made by genetic engineering techniques. Thus genetic material (DNA) coding for one polypeptide cell regulator, peptide linker segment and the other polypeptide cell regulator is inserted into a suitable vector which is used to transform bacteria, yeast or mammalian cells. The transformed organism is grown and the protein isolated by standard techniques. The resulting product is therefore a new protein which has two complementary cell regulatory regions joined by a peptide linker segment as shown in the formula R1 --L--R2, wherein R1 and R2 represent polypeptide cell regulator regions and L represents the peptide linker segment.
Table 1 shows the origin and identification of the plasmids used in the construction of polypeptide cell modulators.
Table 2 shows expression and molecular weight data for IFN X601.
Table 3 shows a comparison of the antiviral activity of IFN X601 with that of the parental IFNs.
Table 4 shows a comparison of the antiproliferative activity of IFN X601 on Daudi lymphoblastoid cells and HEp-2 carcinoma cells with that of the parental IFNs.
Table 5 demonstrates synergy between human IFN-gamma and IFN X430.
Table 6 shows the antigenic properties of IFN X601 as judged by enzyme-linked immunoadsorbent assay (ELISA).
Table 7 shows a comparison of the binding to Daudi cell IFN alpha 2 receptors of IFN X601 with that of the parental interferons, IFN X918 and IFN X430.
Table 8 shows the antiviral, antiproliferative and HLA DR inducing activity of IFN X601 eluted from monoclonal antibody affinity columns.
Table 9 shows the antiviral, antiproliferative, HLA DR inducing and ELISA activity of IFN X602 compared with IFN X601.
Table 10 shows the antiviral, antiproliferative, HLA DR inducing and ELISA activity of IFN X603.
FIG. 3 depicting Chart 1A shows the path to construction of the plasmid vector pGC269, which expresses IFN X601. FIG. 4 and FIG. 5 depicting Charts 1Aa and 1Ab show preparation of starting plasmid pAP8.
FIG. 6 depicting Chart 1B shows the path to construction of the plasmid vector pZZ102, which expresses IFN X603.
FIG. 7 depicts Chart 2; Chart 2A shows the ligated DNA duplex coding for the spacer amino acids and used to prepare an intermediate plasmid (pGC262) in the construction of pGC269. Chart 2B shows the DNA duplex coding for (Ala--Gly--Ser)7, an alternative spacer for linking IFN X918 to IFN X430.
FIG. 8 and FIG. 9 depicting Chart 3 shows the complete nucleotide and amino acid sequences of the IFN X601 gene and IFN X601, respectively.
FIG. 10 and FIG. 11 depicting Chart 4 shows the complete nucleotide and amino acid sequences of the IFN X602 gene and IFN X602, respectively.
FIG. 12 and FIG. 13 depicting Chart 5 shows the complete nucleotide and amino acid sequences of the IFN X603 gene and IFN X603, respectively.
FIG. 14 and FIG. 15 depicting Chart 6 shows the complete nucleotide and amino acid sequences of the IFN X604 gene and IFN X604, respectively.
FIG. 16 and FIG. 17 depicting Chart 7 shows SDS-PAGE analysis of immunoprecipitates of 35 S-labelled E. coli extracts made with anti IFN-β and anti IFN-γ monoclonal antibodies.
FIG. 18 and FIG. 19 depicting Chart 8 shows Western blotting confirmation of co-identity of IFN-β immunoreactivity with IFN X601 36 kd protein.
FIG. 1 shows the enhanced antiproliferative activity of IFN X601 and a mixture of IFN X918 and IFN X430 against HEp-2 carcinoma cells.
FIG. 2 shows the activity of IFN X601 in inducing HLA DR expression on human fibroblasts in comparison with the parental IFNs used either individually or as a mixture.
Polypeptide cell modulators include soluble protein modulators released by differentiated cells which have their principle effect on other cell types and include lymphokines, monokines, peptide hormones or peptide growth factors.
Among the polypeptide cell modulators are cytokines, that is, all soluble protein modulators released by a differentiated cell that have their principal effect on other cell types. Included within this cytokine class are lymphokines, monokines, products of the endocrine, paracrine or autocrine hormone systems and polypeptide growth factors.
Specifically included within this cytokine class are the following polypeptides: interleukins 1, 2 and 3, alpha interferons (all types), beta interferon, gamma interferon, lymphotoxin, tumour necrosis factor, epidermal growth factor or urogastrone, B-cell growth factor, insulin like growth factors I and II, bone-derived growth factor, chondrocyte growth factor, T-cell growth factors, endothelial-derived growth factors, nerve growth factor, macrophage-derived growth factor, platelet-derived growth factor, neurotrophic growth factors, transforming growth factor (Type I or II), transforming growth factors, T-cell replacing factor, cartilage-derived growth factor, growth hormone, colony-stimulating factors, insulin, endothelial-cell growth factors, placental lactogen, erthropoietin, plasminogen activators, eye-derived growth factor, prolactin, fibroblast-derived growth factor, relaxin, fibroblast growth factors, thrombin, glial growth factor, transferrin, osteosarcoma-derived growth factor, vasopressin, thymosin, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, calcitonin, adrenal corticotropin, melanocyte stimulating hormone, parathyroid hormone, oxytocin, glucagon, secretin, cholecystokinin, gastrin, angiotensin, angiogenin and the polypeptide releasing factors from the hypothalamus.
Those skilled in the biochemical arts will recognize that modification of the polypeptide cell modulators such as changing amino acid sequences and derived or synthetic portions or regions of active cell modulators are equally useful as polypeptide cell modulators and are included as polypeptide cell modulators.
These polypeptide cell modulators are either linked directly or through a peptide linker segment. The peptide linker segment is generally a polypeptide derived from 1 to 500 amino acids. Other peptide linker segments such as dicarboxylic acids, diaminoalkyls and the like are useful for chemically linking polypeptide cell modulators. Peptide linker segments from the hinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgE provide an angular relationship between the attached polypeptide cell modulators. Especially useful are those hinge region sections where the cysteines are replaced by serines.
Since the preferred methods for preparing these linked polypeptide cell modulators are through genetic engineering, it is understood that variations in the genetic code can produce polypeptide cell modulators which have the general structure of
which is a peptide in which R1 and R2 are regions which have sequences which have the above described polypeptide cell modulator activity and L is a peptide linker segment. Large numbers of variations will produce equivalent results. The invention also encompasses glycosylated proteins which for example are produced as a result of expression in yeast or mammalian cells. Also encompassed are variations in the composition of oligasaccharide chains attached to the protein through specific amino acid glycosylation sites. Such variations can be introduced by expression in cells or organisms of varying type of by modification of amino acid glycosylation sites by genetic engineering techniques.
Plasmids used in the construction of, or expression of linked polypeptide cell modulator genes are listed in Table 1. One preferred embodiment of the present invention is plasmid pGC269 which codes for IFN X601 (Chart 3) and was derived from plasmids pGC262 (Chart 1A) and pJA39 (Chart 1A). Plasmid pGC262 was derived from plasmid pCC203 (deposited at ATCC no. 39,494) via plasmid pJB9 (Chart 1A); pJA39, which codes for the IFN X430 gene, was derived from plasmid pAP8.
Another preferred embodiment of the present invention is IFN X601 which is composed of sequentially from the N-terminus 1. IFN-gamma in which the N-terminal cys-tyr-cys has been replaced by met (designated IFN X918; Chart 3); 2) a 22 amino acid peptide linker segment coded by synthetic DNA (Chart 2A), related to the mouse IgG 2b "hinge" region (Chart 3, amino acids 145 to 167; and Nature 283, 786, 1980), except that the four cysteines are replaces by serines (Chart 3; serine residues 156, 159, 162 and 166); 3) IFN X430, which is identical to human IFN-beta, except that amino acid residues 36 to 48 inclusive are replaced by the equivalent residues from human IFN-alpha 1 (Chart 3, residues 202 to 214).
The plasmid pGC269 of example 1 below (Chart 1A; Table 1) was used in the expression of a polypeptide cell modulator (IFN X601) of example 2 having the antiviral, antiproliferative and immunomodulatory properties described in example 3.
IFN X918 is just one version of IFN-gamma which may be used (i.e., the N-terminal cys-tyr-cys may be present). IFN X430 is just one example of a type I IFN which may be linked to IFN-gamma, or a modified IFN-gamma, such as IFN X918. Other type I IFNs which may be used include IFN-beta or any IFN-alpha (e.g., IFN-alpha 2; Streuli, M. et al. Science 209, 1343, 1980).
Any suitable peptide linker segment may be used which correctly aligns and separates the two polypeptides comprising the polypeptide cell modulator, for example, the mouse IgG gamma 2b "hinge" region (Nature 283, 786, 1980) with the four cysteines converted to serines (e.g., Chart 3; residues 145 to 167); or a seven times repeated unit coding for alanine-glycine-serine (Chart 2B; and Chart 4; residues 145 to 165) which separates IFN X918 and IFN X430, giving rise to IFN X602 (Chart 4).
A further embodiment is expression plasmid pZZ102 of example 1 which codes for IFN X603 (Chart 5), which was derived from plasmids pZZ101 and pLT101 (Chart 1B and Table 1). Plasmid pZZ101 was derived from plasmid pJB9 by insertion of a 106 bp peptide linker segment coding for the C-terminus of IFN X918 and the amino-terminal 21 amino acids of hLT (Chart 5; residues 132 to 166); plasmid pLT 101 contains a synthetic human lymphotoxin gene (i.e., amino acid residues 146 to 316; Chart 5) cloned between the ClaI and BamHI sites of plasmid pAT153 (Twigg, A. J. Nature 283, 216, 1980). IFN X603 is composed of sequentially from the N-terminus; 1) IFN X918; a single methionine; and 2) human lymphotoxin (Chart 5).
Alternatively, any suitable peptide linker segment may be used which results in significant potentiation of biological activity, but preferably the mouse IgG gamma 2b "hinge" with the four cysteines converted to serines. This modified hinge region may be inserted between IFN X918 and hLT (Chart 6).
It must be appreciated that the DNA sequences coding for IFN X601, IFN X602, IFN X603 and IFN X604 disclosed in charts 3 to 6, are examples of many possible combinations given that alternative triplet codons exist for all amino acids except methionine and tryptophan. Other DNA sequences can code for the amino acid sequences defined in the charts (e.g., Gln-2 in IFN X601 in Chart 3 may be coded by CAG or CAA, etc.).
Expression of polypeptide cell modulators, as in example 2, may be in E. coli K12 HB 101, or other E. coli strain; from any strong promoter and ribosome binding site combination of prokaryotic or eukaryotic origin, but preferably the E. coli strain; from any strong promoter and ribosome binding site combination of prokaryotic or eukaryotic origin, but preferably the E. coli trp promoter minus attenuator (Patent applications EP 130 564 and EP 130 564 A) linked to the following ribosome binding site sequence: ##STR1## where S.D. is the Shine Dalgarno region and I.C. is the Initiation codon of IFNsX601, or X602, or X603 or X604.
The novel, polypeptide cell modulators of the present invention can be formulated by methods well known for pharmaceutical compositions, wherein the active chimaeron is combined in admixture with a pharmaceutically acceptable carrier substance, the nature of which depends on the particular mode of administration being used. Remington's Pharmaceutical Sciences by E. W. Martin, hereby incorporated by reference, describes compositions and formulations suitable for delivery of the compounds of the present invention. For instance, parenteral formulations are usually injectable fluids that use phsiologically acceptable fluids such as saline, balanced salt solutions, or the like as a vehicle.
The novel, polypeptide cell modulators of the invention may be administered to humans or other animals on whose cells they are effective in various ways such as orally, intravenously, intramuscularly, intraperitoneally, intranasally, intradermally or subcutaneously. Administration of the polypeptide cell modulators is indicated for patients with malignancies or neoplasms, whether or not immunosuppressed, or in patients requiring immunomodulation, or antiviral treatment. Dosage and dose rates may parallel those employed in conventional therapy with naturally occurring interferons--approximately 105 to 108 antiviral units daily. Dosages significantly above or below these levels may be indicated in long term administration or during acute short term treatment. A novel, polypeptide cell modulators may be combined with other treatments or used in association with other chemotherapeutic or chemopreventive agents for providing therapy against the above mentioned diseases and conditions, or other conditions against which it is effective.
a) Chemical Synthesis of Oligonucleotides
Oligodeoxyribonucleotides were synthesized by the phosphoramidite method (M. H. Caruthers, in "Chemical and Enzymatic Synthesis of Gene Fragments", ed. H. G. Gasen and A. Lang, Verlag chemie, 1982, p. 71) on controlled pore glass (H. Koster et al., Tetrahedron, 1984, 40, 103). Fully protected 2'-deoxyribonucleotide 3'-phosphoramidites were synthesized from the protected deoxyribonucleotide and chloro-N, N-(diisopropylamino) methoxyphosphine (L. J. McBride and M. H. Caruthers, Tetrahedron Lett., 1983, 24, 245 and S. A. Adams et al., J. Amer. Chem. Soc., 1983, 105, 661). Controlled pore glass supports were synthesized as described (F. Chow et al., Nuc. Acids Res., 1981, 9, 2807) giving 30-50 umol deoxynucleoside per gram.
After completion of the synthesis, the protecting groups were removed and the oligomer cleaved from the support by sequential treatment with 3% (v/v) dichloroacetic acid/dichloromethane (120s), thiophenol/triethylamine/dioxane 1/1/2v /v) (1 hour) and concentrated ammonia at 70� C. (4 hour). The deprotected oligonucleotides were purified either by HPLC on a PartisilR 10 SAX column using a gradient from 1M to 4M triethylammonium acetate pH4.9 at 50� C. or by electrophoresis on a denaturing 15% polyacrylamide gel (pH8.3).
b) Ligation of Oligonucleotide Blocks
500 pmole aliquots of the oligonucleotides were phosphorylated with 1 unit of T4 induced polynucleotide kinase in 20 ul of a solution containing 1000 pmole [32 p]gamma-ATP (2.5 Ci/mMole), 100 uM spermidine, 20 mM DTT, 10 mM MgCl2, 50 mM Tris-HCl (pH9.0) and 0.1 mM EDTA for 60 minutes at 37� C. The mixtures were then lyophilized and each oligonucleotide purified in a denaturing 15% polyacrylamide gel (pH8.3). After elution from the gel, the recovery was determined by counting the radioactivity.
Blocks (length 30-50 bases were assembled by combining 25 pmole of each phosphorylated component with equimolar amounts of the unphosphorylated oligomers from the complementary strand. The mixtures were lyophilized and then taken up in 15 ul water and 2 ul 10� ligase buffer (500 mM Tris-HCl pH7.6, 100 mM mgCl2). The blocks were annealed at 90� C. for 2 minutes, then slowly cooled to room temperature (20� C.). 2 ul 200 mM DTT and 0.5 ul 10 mM ATP were added to give final concentrations of 20 mM DTT and 250 uM ATP in 10 ul. 1.25 units of T4 DNA ligase were also added. After 18 hours at 20� C., the products were purified in a 15% polyacrylamide gel under denaturing conditions.
The final duplexes were then constructed from the single-stranded pieces. 1.5 pmole of each piece was taken and the mixtures lyophilized. Annealing was carried out in 15 ul water and 2 ul 10� ligase buffer at 100� C. for 2 minutes, then slowly cooled to 10� C. 2 ul 200 mM DTT, 0.5 ul 10 mM ATP and 1.25 units T4 DNA ligase were added. The reaction was left at 10� C. for 18 hours. The final products were then purified in a 10% native polyacrylamide gel.
c) Plasmid Constructions
(i) Plasmid pGC269 (Table 1)
DNA corresponding to the amino-terminal cys-tyr-cys of human IFN-gamma in the plasmid pCC203 (ATCC No. 39, 494) was deleted by ClaI/BamHI double restriction enzyme digestion as in Chart 1A (Methods in Molecular Cloning, a Laboratory manual, eds. Maniatis et al., Cold Spring Harbor Laboratory, 1982). The resultant expression plasmid, pJB9, codes for IFN X918 which has the cys-tyr-cys replaced by methionine (PCT No. 83/04053).
A 171 bp chemically synthesized duplex (Chart 2A) coding for the C-terminal 13 amino acids of IFN X918, 22 amino acids of the mouse immunoglobulin gamma 2b "hinge" region (cys- ser) and 20 N-terminal amino acids of IFN X430, was ligated to the Bg1II to SalI large vector fragment of pJB9 (Chart 1A). The resultant plasmid, pGC 262 (table 1) contains a HindIII site for insertion of the remainder of the IFN X430 gene.
To create an IFN X416 gene (European Patent application No. 85105914.7) with a unique HindIII site, plasmid pAP8 was cut with ClaI and XhoI (chart IA), and the 230 bp fragment replaced by an identical chemically synthesized fragment except that codons 19 and 20 are AAGCTT (HindIII) instead of AAGCTC. The resultant plasmid was designated pJA39 (Table 1).
Since IFN X416 and IFN X430 are identical except at amino acid position 17, the HindIII to SalI 719 bp fragment from pJA39 (equivalent to amino acids 19 to 166 of IFN X430 or IFN X416) was ligated to the large HindIII/SalI vector fragment of pGC262 to give plasmid pGC269, which codes for the IFN X918 - IFN X430 polypeptide cell modulator, designated IFN X601 (Chart 3).
(ii) Plasmid pZZ102 (Table 1)
A similar strategy was used to construct pZZ102.
Plasmid pJB9 (Chart 1B) was cut with Bg1II and SalI and a 106 bp chemically synthesized duplex, coding for the C-terminal 13 amino acids of IFN X918 (as in Chart 2A); and a single methionine followed by the 21 N-terminal amino acids of human lymphotoxin (Chart 5; residues 132 to 166) was ligated to the Bg1II to SalI large vector fragment of pJB9 (Chart 1B). The resultant plasmid, pZZ101, contains an NsiI site at hLT condons 20 and 21 (Gray, P. W. et al. Nature 312, 721, 1984) for insertion of the remainder of the hLT gene, i.e. ##STR2##
Plasmid pZZ101 was cleaved with NsiI and SalI and the large vector fragment isolated in preparation for insertion of the remainder of the hLT gene, which was isolated from pLT101 (Table 1; chart 1B).
pLT101 contains a complete synthetic hLT gene modified from Gray, P. W. et al. Nature 312, 721, 1984 (equivalent to amino acid residues 145 to 316 in Chart 5). The hLT gene in pLT 101 was cloned on a ClaI to BamHI fragment in the ClaI/BamHI sites of plasmid pAT153. The nucleotide sequences of the ClaI and BamHI junctions are, respectively: A T C G A T A A G C T A T G. and T A G A G G A T C C (ATG=initiation codon, TAG=termination codon).
Plasmid pLT101 was cleaved with NsiI and SalI and the resultant 725 bp small fragment was ligated to the NsiI and SalI large vector fragment of ppZZ101 (Chart 1B) to give plasmid pZZ102, which codes for the IFN X918-lymphotoxin polypeptide cell modulator, designated IFN X603 (Chart 5).
a) Expression of plasmids coding for IFN X601, X602, X603 and X604
Overnight cultures (10 ml.) of transformed bacteria were grown in M9/casamino acids medium (EP 131 816A) supplemented with tryptophan (40 ug/ml) and ampicillin (100 ug/ml). Inocula (0.5 ml.) were added to 50 ml. M9/casamino acids medium containing 100 ug/ml. ampicillin. Growth was continued at 37� C. until the A 670 nm had reached 0.5, at which time the cultures were made 20 ug/ml. with respect to beta -indole acrylic acid in order to induce the synthesis of polypeptide cell modulators. Growth was at 37� C. with vigorous shaking, and samples for biological assay (as described in example 3 below) and electrophoretic analysis were removed at 4 hours after induction.
b) SDS-polyacrylamide gel electrophoresis of total E. coli proteins for estimation of expressed protein content
The volume of cells equivalent to 0.5 optical density units at 670 nm was removed from the culture immediately and at 4 hours after adding IAA, and the bacteria recovered by centrifugation. The cells were immediately resuspended in 50 ul of 60 mM tris-HCl pH6.8, 0.05% bromophenol blue, 5% glycerol, 1% sodium dodecylsulphate, 0.5% 2-mercaptoethanol, heated at 100� C. for 3 min. and quick frozen on dry ice. The boiling-freezing cycles were repeated 2-3 times to reduce the viscosity of the sample before a final boiling 5 minutes prior to loading 7.5 μl on a 15% SDS- polyacrylamide gel (Molecular Cloning, A Laboratory Manual, ibid.). The gel was stained with coomassie brilliant blue and dried. The dried gel was scanned with a Joyce-Loebl `chromascan 3` gel scanner, which computes the percentage of total protein for each polypeptide band.
Table 2 shows that for IFN X601, a polypeptide of approximately the size expected for an IFN X918/hinge/IFN X430 fusion is expressed in the range 5.4 to 10% of total bacterial protein.
This polypeptide is absent from cultures of E. coli K12 HB 101 harbouring plasmid pJB9 expressing IFN X918 (˜17K) or pIL201 expression IFN X430 (˜19K).
c) Preparation of bacterial extracts for biological assay
10 to 20 ml. of bacterial culture was removed at the optical density (670 nm) of 1.5-2.0 (middle to late log phase of growth) and centrifuged to recover the cells. After suspension in 25 mM tris-HClpH 7.5, 50 mM NaCl (1 ml.) and 1 mM EDTA (1.4 ml.) at 0� C., 28 ul lysozyme was added to a final concentration of 50 ug/ml and the suspension incubated at 0� C. for 30 min. The suspension was sonicated for 24 sec., the cell debris removed by centrifugation and the supernatants assayed for biological activity as described in Example 3 or gel analysis as described in Example 2.
Alternatively, lysis without sonication was used as follows. 10 ml. culture was centrifugated and the bacterial pellet resuspended in 2 ml. 30 mM NaCl, 50 mM tris-HCl pH 7.5, 0.05 to 1 mg/ml lysozyme. Following incubation at 25� C. for 10 min. and 0� C. for 15-30 min. three freeze-thaw cycles were performed (-70� C.). The supernatant from a 15,000 rpm, 15 min. centrifugation was divided for gel analysis, protein estimation and assay.
a) Antiviral assay
The cellular extract prepared as in Example 2 (together with 1 log dilutions to 10-6) was assayed for antiviral activity by monitoring the protection conferred on Vero (African Green Monkey) cells against the cytopathic effect of encephalomyocarditis (EMC) virus infection in an in vitro microplate assay system; for example, Dahl, H. and Degre, M. Acta. Path. Microbiol. Scan., 1380, 863, 1972.
A comparison is made in Table 3 of the antiviral (AV) activity in crude bacterial extracts of IFN X601 and the parental IFNs, derived from equivalent numbers of bacterial cells. IFN X601 consistently exhibited 2.5-3.0 fold higher AV activity than IFN X430 and a 4-6 fold higher AV activity than IFN X918, despite a ˜2- fold lower level of protein expression (Table 2).
A 1:1 mixture of the separately expressed IFNs X918 and X430 also exhibited a significantly enhanced AV activity, which was 4 fold higher than the value expected if the AV activities of the individual IFNs X918 and X430 were additive (Table 3). This is a reflection of the known synergy between Type I and Type II IFNs (Czarniecki, C. W. et al. J. Virol. 49, 490, 1985; and EP 0107 498).
In conclusion, the polypeptide cell modulator IFN X601 displayed a significant enhancement of AV activity compared with the parental IFNs, which was similar to that of equimolar mixtures of IFN X918 and IFN X430.
b) Antiproliferative assays
(i) Daudi (lymphoblastoid) cells
Antiproliferative (AP) activity was assessed by the ability of the polypeptide cell modulator to inhibit the replication of Daudi (lymphoblastoid) cells (Horoszewicz et al. Science 206, 1091, 1979). Daudi cells in log phase were cultured for 6 days in 96 well plates in the presence of various dilutions of chimaeron or IFN. The phenol red in the medium changes from red to yellow (more acid) with progressive cell growth. Liquid paraffin was added to prevent pH change on exposure to the atmosphere, and the pH change in the medium measured colorimetrically on a Dynatech plate reader. Inhibition of cell growth is reflected by a corresponding reduction in the colour change.
A comparison is made in Table 4A of the Daudi lymphoblastoid cell antiproliferative activity in crude bacterial extracts of IFN X601 and the parental IFNs derived from equivalent numbers of bacterial cells. Daudi cells are known to be unresponsive to IFN-gamma and in a similar fashion did not respond to the antiproliferative action of IFN X918, being more than 100X less sensitive to IFN X918 than to IFN X430 (Table 4A). By contrast, IFN X601 exhibited similar activity to that of IFN X430. Mixtures of IFN X918 and IFN X430 gave a lower titre than with IFN X430 alone i.e., synergy was not evident. These results are expected as the Daudi cell line is capable of responding to the antiproliferative effect of only the IFN X430 portion of the polypeptide cell modulator. These results also indicate that the IFN X430 portion of the polypeptide cell modulator functionally active, contributing to its biological activity (Tables 3 and 4B).
Consistent with these findings is the observation that there is a similar level of binding of IFN X430 and IFN X601 to Daudi receptors (Table 7), while the lack of AP activity of IFN X918 correlates with very low receptor binding.
(ii) HEp-2 (human laryngeal carcinoma) cells
Antiproliferative activity was also assessed in HEp-2 cells Growth inhibition was measured by methylene blue staining of the cell monolayer by a modification of the method of Ito. (Ito, M. J. Interferon Res. 4, 603, 1984). Inhibitory concentration (IC50) end point is the log dilution giving 50% reduction of methylene blue staining.
A comparison is made in Table 4B of the HEp-2 antiproliferative activity in crude bacterial extracts of IFN X601 and the parental IFNs, derived from equivalent numbers of bacterial cells. IFN X601 consistently displayed a 3 fold higher AP activity than IFN X430 and a 15 fold higher AP activity than IFN X918, despite a ˜2-fold lower level of protein expression (Table 2). Furthermore, when equivalent antiviral units of these interferons were compared it was seen that IFN X601 had an enhanced antiproliferative effect as shown in FIG. 1. For the individual IFNs X430 and X918 there is a maximum achievable level of growth inhibition which cannot be increased despite adding a hundredfold excess of interferon. This is not seen with IFN X601 where a markedly increased level of growth inhibition is seen.
These properties of IFN X601 are reminiscent of the antiproliferative effect of mixtures of IFN X430 and IFN X918. For example, Table 4B shows the equivalent concentrations of these two IFNs mixed together gave 1.8-8.6 fold higher AP activity than either alone. In this case, AP activity was almost 3 fold higher than the value expected if the AP activities of the individual IFNs X918 and X430 were additive (Table 4B). Further, like IFN X601, equimolar mixtures of IFN X918 and IFN X430 have enhanced antiproliferative activity against HEp-2 cells (FIG. 1).
Potentiation of AP activity by mixtures of IFN X918 and IFN X430 is a reflection of the synergy which can be demonstrated between IFN-gamma (equivalent to IFN X918) and IFN X430 and is illustrated by the results presented in Table 5. Where the FIC index (as defined in Table 5) is less than 0.5, synergy is evident. Maximum synergy was observed at equivalent numbers of antiviral units of IFN-gamma and IFN X430 (10 U/ml). Since the specific activities of IFN-gamma and IFN X430 differ only by a factor of approximately two, similar amounts of IFN protein are also present.
Taken together, these results indicate that (i) a covalent combination of IFN X918 and IFN X430 via a peptide linker segment potentiates cytotoxicity in a manner analogous to simple mixtures; (ii) a covalent combination of IFN X918 and IFN X430 is a suitable ratio to potentiate biological activity; (iii) the IC50 end point on HEp-2 cells for IFN X601 was significantly higher than the values for the parental IFNs. Potentiation was similar to that observed with synergistic mixtures of IFN X918 and IFN X430.
c) HLA-DR Antigen presentation on human fibroblasts
IFN-gamma, but not IFN-beta or IFN X430, induces the expression on the surface of normally DR-negative human foetal lung fibroblasts (17/1 strain). This is detected and measured by the binding of monoclonal antibody against HLA-DR.
Fibroblasts are grown to confluence in DMEM/10% FCS (Dulbecco's Modified Eagles Medium) in 96-well tissue culture plates. IFN-gamma or modified IFN is serially diluted in DMEM/0.1% BSA and dilutions are added to the medium on the fibroblasts. The fibroblasts are incubated at 37� C. for a further 3 days and then the medium is removed and the cells are washed once with PBS. Admixtures in Herpes-buffered DMEM of a monoclonal antibody directed against HLA-DR and peroxidase conjugated antibody against mouse IgG, is added to the cells and incubated at room temperature for 2 hours. The cells are washed five times with PBS and then the amount of anti-DR antibody bound to the cells is measured by assaying for bound peroxidase using tetramethyl benzidine (TMB) as a chromogen. The colour generated is measured with a Dynatech™ microelisa reader.
IFN X601 and IFN X918 clearly caused expression of HLA-DR antigens on the surface of 17/1 fibroblasts while IFN X430 did not (table 9). The level of HLA DR induction by IFN X601 was markedly lower than that induced by equivalent antiviral units of IFN X918. This may be due to suppression by the IFN X430 domain because the HLA DR induction by IFN X918 was seen to be reduced in a 1:1 mixture with IFN X430. The HLA DR induction by IFN X601 can be increased more than ten fold by blocking the activity of the IFN X430 domain with anti IFN-β monoclonal antibody. These results demonstrate that IFN-gamma biological activity is present in the polypeptide cell modulator IFN X601.
d) Analysis of IFN X601 with Antibodies Against beta and gamma -IFNs
i) Enzyme linked immunoadsorbent assay (ELISA) for interferon
The ELISA for both beta and gamma interferons utilizes an indirect two site sandwich technique. Dilutions of the interferon samples (or standards) are allowed to bind to interferon antibodies attached to the wells of a 96 well microplate. A second antibody to interferon, but raised in a different species from that attached to the plate, is included in the incubation mixture, which then binds to a second epitope on the interferon molecule. After washing away the unbound molecules, an enzyme labelled antispecies antibody is added which binds to the second interferon antibody. The presence of bound enzyme is detected by adding a substrate which changes color in the presence of enzyme. The amount of color produced is proportional to the amount of interferon, since the other reagents are present in excess.
For the beta and gamma interferon ELISA's, two antibodies against the corresponding interferon are used, while for a hybrid ELISA, an antibody directed against beta interferon is bound to the plate, while the second antibody used is one directed against gamma interferon.
The general scheme of the assay is illustrated below:
96 well microplates (Nunc Immunoplate 1) are coated with a goat anti human beta interferon antibody (Rega Institute). To each well of a microplate, is added 100 microliter of a 5 microgram/ml solution of immunoglobulin (obtained by a 40% ammonium sulphate precipitation of the interferon antibody) in 0.05M sodium carbonate buffer, pH 9.8, and incubated for two hours at room temperature. After removal of the well contents, unoccupied binding situes are blocked by incubation with 100 microliters of phosphate buffered saline containing 0.5% casein (PBS/C), for 30 minutes at room temperature. The plates are then washed six times with phosphate buffered saline containing 0.05% Tween 20 (PBS/T), and stored at +4� C. in a covered moist box until required.
Serial dilutions of interferon samples are made in the plates, by dilution in PBS/C containing a mouse monoclonal antibody to beta interferon at a 1/100 dilution. Each plate also contains an internal standard which has been calibrated against the International Reference Standard. After incubation overnight at +4� C., the well contents are removed and the plates washed six times with PBS/T.
100 microliters of peroxidase conjugated goat anti-mouse immunoglobin (Sigma a7282, diluted 1/2000 in PBS/T), are added to each well and incubated for thirty minutes at room temperature. The well contents are removed and the plates are washed six times with PBS/T. 100 microliters of TMB (Tetramethyl benzidine, Sigma, 50 mcg/ml in 0.1N acetate/citrate buffer pH 6.0, containing 0.0022% hydrogen peroxide) are added and incubated for one hour at room temperature. 25 microliters of 2.5M sulphuric acid is added to stop the reaction and the optical density read at 450 nm in an automatic plate reader (Titertek Multiscan MC). Data is fed into a computer and the 50% end points determined by linear regression analysis of the logic log transformed data. Corrections are then made to the internal standard included on each plate.
This assay is carried out in the same way as the beta ELISA, with the following changes: the plates are coated with a mouse monoclonal antibody to gamma interferon (Meloy Laboratories) at 1/200 in carbonate buffer. Serial dilutions of the gamma interferon samples are made in PBS/C containing a rabbit antiserum to human gamma interferon (Immunomodulator laboratories, diluted to 1/5000). A peroxidase conjugated goat anti rabbit immunoglobulin (Tago Laboratories, diluted to 1/3000) is used as the indicator molecule.
The only difference from the beta ELISA is that the interferon samples are diluted in PBS/C containing a mouse monoclonal to human gamma interferon (Meloy Laboratories, at a dilution of 1/1000). This assay will only detect interferon molecules containing both a beta and a gamma epitope.
The results of testing the polypeptide cell modulator IFN X601 and the appropriate controls in the beta, gamma and hybrid ELISA's are given in Table 6. In the beta ELISA, IFN X430 (equivalent to beta) reacts, the gamma interferon shows no sign of cross reactivity, while a 50/50 mixture of the two gives a titre reduced by 0.4 log unit/ml, close to the expected 0.3 reduction. The IFN X601 also reacts strongly, showing that the two beta interferon epitopes are still available to bind antibodies.
In the gamma ELISA, the gamma interferon reacts, the IFN X430 shows no cross reactivity, while a 50/50 mixture of the two gives a titre reduced by the expected 0.3 log units/ml. IFN X601 also reacts, though with a reduced titre compared to the other positive reactions, which might indicate that one of the gamma epitopes is slightly sterically affected by the presence of the beta hybrid interferon.
In the hybrid ELISA, the only sample to react is IFN X601, which conclusively demonstrates that the molecule contains both beta and gamma epitopes covalently bonded to each other. Quantitatively the results from this assay cannot be compared to the other two ELISA's since there is no standard available and the 50% end points are dependent on relative affinities and concentrations of the various reagents used, which differ for the three assays used. However, the results indicate that a substantial proportion of the polypeptide cell modulators is present in the covalently linked state in sample X601.
(ii) Immunoprecipitation
Interferons were labelled by including 35 S-methionine in bacterial growth medium and extracts were prepared by treatment by lysozyme and sonication. 35 S-labelled E. coli extracts were immunoprecipitated with either monoclonal antibodies directed against IFN-β or IFN-γ and the immunoprecipitates were analyzed by SDS-PAGE.
The results in Chart 7 show that anti IFN-β monoclonal antibody precipitates IFN X430 but not IFN X918, anti IFN-γ monoclonal antibody precipitates IFN X918 but not IFN X430 while both monoclonal antibodies precipitate a ˜36 kd protein in the IFN X601 extract. The material precipitated from the IFN X601 extracts by both antibodies therefore has the predicted molecular weight for the chimaeric protein and has both X430 and X918 antigenic activity.
(iii) Western Blot Analysis
Bacterial extracts containing IFNs were run out on SDS-PAGE and analyzed by Western blotting with anti IFN-β monoclonal antibody.
Chart 8 shows that anti-IFN-β monoclonal antibody detects IFN X430 in lanes A, does not recognize IFN X918 in lanes B and recognizes a ˜36 kd band in the IFN X601 extract in lanes C. This again demonstrates that a band in the IFN X601 extract which is recognized by anti-IFN-β monoclonal antibody has the predicted MW for the chimaeric protein IFN X601.
(iv) Monoclonal antibody affinity column purification
Bacterial extracts containing IFN X601 were loaded on to monoclonal antibody affinity columns consisting of either anti-IFN-β bound to CNBr sepharose or anti-IFN-γ bound to CNBr sepharose (Celltech MAb). The loaded columns were extensively washed, bound material was eluted and fractions were assayed for antiproliferative activity against Daudi and HEp-2 cells and for HLA DR inducing activity on human lung fibroblasts.
The results in Table 8 demonstrate that material from an E. coli lysate containing IFN X601 can be bound to and eluted from both anti-IFN-β and anti-IFN-γ affinity columns. The material eluted from the anti-IFN-β column must have IFN X430 antigenicity and has been shown to have IFN X430 biological activity (Daudi antiproliferative assay) as well as IFN X918 activity in the HLA DR induction assay. The material eluted from the anti-IFN-γ column must have IFN X918 antigenicity and has been shown to have IFN X918 biological activity (HLA DR induction activity) as well as IFN X430 activity in the Daudi antiproliferative assay. In addition, eluted material from both columns showed enhanced antiproliferative activity against HEp-2 cells which is taken to indicate that both the IFN X430 and IFN X918 domains are biologically active.
Biological Activity of IFN X602 (IFN X918 (AGS), IFN X430)
Table 9 shows X602 to have similar biological properties as X601.
Biological Activity of IFN X603 (IFN X918-hLT)
Table 10 shows that IFN X603 retains both lymphotoxin and interferon-like activities. Antiproliferative activity against mouse L cells is characteristic of LT activity, while AV, HLA DR and ELISA give characteristic IFN-gamma activities. (HEp-2 antiproliferative activity could be due to IFN-gamma or lymphotoxin/IFN-gamma combination but not to lymphotoxin alone.)
Charts 1Aa and 1Ab illustrate the path to constructing a high level expression vector for IFN-β[β(36-48)→α1 (34-46)][cys17 →ser17 ], also referred to as IFNX416, in the host E. coli HB101 (European Patent No. 85105914.7). The starting vector was p1/24C (˜4,440 bp) which was identical to plasmid pl/24 U.K Patent 8,102,051, except for the underlined sequences which follows: ##STR3##
Step 1 (Chart 1Aa)
The subcloning of the natural human IFN-β gene from plasmid p1/24C (Taniguchi et al., Gene, 10, 11, 1980) in phage M13mp8 (Sanger, F. et al., J. Mol. Biol., 143, 161, 1981) was performed, and the presence of the whole fragment was confirmed by restriction endonuclease mapping of M13 plasmid mAP2.
Step 2 (Chart 1Aa)
The technique of "site-directed mutagenesis" (Zoller and Smith, Nucl. Acids Res., 10, 6487, 1982) was employed to introduce two base changes, one each in the IFN-β codons 74 and 75 so as not to change the encoded amino acid sequence. Supercoiled DNA resulting from transcription/ligation was separated from non-ligated DNA in a 1% agarose gel and used to transform E. coli JM101. Total plasmid DNA was prepared.
Step 3 (Chart 1Aa)
Mutant DNA bearing a unique XhoI site was separated from non-mutant DNA by XhoI restriction and electrophoresis in 1% agarose. The linear DNA was electroeluted from the agarose (Molecular cloning, A Laboratory Manual, eds. Maniatis et al., p. 168, Cold Spring Harbor Laboratories). Following self-ligation of the linear DNA and transformation of E. coli JM101, M13 clones were obtained all of which had a unique XhoI site, one of which was designated mAP3.
Step 4 (Chart 1Ab)
The complete IFN-β gene with an XhoI site spanning codons 74-76 was recloned back in pAT153. This generated a vector (pAP4) similar to p1/24C, except for the changed codons 74 and 75 and the deletion of the ˜546 base pair Bg1II-BamHI fragment, originally lying 3' to the IFN-β coding sequence. The new sequence of the Serine codons 74 and 75 is given in Chart 1Aa.
Step 5 (Chart 1Ab)
The ˜230 bp synthetic DNA fragment, assembled as described above, was cloned in the ClaI-XhoI sites of plasmid pAP4 to give pAP8 (Chart 1Ab), a plasmid expressing IFNX416 in the host E. coli HB101.
Modifications of the above described mode for carrying out the invention such as, without limitation, use of alternative vectors, alternative expression control systems, and alternative host micro-organisms and other therapeutic or related uses of the novel polypeptide cell modulators that are obvious to those of ordinary skill in the biotechnology, pharmaceutical medical and/or related fields are intended to be within the scope of the following claims.
TABLE 1______________________________________Table of PlasmidsPlasmid Properties Source______________________________________pAP8 Expression vector coding for EP 85105914.7 IFN X416 gene U.K. Patent 8,102,051, Chart 1Aa and 1Ab and example 4pJA39 Expression vector containing Amino acids 19/20 IFN X416 gene plus HindIII coded by AAG.CTT site instead of AAG.CTC (pAp8)pGC262 Intermediate vector in Chart 1A construction of pGC269 - codes for IFN-gamma + 22 amino acid mouse gamma 2b IgG "hinge"pCC203 Expression vector containing Chart 1A and synthetic human IFN-gamma PCT 83/04053 genepJB9 Expression vector containing Chart 1A and synthetic IFN-gamma gene PCT 83/04053 with DNA coding for N-terminal Cys--Tyr--Cys deleted and replaced by Met. (IFN X918)LT3/1 Expression vector containing Charts 1A, 3 synthetic human lymphotoxin Nature 312, 721, gene 1984pGC279 Intermediate vector in Chart 1B construction of pZZ102; codes for IFN X918 plus 22 N-terminal amino acids of lymphotoxinpZZ102 Expression vector containing Charts 1B, 5 IFN X603 gene (IFN X918 - met-lymphotoxin polypeptide cell modulator).pGC269 Expression vector containing Charts 1A, 3 IFN X601 gene.______________________________________
TABLE 2______________________________________Molecular Weight and Expression in E. coli of IFN X601Inter- Molecular weight Range of expressionferon (from polyacrylamide gel) (% of total bacterial protein)______________________________________X918* 17,000 13.6-15.6 (N = 14.6)X430+ 19,000 12.3-17.0 (N = 14.65)X601 37,500 5.4-10.0 (N = 7.7)______________________________________ *IFN-gamma with Nterminal cys--tyr--cys deleted and replaced by met (Char 3) + IFNbeta with amino acids 36 to 48 inclusive replaced by amino acid 34 to 46 inclusive from IFNalpha 1. N mean.
TABLE 3______________________________________Antiviral Activity of IFN X601 Antiviral activity Increase compared I.U/ml at 10 A670 with:Interferon � 10-6 IFN X430 IFN X918______________________________________.sup. X9181 0.59 (0.5X) --X430 1.1 -- 2.9XX601 2.87 2.6X 4.9XX918 + X4302 3.47 3.2X 5.9X______________________________________ *IU/ml 10 A670 � 10-6. Mean of 3 determinations in 2 separate experiments: 1. IFNgamma with Nterminal Cys--Tyr--Cys replaced by Met (chart 3). 2. Approximately 1:1 mixture of each IFN (protein).
TABLE 4______________________________________ Increase compared Antiproliferative with:Interferon Activity* IFN X430 IFN X918______________________________________A. Daudi lymphoblastoid cellsX918 0.004 -- --X430 2.7 -- --X601 3.3 1.2X --X918 plus X4301 1.9 (0.7X) --B. HEp-2 carcinoma cellsX918 0.57 (0.2X) --X430 2.8 -- 4.9XX601 9.0 3.2X 15.8XX918 plus X4301 4.9 1.8X 8.6X______________________________________ *Units/ml � 10-4 = dilution of IFN at 50% cell growth inhibition. Mean of 2 determinations. 1 Mixture 1:1 w/w
TABLE 5______________________________________IFN X430/IFN-gamma synergy on HEp-2 carcinoma cellsA. IFN X430 B. IFN-gamma+Antiviral FIC* Antiviral FIC* FIC IndexUnits/ml "A" units/ml "B" ("A" + "B")______________________________________168 1.000 0 0.000 1.00056 0.334 0.3 0.003 0.33740 0.230 1.0 0.009 0.23932 0.188 3.1 0.029 0.21710 0.059 10 0.094 0.1533.1 0.018 27 0.252 0.2702.2 0.013 32 0.298 0.3111.0 0.006 81 0.767 0.7730.8 0.004 100 0.940 0.9440 0 106 1.000 1.000______________________________________ *FIC. Fractional Inhibitory Concentration Ratio: antiviral units at 50% cell growth inhibition of a given IFN (e.g. `A`) in combination with another IFN (e.g. `B`) to antiviral units of IFN`A` alone. Concentration of IFN alone or in combination required to produce 50% inhibition of HEp2 growth. Synergy is present when FIC index is equal to or less than 0.5
TABLE 6______________________________________ ACTIVITY (LOG UNITS/ML) Beta Gamma Hybrid ELISA ELISA ELISA E F E F E F______________________________________A Gamma interferon ND ND 4.47 5.44 ND NDB IFN X430 (=beta) 3.95 5.84 ND ND ND NDC Interferon X601 4.13 6.02 2.98 3.95 3.73 --D Mixture of A and 3.59 5.48 4.16 5.13 ND ND B (1:1)______________________________________ Notes 1. E represents the 50% end points 2. F represents the corrected activities 3. ND is not detectable activity
TABLE 7______________________________________COMPETITION BY IFN X601 FOR THE BINDINGOF 125 I-IFN alpha 2 TO DAUDI CELL RECEPTORSIFN Activity Log U/ml.*______________________________________X430 7.0X918 3.6X601 6.6______________________________________ *IFN α2 antiviral unit equivalents. The activity in each sample was calculated by interpolation from a standard dose curve of the competition by IFN α2 for the binding of 125 IIFNα2.
TABLE 8______________________________________MONOCLONAL ANTIBODY AFFINITYPURIFICATION OF CRUDE LYSATES OF IFN X601 IFN Activity*Fraction Daudi HEp-2** HLA DR______________________________________Anti IFN-Beta Column3 3.00 Not done 2.34 3.25 2.89 2.35 4.25 3.79 2.476 4.20 3.85 2.657 3.82 3.25 Not doneAnti IFN Gamma Column3 3.24 2.72 2.34 3.72 4.31 2.45 3.70 4.15 2.36 3.28 3.95 2.37 3.22 3.67 Not done______________________________________ *Log units/ml = dilution of IFN at 50% assay end point. **Enhanced antiproliferative activity seen.
TABLE 9__________________________________________________________________________BIOLOGICAL ACTIVITY OF IFN X602COMPARED WITH IFN X601Antiviral Antiproliferative HLA DR Induction ELISAIFN EMC/Vero HEp-2 Daudi Lung Fibroblasts Beta Gamma Mixed__________________________________________________________________________X601 6.49 4.74* 4.28 3.30 5.93 4.08 3.50X602 6.46 3.89* 3.55 2.81 5.94 3.46 2.75__________________________________________________________________________ Antiviral plus Beta and Gamma ELISA activities expressed as Log IU/ml/10 A670. Antiproliferative, HLA DR and Mixed ELISA activities expressed as Log dilution/ml/10 A670 at 50% end point. 1. Assayed in presence of anti IFN beta monoclonal antibody to overcome inhibitory activity of the X430 domain. *Enhanced growth inhibitory activity typical of IFN gamma/IFN X430 mixtures.
TABLE 10__________________________________________________________________________BIOLOGICAL ACTIVITY OF IFN X603 Antiproliferative HLA DR Induction ELISAIFN Anitviral EMC/Vero HEp-2 L Cell Lung Fibroblasts Gamma__________________________________________________________________________X603 4.47 3.19 4.02 2.80 4.31__________________________________________________________________________ Antiviral and Gamma ELISA activities expressed as Log IU/ml/10 A670. Antiproliferative and HLA DR activities express as Log dilution/ml/10 A67 at 50% end point.
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