text:
Intruder Reviews
|Ken S
Super Reviewer
May 20, 2007
If you've seen damn near every slasher movie, Intruder is a pleasantly solid entry in the genre. GREAT gore and kills.
Super Reviewer
January 4, 2012
Scott Spiegel's Intruder is one of the 1980's last great Slasher flick that came out at the end of the decade. While many Slasher franchises were recycling ideas, and many countless ripoffs were filmed, Intruder is one of the better of the bunch. Of all the slashers that in the last years of the 80's, Intruder has got to be the most original, the most grotesque and overall the best of the decades most popular genre of horror. Intruder was directed by Scott Spiegel who previously worked with Sam Raimi on Evil Dead 2. Like I previously stated, Intruder is a great slasher film, and is one of final slasher greats of the 80's. The film boasts some very impressive kills scenes with plenty of blood and gore to appeal to the most die hard Slasher film fan. The key aspect of the film is the special effects, I personally think that these are some of the best special effects I've seen in a slasher film in quite some time. Like I said, the kills are great and will definitely appeal Slasher fans. There's also a good mix of suspense and humor in the films plot, and Scott Spiegel has crafted a solid Slasher film. Intruder is a fine Slasher film that signaled the end of 1980's slashers. The film is great, and if you've seen the countless early 80's Slashers, and want something refreshing, then this is a great film to watch. This is a definite Slasher classic that is a must see for every horror fan, and with great gore effects, and awesome kills; it really makes Intruder a film that separates itself from most Slashers. This film is an awesome experience for horror fans.
Super Reviewer
December 28, 2011
I was actually quite surprised by how well-made Intruder turned out to be when I finally got around to watching it. In the back of my mind, I pictured a grainy 16mm movie with hardly any real actors, lots of gore and great ethusiasm. That's actually a nearly perfect description for the film (minus the 16mm part), but the difference is that these people know what they're doing. There's some great visual gags and moments, as well as a very nice score that's put to effective us. The gore and special effects are wonderful, courtesy of the KNB guys (before KNB was established). In particular, the band saw scene, which is the strongest effect in the entire film. The acting isn't amazing or anything, but it works in the context of what it is. I think it's rather original to set a horror movie entirely in a grocery store for the length of the film. I can't say whether or not that's a first, but it works well. There are several things about the movie that don't make sense, as there are plot holes abound, but it's effective nonetheless. Definitely a great slasher movie.
Super Reviewer
December 20, 2011
Worth watching for completists and rabid fans of the Raimi brothers and Bruce Campbell, but ultimately it's nothing special.
Super Reviewer
February 28, 2011
A good fun slasher all set in a supermarket where the night staff end up getting butchered by a maniac. It's limited in what it has to offer but it serves up a good piece of entertainment for fans of the slash 'em up. The kills are gory and bloody and frequent after having to wait 35 minutes for the first one to occur. It's not one that takes itself too seriously either with the cheesy acting and goofy characters all providing the odd laugh along with some quirky camera angles. All in all a good late nights viewing.
|Cory T
Super Reviewer
October 16, 2011
This may be the first example of a product-placement horror films where all the brand names are displayed front and center. As for the film itself, it's a pretty pedestrian slasher movie that is only notable for the ostentatiously gory dismemberment by KNB. The whodunit aspect is strictly 'Scooby Doo' cheesiness with the villain overacting to a cartoonish degree after he is unveiled. Writer-director Spiegel was a frequent collaborator with the Raimi brothers and they are present in the film as well. In fact, most of the convex, off-kilter cinematography is directly pilfered from Sam Raimi (ex. Inside-the-cart shot and through the murky glass of a whiskey bottle). What begins as suitably spooky quickly devolves into a homogenized stalking-killer flick with eye-gouging and skull compacting.
Super Reviewer
March 10, 2011
This film came out in 1989, and though most drive-in's were closed by then the ending scene is excellent for the drive in screen. Another late eighties slasher film, that had the titles final checkout, and night crew, besides Intruder. A crazy old boyfriend shows up at a store, the cashier , beautiful Elizabeth Cox ( Who had such a short career) broke up with over a year ago, he has been in prison for past year. He is thrown out of the store, and after closing people start dying. But is the boyfriend the killer. Didn't take me long to figure this one out. As good as any slasher film from that time period. But is full of mistakes, like music cuts and spliced in changing scenes. Beyond Infinity Productions didn't spend much on this one, but still its an enjoyable slasher film fest weekend film. 3 1/2 stars
|Cassandra M
Super Reviewer
April 6, 2008
Right at the end of the 80's, a horror decade ruled by violent slashers, comes one of the coolest independent productions ever unleashed upon us, gorehounds: Scott Spiegel's "Intruder". It looks like the most basic and primitive slasher ever made (the story can be summarized in one phrase, namely, a group of supermarket employees are all brutally murdered by an unidentified maniac) but if you observe "Intruder" more closely, you'll find plenty of peculiar aspects that proves that this film definitely outshines the majority of late 80's horror films. First and foremost, the names involved in the production. Sam Raimi (by then very famous already for his "Evil Dead" films) and Lawrence Bender helped realizing "Intruder" and, even though the budget possibilities were still very limited, their presence is an added value. Secondly, there's the truly ingenious camera-work! The film takes entirely place in a familiar setting ? a local supermarket like every town has one ? but all the different and innovating camera angles add an unexpected amount creativity and playfulness. But the obvious element to love is, of course, the very high level of nasty and unhinged gore. One by one, all the cast members are killed in the most repulsive ways imaginable, using typical supermarket-attributes like meat hooks and garbage chutes. The murders are graphically shown, so this film isn't for the faint-hearted, but there's also a large amount of pitch-black humour to enjoy, as well as shockingly unhappy ending! "Intruder" is a must-see for all you sick puppies out there, just make sure you purchase an uncut version!!
Super Reviewer
July 12, 2007
Not much on plot but who cares? The film has great cinematography as well as some nasty gore effects.
|Francisco G.
Super Reviewer
November 16, 2013
A very bloody and gory slasher with amazing dry jokes and super fun mood right until the end, plagued by severe logic actions and over the top acting.
|Sylvester K
Super Reviewer
July 19, 2011
One of the best slasher films that defined the 80s
Super Reviewer
August 7, 2007
Gory, fun, and entertaining low budget slasher with come stylish direction and cool death scenes.
A must see for slasher/horror fans.
|Christopher B
Super Reviewer
February 17, 2007
Gory fun. One of my favourite slasher flicks with some great P.O.V. shots before that was all Spiegel did. Great kills, fun story and funny characters, bloody KNB effects, with the Raimis and Campbell popping up to add to it's awesomeness. Don't be scared off by the Full Moon release, this film has nothing to do with Charles Band, but has everything to do with kicking ass.
|Anthony A
January 5, 2014
This 80's slasher movie is quite inventive. Some of the old-school physical gore effects were great and some just did not stand up.
October 10, 2013
Both Bruce Campbell and Sam Raimi appear in this low budget slasher flick. It takes place after closing at a supermarket while the crew is stocking and cleaning up. Some cool camera angles ala Evil Dead (well, Ted Raimi, Scott Spiegel and Danny Hicks were also part of this one).
|Bill B
January 24, 2012
This film is one of those notorious films that I've always heard about in association with the Evil Dead films, and having finally checked it out I can say I was amused by the gore, as well as how much you can see the style that was passed around between Raimi and hos cronies, as the camera hits so many POV shots that it becomes slightly distracting, which I found a bit off-putting to be perfectly honest.
I am happy to have seen this one finally, as it has a certain earnestness of the time that I can appreciate, which made the camera work slightly less distracting, so it's a fun little watch.
Worth a look, give it a rental.
November 30, 2010
Fun, low-budget 80's slasher flick from Scott Spiegal. Not much of a plot, but the gore effects/cinematography are top notch. Sam Raimi, Ted Raimi, and Bruce Campbell also have small cameo roles.
|Blake J
April 29, 2010
Great. Makes me want to pick up a camera and make movies again. Lots of fun with angles and simple filmmaking techniques. Almost reminds me of a sort of precursor to the Final Destination movies.
October 21, 2009
One of the most fun and gory slasher films of the 80's comes right at the end of the decade thanks to director Scott Spiegel and Producer Lawrence Bender. On display here are the talents of a fledgling KNB in the form of amazing gore effects. The fact that Danny Hicks from Evil Dead 2, Sam Raimi and Ted Raimi are in the flick (along with cameo from Bruce Campbell) certainly helps the humor element. One of my favorite B flicks of all time.
October 20, 2009
Great 80's flick. Bruce Campbell makes a cameo (brief, don't know why he's listed as the #1 actor in this movie...).


text:
'4G LTE capability & a zippy dual-core processor'
'Super-fluid performance'
'State-of-the-art everything'
'Excellent battery life'
'Features a fast processor, a lovely screen, 4G LTE'
'Remarkable compact size, thanks to the almost bezel-less display'
'Great performance for the money'
'Affordable and compact'
'Can take a bath, but not a beating'
'Dirt cheap under contract, has 4G LTE'
'Nice big screen, a large battery'
'Attractive appearance'
'Good price, Fast performance, World phone'
'Abysmal battery life, Runs older Android OS'
'Competitively priced, waterproof, and has 4G LTE capabilities'
'Comfortable five-row keyboard, NFC'
'Compact to hold and carry around'
'Comfortable, roomy hardware keyboard'
'Decent choice -- the keyboard and business features'
'Stunning detail with its 5-inch 1080p display'
'Seriously beefy specs'
'No better smartphone out there right now'
'Breathtakingly-fast processor'
'Pretty good 5-megapixel camera'
'Inexpensive, Solid performance, Good LTE speeds'
'Its decent camera includes plenty of features'
'Fast, fluid performance'
'Solid performance, Comfortable keyboard'
'Can record high-definition 1080p video'
ReviewGist helps you find the best products to buy by making sense of online reviews. We are currently aggregating 368,204 reviews covering over 29,103 products. See what the press says about ReviewGist here.
The Best Cellphone Buying Guide for 2014 is the definitive starting point for your next cellphone purchase.

subset: commoncrawl


text:
Williams Lake Stampede
This is a stampede where many people from all around the area such as: states, Vancouver, Prince George, and ect. There are so many things going on during this weekend such as fair grounds, baseball tournament, parade, and ect.
- Adventure Travel
- Casino and Gambling
- Theme Park Trips
The Williams Lake Stampede
Every year in July, Williams Lake is host to the second largest rodeo and stampede in Canada...only the Calgary Stamped is larger. The Stampede grounds are right beside the highway as you head down Hy 20 towards Bella Coola. When you're in Cariboo Country, you're in the middle of cattle and horse country.
- Road Trip
- Adventure Travel
- Horse Riding
- 1
Show Prices
Show Prices
Show Prices
Show Prices
Instant Answers: Williams Lake
Get an instant answer from local experts and frequent travelers
- Save up to 60% on hotels.
- Hotwire.com Hotwire: Get a 4-star hotel for a 2-star price.
- Save up to 50% off Hotels Everyday
- Expedia.com Photos, Reviews and the Guaranteed Lowest Prices
Sponsored Links


text:
Legal Insight
Corporate Governance
Understand the basic legal framework.Read More
The March 2015 edition of Quality Magazine includes this years Quality's 2015 Plant of the Year.
With access to over one million professionals and more than 60 industry-specific publications,Clear Seas Research offers relevant insights from those who know your industry best. Let us customize a market research solution that exceeds your marketing goals.


text:
Press release from Business Wire
'The Messenger' Has the Only Cure for Lottery Fever: Thousands of Free Mega Millions©

 Progressive® Insurance character 'saves it forward,' by giving away chances to win more than $500 million

Friday, March 30, 2012
'The Messenger' Has the Only Cure for Lottery Fever: Thousands of Free Mega Millions©08:24 EDT Friday, March 30, 2012 MAYFIELD VILLAGE, Ohio (Business Wire) -- Lottery fever has reached an even more frenzied pitch with news that The Messenger, featured in ads from Progressive auto insurance, is randomly giving away 1,937 Mega Millions© tickets to people throughout New Orleans. The 1,937 tickets will help mark Progressive's 75th anniversary, as a nod to the year the company was founded – 1937. The Messenger displays the tickets. (Photo: Business Wire) "I'm already 'saving it forward' by giving people across the country opportunities to save time and save money," said The Messenger. "Now I'm giving them a chance to live in a way most of us can only dream about. If one of those tickets holds the lucky numbers, I hope the winner also saves it forward and spreads their good fortune with others." The Messenger purchased the tickets at Esplanade Discount One on W. Esplanade Ave. "I've never sold so many lottery tickets at one time," said Shobha Movva, the store's owner. "And when he said he was giving them all away I thought he was crazy. He just told me he's 'saving it forward.' And I guess we could all use a little bit of savings." The Messenger has been in New Orleans for the past two days, taking the money he saved by switching his insurance to Progressive and saving it forward to people he meets. He's already bought locals their morning beignet, helped cover their tax payments, and has made "positive pullovers" that rewards drivers on the spot for their safe driving habits. His visit to New Orleans is part of a nationwide journey, which has included stops in Tampa, Fla., Cleveland, Colorado Springs, Colo., Phoenix, Ariz., Austin, Texas, Los Angeles, Portland, Ore., and Seattle. Along the way he's filled up tanks of gas for people, paid for their parking, covered their airport baggage fees, waited in line to get their morning coffee, gave away hard-to-get concert tickets, and much more. Hundreds of people have been touched by The Messenger's random acts of savings so far, and his journey can be followed on Facebook at facebook.com/progressivemessenger. NOTE TO EDITORS:The Messenger will be giving away lottery tickets on 3/30 at 9:30 a.m. on the corner of Decatur and Canal Street.He will be available for photographs and video at that time.About Progressive The Progressive Group of Insurance Companies makes it easy to understand, buy, and use auto insurance. Progressive offers choices so consumers can reach us whenever, wherever, and however it's most convenient for them—online at progressive.com, by phone at 1-800-PROGRESSIVE, or in-person with a local agent. Progressive offers insurance for personal and commercial autos and trucks, motorcycles, boats, recreational vehicles, and homes. It's the fourth largest auto insurer in the country, the largest seller of motorcycle insurance, and a leader in commercial auto insurance. Progressive also offers car insurance online in Australia at http://www.progressiveonline.com.au. Founded in 1937, Progressive continues its long history of offering shopping tools and services that save customers time and money, like Name Your Price®, the Snapshot Discount®, and a concierge level of claims service. The Common Shares of The Progressive Corporation, the Mayfield Village, Ohio-based holding company, trade publicly at (NYSE:PGR). Photos/Multimedia Gallery Available: http://www.businesswire.com/cgi-bin/mmg.cgi?eid=50223557&lang=en Progressive PRSteve Kaczynski, firstname.lastname@example.com


text:
+ - When does a cyber-attack lead to conventional-war?
Submitted by Nymz
Nymz (905908) writes "A recent wave of cyber-attacks has been directed at multiple Estonian institutions, including government ministries, political parties, news organizations, and banks. The scope of attacks suggest the entire country is the target, and with no clear solution for dealing with this type of situation, one has to wonder if an escalation to a conventional-attack or war is far off."


text:
hedges side tableSKU: #H5220
Our hand-cast aluminum hedges collection recreates the look of classic English cast iron without the weight. Black powder-coated enamel provides a rust-free, UV-resistant finish that is extremely durable. Chair and sofa backs feature a Union Jack criss-cross motif with a center medallion. This British flag pattern is repeated in all the table tops.
Quick ship cushions available in Sunbrella® ginkgo or dupione bamboo or choose from made-to-order Sunbrella® and other premium performance fabrics.
Signature or premium cushion sales are final and ship in 2-3 weeks.
- item
- H5220 hedges side table
- shipping
- standard and 2-DAY shipping options available at checkout
- overall
- 24w 24d 21h
- weight
- 27 lbs
- status
- in stock This item typically leaves the warehouse within 1-2 days. Expedited or white glove delivery options may be available at checkout.
- collection
- hedges
- assembly
- some assembly required view PDF
- materials
- powder-coated aluminum


text:
PKD Store
Make a Gift
Read Our Blog
Sign In
Fundraise Your Way: Take the fight against polycystic kidney disease into your own hands!
Are you passionate about finding treatments and a cure for PKD? This winter, take the fight against this disease into your own hands by organizing a local fundraiser. You can do just about anything to raise dollars for PKD. Here are some ideas to get you started:
Organize a Polar Bear Plunge, like Sean.Host a chili cookoff.Shovel driveways for donations.Sell hot chocolate at your workplace.Make homemade scarfs and sell them for donations.
Get started today with these 3 easy steps:
Help end PKD. The research, advocacy, education, support and awareness the PKD Foundation offers would not be possible without you.
Create your fundraiser today!
Questions? Email us at email@example.com.
2000 Daniel Island Drive, Charleston SC 29492
Phone: 800.443.9441 | Fax: firstname.lastname@example.com
©2015, PKD Foundation ·The PKD Foundation is a 501 (c)(3), 509 (a)(1) public charity.


text:
Thanks for redeeming.{{else}}
Success! Thanks for repurchasing.{{/if}} {{else}} {{#if Expired}}
Increase my visibility!{{else}} {{#if IsMultiPack}}
{{#if HasMultiPack}} You have {{MultiPackCount}} top spot(s) left. Now loading you into the search listings... {{else}} You have used all top spots. Now loading you into the search listings... {{/if}}{{else}}
We are loading you into the search listing pages...{{/if}} {{/if}} {{/if}} {{/if}}
{{Current}} just saw you!{{/if}} {{#if LastTopSpotPurchaseIsMulti }} {{#if HasAnyTopSpot}}
{{#if HasFreeTopSpot }} {{ UnclaimedTopSpotFree }} Free {{/if}} {{#if HasPaidTopSpot }} {{#if HasFreeTopSpot }} & {{/if}} {{ MultiPackCount }} {{/if}} Top Spot{{#if Plural}}s{{/if}} Left{{else}}
0 Top Spot{{#if Plural}}s{{/if}} Left{{/if}} {{else}} {{#if HasFreeTopSpot }}
{{ UnclaimedTopSpotFree }} Top Spot{{#if Plural}}s{{/if}} Left{{/if}} {{/if}} {{else}} {{#if HasResults}}
{{Current}} see you now!{{else}} {{/if}} {{/if}}
You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
29 year old man | Saint Louis, MO, USA | Seeking women 24-29 within 20 miles
I do say, Buzz Killington is one dandy fellow!
Some of my friends have described me as a hipster given the fancy hat and awesome glasses. Now before you get all preachy, the only thing that I admit before it was cool was biting into a piece of pizza and burning my mouth in the process. I'm a senior software engineer and consultant for one of the more well known consulting firm in the city. Call me a code monkey if you will, but I do a lot more than code! I'm constantly trying to better myself by learning new things and seeking new experiences. I'm a glutton for education. The coursework for my second Masters degree has almost come to an end and the dawn of the third will soon rise. Activities for the spending of free time include but are not limited to: tai chi (fan and sword), riding around on the motorbike, improving my repitoire of karaoke tracks, trying out new clubs, and hangin' with Mr. Cooper.
Camping , Business networking , Movies/Videos , Music and concerts , Exploring new areas , Nightclubs/Dancing , Performing arts , Playing cards , Playing sports , Political interests , Video games , Volunteering
Aerobics, Baseball, Billiards / Pool, Bowling, Cycling, Dancing, Inline skating, Martial arts, Other types of exercise, Skiing, Soccer, Swimming, Weights / Machines, Yoga
I exercise 3-4 times per week.
I have Other
I'd definitely like a Husky or Welsh Corgi when I finally get around to buying a bigger house.
Middle of the Road
The Loop and Main Street are my favorite night spots currently. I haven't had too much exposure to South City, CWE, or the Landing despite working downtown. I also like to visit Newtown once in a blue moon to revisit Beatnik's (awesome microbrew).
University of Missouri–Columbia, Columbia, MO
Music: deadmau5, Electric Six, They Might Be Giants, Gorillaz, Paul Van Dyk, MYLO, The Killers, Apoptygma Berserk, Weird Al, Fleetwood Mac, Beatles, etc TV Shows: Big Bang Theory, Dexter, Breaking Bad, Doctor Who, Torchwood, Phineas and Ferb etc
By the time I get off work it's time for tai chi or kung fu. I also try do the best a white guy can when trying out new dance clubs. How has anyone not caned me off the dance floor yet? I also like to get around on my "Batman lookin'" motortrike.
Honestly I'm not much of a reader but the last thing I re-read was House of Leaves which could be best described as an awesome horror romance novel. Other books I enjoy are anything by Kurt Vonnegut, Piers Anthony, Asimov, or Orson Scott Card
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|6'1" (185cms)
|3'0" (91cms) to 8'11" (271cms)
|Body type:
|About average
|Slender, About average, Athletic and toned
|Black, Blue, Brown, Grey, Green, Hazel
|Dark brown
|No preference
|No Way
|No Way, Occasionally, Yes, but trying to quit
|Social Drinker
|Social Drinker, Moderately
|Technical / Science / Computers / Engineering
|No preference
I'm a software engineer that spends most of his time eating Fritos and drinking Tab and Mountain Dew because code monkey very simple man with warm fuzzy secret heart..... (code monkey like you...) Jon Coulton anyone?
|$100,001 to $150,000
|No preference
|Never Married
|No preference
|Have kids:
|Wants kids:
|Definitely, Someday, Not sure
|White / Caucasian
|Asian, Black / African descent, East Indian, Latino / Hispanic, Middle Eastern, Native American, Pacific Islander, White / Caucasian, Other
Throw in some Scottish, a little bit of Polish, a dash of German and Cherokee and you get... well I guess you get my bloodline.
|Spiritual but not religious
|No preference
I believe in certain forces that govern this universe. Thus, I don't subscribe to any organized religion though I observe parts in many of them in my day to day life. I respect others' beliefs but don't fancy those who blindly follow them.
|Chinese, English, Japanese
|Graduate degree
|Bachelors degree, Graduate degree, PhD / Post Doctoral
I have a Bachelors in Information Technology and Computer Science and two Masters in Business Administration and Information Technology Management.


text:
First Citizens Bank and Trust Company, Inc. v. Island Time, LLC et al
|Jacqueline F. Jenkins, Island Time, LLC and Julian C. Bradham, Jr.
|First Citizens Bank and Trust Company, Inc.
|Case Number:
|April 17, 2013
|Georgia Northern District Court
|XX US, Outside State
|Presiding Judge:
|Charles A. Pannell
|Nature of Suit:
|Negotiable Instrument
Access additional case information on PACER
Use the links below to access additional information about this case on the US Court's PACER system. A subscription to PACER is required.
Access this case on the Georgia Northern District Court's Electronic Court Filings (ECF) System
- Search for Party Aliases
- Associated Cases
- Attorneys
- Case File Location
- Case Summary
- Docket Report
- History/Documents
- Parties
- Related Transactions
- Check Status


text:
FIRST LETTER TO THE CORINTHIANS Background Paul established a Christian community in Corinth about the year 51, on his second missionary journey. Corinth, a commercial crossroads, was a cosmopolitan city full of devotees of various pagan cults and marked by a measure of moral depravity not unusual in a great seaport. The Acts of the Apostles suggests that moderate success attended Paul's efforts among the Jews in Corinth at first, but that they soon turned against him (Acts 18:1-8). More fruitful was his year and a half spent among the Gentiles (Acts 18:11), which brought to the faith many among the poor and the underprivileged (1 Cor 1:26). Occasion and Purpose While Paul was in Ephesus on his third journey (1 Cor 16:8; Acts 19:1-20), he received information about the situation in Corinth. The community was displaying open factionalism, as certain members were identifying themselves exclusively with individual Christian leaders and interpreting Christian teaching as a superior wisdom for the initiated few (1 Cor 1:10-4:21). The community lacked the firmness to take appropriate action against one of its members who was living publicly in an incestuous union (1 Cor 5:1-13). Other members engaged in legal conflicts in pagan courts of law (1 Cor 6:1-11); still others may have participated in religious prostitution (1 Cor 6:12-20) or temple sacrifices (1 Cor 10:14-22). The community's behaviour was reflected in its liturgy. In the celebration of the Eucharist certain members discriminated against others and denied Christian social courtesies to the poor among the membership (1 Cor 11:17-22). Charisms such as ecstatic prayer, attributed freely to the Holy Spirit, were more highly prized than works of charity (1 Cor 13:1-2, 8), and were used at times in a disorderly way (1 Cor 14:1-40). Still other problems concerned matters of conscience: the eating of meat that had been sacrificed to idols (1 Cor 8:1-13), the use of sex in marriage (1 Cor 7:1-7), and the attitude to be taken by the unmarried toward marriage in view of the possible proximity of Christ's second coming (1 Cor 7:25-40). There was also a doctrinal matter: some members of the community, despite their belief in the resurrection of Christ, were denying the possibility of the resurrection of the body. All these problems of which they wanted Paul's advice were brought to his attention. It was against this background that Paul writes to the community with confidence in the authority of his apostolic mission, and he presumes that the Corinthians, despite their deficiencies, will recognise and accept it. On the other hand, he does not hesitate to exercise his authority as his judgment dictates in each situation. The letter illustrates well the mind and character of Paul. Although he is forced to insist on his office as founder of the community, he affirms that he is only one servant of God among many (1 Cor 3:5-8). He provides us in this letter with many valuable examples of his method of theological reflection and exposition. Certain passages of the letter are of the greatest importance for the understanding of early Christian teaching on the Eucharist (1 Cor 10:14-22; 11:17-34) and on the resurrection of the body (1 Cor 15:1-58). Authorship and Date To treat this wide spectrum of questions, Paul wrote this letter from Ephesus about the year 56. Paul's authorship of 1 Corinthians, apart from a few verses that some scholars regard as later interpolations, has never been doubted. Some scholars have proposed, however, that the letter as we have it contains parts of more than one original Pauline letter. We know that Paul wrote at least two other letters to Corinth (see 1 Cor 5:9; 2 Cor 2:3-4) in addition to the two that we now have; scholars believe that the additional letters are actually contained within the two canonical ones. Most commentators, however, favour the unity of the letter and treat it as a single coherent work. Structure of the First Letter to the Corinthians 1. Greetings and Thanksgiving (1 Cor 1:1-9) 2. Dissensions in the Corinthian Community (1 Cor 1:10-6:20) - Divisions in the Church (1 Cor 1:10-4:21) - Moral Disorders (1 Cor 5:1-6:20) 3. Answers to the Corinthians' Questions (1 Cor 7:1-11: 1) - Marriage and Celibacy (1 Cor 7:1-40) - Offerings to Idols (1 Cor 8:1-11:1) 4. Problems in Liturgical Assemblies (1 Cor 11:2-14:40) - Women's Headdresses (1 Cor 11:3-16) - The Lord's Supper (1 Cor 11:17-34) - Spiritual Gifts (1 Cor 12:1-14:40) 5. The Resurrection (1 Cor 15:1-58) - The faith of the Church (1 Cor 15:1-11) - The Resurrection of the Dead (1 Cor 15:12-34) - The transformation at the Resurrection (1 Cor 15:35-58) 6. Conclusion (1 Cor 16:1-24) Themes of the Letter Called to Be Saints The basic message of the First Letter to the Corinthians is already announced in the opening greetings in which there is the emphasis on the holiness of life. The Corinthians are to understand that in virtue of their baptism they were sanctified and "set aside" to be holy and to live their lives in Christ. Because of this common call to baptism there is a unity among them and all who "call on the name of Our Lord Jesus Christ". You are God's People Paul uses the image of "the field" to challenge the Corinthians to depend on God and be at its disposal. Then Paul uses another metaphor, "the building" to underline that the building belongs to God who has constructed it according to His plan and has gifted it with gifts needed for His purposes. Then Paul introduces another metaphor of "the temple" to remind the Corinthian community and each Christian that we are the place of God's presence, set apart as something holy and called to be a sign of God and to reflect in their way of living this great privilege. Having said all this Paul deals with a moral aberration among the Corinthian community according to the information he has received. He instructs them on incest, law-suits in pagan courts and adultery and prostitution. Marriage and Celibacy Paul answers to the questions put forward to him by the Christians of Corinth concerning sexual relations in marriage. Paul manifests his preference for the celibate state, but does not agree with the tendency of some Christians in Corinth who were escaping from marriage commitments already made with the excuse of their baptism and the pretext of asceticism, sustaining that marriage had no place in the eschatological kingdom. With this attitude there was the danger of depriving marriage of its value. Paul accepts a temporary abstention from sexual love in marriage provide it is the result of a mutual agreement and for only a period of time for the purpose of having a closer relationship with God. Regarding to each state of life Paul considers it as a vocation from God: "Only, everyone should live as the Lord has assigned, just as God called each one... Everyone should continue before God in the state in which he was called." (1 Cor 7:17,24) The Lord's Supper and the Gifts of the Spirit At the time of Paul the Eucharistic celebrations took place in someone's home. Christians used to bring food and shared it with those taking part in the Eucharistic meal. Some rich members were sharing food with those of their social status leaving out the poorer members. Paul reproaches such divisions among Christians who come together to share Christ's body and blood and makes them realise that with such behaviour they divide the body of Christ and are therefore not living in holiness. Then Paul speaks about the gifts of the Spirit. There is a "variety of gifts, but the same Spirit" (1Cor 12). As they are all members of the body of Christ, each person is gifted by God for the edification of the community. Paul mentions several gifts of the Spirit but stresses that the greatest among them is love (1 Cor 13). All the other gifts are authentic only if they are motivated and accompanied by love. The Resurrection Some of the Christian Corinthians denied the resurrection of the dead. Paul, referring to this denial, instructs the Corinthians about the resurrection. He first reminds them of the paschal mystery: death, burial and resurrection of Christ as the foundation of our faith. Then Paul deals with the implication that Christ's resurrection has for the Christians. Christ was raised from the dead to a new life so all those who share in Christ's life will be raised to life at the end of time. Missionary Method of Paul Paul's first letter to the church of Corinth provides us with a clear picture of the life of the Christian community of the first generation. Through it we can have a glimpse both of the strengths and of the weaknesses of this small group who had accepted the good news of Christ and were now trying to realise in their lives the implications of their baptism. Paul, who had founded the community and continued to look after it as a father, responds both to questions addressed to him and to situations of which he had been informed. In doing so, he reveals much about himself, his teaching, and the way in which he conducted his missionary task as apostle. _____________________________________________ COLUMN Paul in his own words The hymn on love "If I speak in human and angelic tongues but do not have love, I am a resounding gong or a clashing cymbal. And if I have the gift of prophecy and comprehend all mysteries and all knowledge; if I have all faith so as to move mountains but do not have love, I am nothing. If I give away everything I own, and if I hand my body over so that I may boast but do not have love, I gain nothing. Love is patient, love is kind. It is not jealous, (love) is not pompous, it is not inflated, it is not rude, it does not seek its own interests, it is not quick-tempered, it does not brood over injury, it does not rejoice over wrongdoing but rejoices with the truth. It bears all things, believes all things, hopes all things, endures all things. Love never fails". 1 Cor 13:1-8
Pages to are hidden for
"THE LETTER TO THE PHILIPPIANS"Please download to view full document


text:
Disclosed is a method of inhibiting a neoplasm in a mammal, the method includes administering to the mammal a therapeutically effective amount of recombinant human alpha-fetoprotein.
1. A method of inhibiting a neoplasm in a mammal, said method comprising administering to said mammal a therapeutically effective amount of a compound consisting essentially of recombinant human alpha-fetoprotein (SEQ ID NO:2) or an anti-neoplasm fragment thereof selected from the group comprising at least one of Domain I residues 1 to 197 (SEQ ID NO:3), Domain II residues 198 to 389 (SEQ ID NO:4), Domain III residues 390 to 590 (SEQ ID NO:5), Domain I+11 residues 1 to 389 (SEQ ID NO:6), Domain II+III residues 198 to 590 (SEQ ID NO:7), and rHuAFP Fragment I residues 266 to 590 (SEQ ID NO:8).
2. The method of
3. The method of
4. The method of
5. The method of
6. The method of
7. The method of
8. The method of
9. The method of
10. The method of
11. The method of
12. The method of
13. The method of
14. The method of
15. The method of
[0001] This invention relates to cancer therapeutics and diagnostic methods.
[0002] Cancer is one of the most common of all human diseases, resulting in over 500,000 deaths annually in the United States alone. Cancer typically is detected by the physician as an abnormal growth, or tumor, which causes illness by production of biochemically active molecules, by local expansion, or by invasion into neighboring or outlying tissue sites. The symptoms of the illness depend upon the specific molecular product(s) of the cancer. Thus, each type of cancer has a characteristic developmental history that describes the likely clinical course of the particular neoplastic process. For example, it is known that breast cancer spreads most frequently to the lungs, liver, bone, and brain.
[0003] Early detection and diagnosis of cancer is often necessary for devising an optimal treatment plan for a patient. By determining the presence of early metastatic disease, treatment can often be designed which increases the chance for cure, or delay the development of symptoms if a cure is not achievable. Radiographic imaging is one such procedure widely used for the detection and diagnosis of various disease states, including cancer. For example, radioactive tracers or imaging agents are used for imaging studies to detect sites of human disease. Such tracer molecules are designed to concentrate at a target and define the extent of a tumor or other disease state. Isotopes coupled to monoclonal antibodies, for example, are of clinical interest as applied to cancer screening. Imaging agents are most effective if they show a high specific localization at the target site, i.e., a high target-to-background contrast. The contrast produced by an imaging agent, e.g., a labelled monoclonal antibody, is largely determined by its biodistribution in vivo. Accordingly, to improve the ability to detect abnormalities such as cancer, the development of imaging agents specifically targeted to cancerous cells is considered essential.
[0004] As for cancer treatment, although current therapies such as surgery, biologic therapies, radiotherapies, and chemotherapies have saved and improved countless lives, they remain imperfect solutions. Indeed, a major clinical problem is that many cancers remain unresponsive to these therapies. For example, the capacity to cure disseminated cancer is dependent on combination chemotherapy, alone or together with biologic therapy, surgery, and/or radiotherapy. Moreover, many cancer cells have been found to develop resistance to many anti-cancer drugs attenuating their therapeutic effectiveness. Accordingly, the search has begun for new anti-cancer compounds which can interact with oncogene products, gene regulators, and growth factors and their receptors. Research employing the tools of molecular biology promises to provide a new array of anti-cancer agents.
[0005] In general, the invention features compositions and methods for the protection, treatment, and diagnosis of neoplasia, in particular, cancer. The invention is based on my discovery that unglycosylated recombinant human alpha-fetoprotein made in a prokaryote (e.g., E. coli) is useful for treating and diagnosing mammals with neoplasms, especially malignant tumors, such as breast or prostate carcinomas, and other carcinomas caused by a proliferation of malignant cells which express receptors which are recognized by recombinant human alpha-fetoprotein.
[0006] In one aspect, the invention features a method of inhibiting a neoplasm in a mammal (e.g., a human patient), involving administering to the mammal a therapeutically effective amount of recombinant human alpha-fetoprotein or an anti-neoplasm fragment or analog thereof. Preferably, the neoplasm is a malignant tumor (e.g., a breast tumor or a prostate tumor); and the recombinant human alpha-fetoprotein is produced in a prokaryotic cell (e.g., E. coli) and is unglycosylated. In preferred embodiments, the cells of the neoplasm express a receptor which is recognized by the recombinant human alpha-fetoprotein. Such a neoplasm is generally a carcinoma such as an adenocarcinoma or a sarcoma. In preferred embodiments, the neoplasm proliferates in response to a hormone, e.g, estrogen or androgen. Preferably, administration of recombinant human alpha-fetoprotein inhibits the proliferation of cells of the neoplasm or, alternatively, kills cells of the neoplasm in the mammal. The method further includes administering to the mammal a chemotherapeutic agent.
[0007] In another aspect, the invention features a method of protecting a mammal from developing a neoplasm, involving administering to the mammal a therapeutically effective amount of recombinant human alpha-fetoprotein. Preferably, the recombinant human alpha-fetoprotein is produced in a prokaryotic cell (e.g., E. coli) and is unglycosylated.
[0008] In another aspect, the invention features a hybrid cytotoxin including a recombinant human alpha-fetoprotein (or a fragment or analog thereof) linked to a cytotoxic agent. Examples of such cytotoxic agents include, without limitation, diphtheria toxin, Pseudomonas exotoxin A; ricin and other plant toxins such as abrin, modeccin, volkensin, viscumin; chlorea toxin (produced by Vibrio cholerae bacteria); the so-called �Shiga-like� toxins (produced by E. coli and other enteric bacteria); Salmonella heat-labile enterotoxin; and E. coli heat-labile enterotoxin. In other preferred embodiments, the cytotoxic agent is non-proteinaceous. Examples of such non-proteinaceous cytotoxic agents include, without limitation, anti-cancer agents such as doxorubicin, as well as α-emitting radionuclides such as astatine and β-emitting nuclides such as yttrium. Preferably, the cytotoxic agent of the hybrid cytotoxin is linked by a peptide bond to the recombinant human alpha-fetoprotein, and the hybrid toxin is produced by expression of a genetically engineered hybrid DNA molecule. In other preferred embodiments, the cytotoxic agent of the hybrid cytotoxin is a protein; such a cytotoxic agent is chemically conjugated to the recombinant human alpha-fetoprotein.
[0009] In other aspects, the invention features a detectably-labelled recombinant human alpha-fetoprotein or a detectably-labelled fragment or analog thereof capable of binding to a human neoplastic cell. Preferably such a molecule is labelled with a radionuclide, e.g., technetium-99m, iodine-125, iodine-131, or indium. Other detectable labels include, without limitation, enzymes, fluorophores, or other moieties or compounds which emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate or, alternatively, the detectable signal can be an epitope recognized by an antibody (e.g., an epitope of alpha-fetoprotein or an epitope which is specifically engineered into the recombinant alpha-fetoprotein such as the HA or myc epitopes). Preferably, the molecule targets a malignant tumor (e.g. a breast tumor, a prostate tumor, or a carcinoma) which express a receptor which is recognized by the recombinant alpha-fetoprotein (or fragment or analog thereof). Typically, such recombinant alpha-fetoprotein is produced in a prokaryotic cell (e.g., E. coli) and is unglycosylated.
[0010] Detectably-labelled recombinant human alpha-fetoprotein is useful for methods of imaging a neoplastic cell-containing region in a human patient in vivo. In general, the method involves: (a) providing a detectably-labelled molecule of recombinant human alpha-fetoprotein (or a fragment or analog thereof); (b) administering the molecule to the patient; (c) allowing the labelled molecule to bind and allowing unbound molecule to be cleared from the region; and (d) obtaining an image of the neoplastic cell-containing region. Preferably, the region is the breast or is the prostate. In other preferred embodiments, the region, without limitation, is liver tissue, is lung tissue, is spleen tissue, is pancreatic tissue, is brain tissue, is lymph tissue, or is bone marrow. Preferably, the image is obtained using dynamic gamma scintigraphy.
[0011] Detectably-labelled recombinant human alpha-fetoprotein (or fragment or analog thereof) can also be used in a method for diagnosing a neoplasm in a mammal (e.g., a human patient). Such a method includes: (a) contacting the biological sample with the detectably-labelled molecule of recombinant human alpha-fetoprotein; and (b) detecting the label bound to the sample, where the detection of label above background levels is indicative that the patient has a neoplasm. Preferably, the method involves a biological sample including cells fixed and sectioned prior to the contacting step, and the label bound to the sample is bound to areas corresponding to the cell membrane of the cells. In preferred embodiments, the biological sample is from the breast or prostate of a human patient.
[0012] Detectably-labelled recombinant human alpha-fetoprotein (or fragment or analog thereof) can also be used in a method for detecting a neoplasm a mammal in vivo. Such a method includes: (a) administering a diagnostically effective amount of the detectably-labelled molecule of recombinant human alpha-fetoprotein; and (c) detecting the presence of the detectable label bound to a tissue of the mammal, where an amount of label above background levels is indicative of the presence of the neoplasm in the mammal. In preferred embodiments, the method involves a human patient suspected of having a breast cancer, and the tissue is breast tissue. In other preferred embodiments, the method involves a human patient suspected of having a prostate cancer, and the tissue is prostate tissue. Preferably, the detectably labelled recombinant human alpha-fetoprotein is linked to a radionuclide (e.g., technetium-90) and the detection step is accomplished by radioimaging (e.g., dynamic gamma scintigraphy).
[0013] In another aspect, the invention features kits for detecting a neoplasm or any cell expressing a receptor which is recognized by recombinant human alpha-fetoprotein (or a fragment or analog thereof) in vivo, in situ or in vitro. In general, the kits include a recombinant human alpha-fetoprotein which is recognized by a neoplasm, and which may be detectably labeled. If the recombinant human alpha-fetoprotein is unlabelled, a second reagent containing a detectable label (e.g. a radionuclide such as technetium-90, iodine-125, iodine-131, or indium) is preferably included. Where the detectable label is an enzyme, the kit further includes a substrate reagent for the enzyme. The kit may also include a reagent for linking the detectable label to the recombinant alpha-fetoprotein. In another embodiment, the kit for detecting a neoplasm or any unwanted cell expressing a receptor which is recognized by recombinant human alpha-fetoprotein (or a fragment or analog thereof) includes a reagent containing an antibody which specifically binds the recombinant human alpha-fetoprotein and a reagent including a detectably labeled recombinant human alpha-fetoprotein that is specifically bound by the anti-alpha-fetoprotein antibody. Preferably, the recombinant human alpha-fetoprotein of the kit is produced in a prokaryotic cell (E. coli) and is unglycosylated.
[0014] By �neoplasm� is meant any unwanted growth of cells serving no physiological function. In general, a cell of a neoplasm has been released from its normal cell division control, i.e., a cell whose growth is not regulated by the ordinary biochemical and physical influences in the cellular environment. In most cases, a neoplastic cell proliferates to form a clone of cells which are either benign or malignant. Examples of neoplasms include, without limitation, transformed and immortalized cells, tumors, and carcinomas such as breast cell carcinomas and prostate carcinomas.
[0015] By �therapeutically effective amount� is meant a dose of unglycosylated recombinant human alpha-fetoprotein or an anti-neoplasm fragment or analog thereof capable of inhibiting the proliferation of a neoplasm.
[0016] By �diagnostically effective amount� is meant a dose of detectably-labelled recombinant human alpha-fetoprotein or a detectably-labelled fragment or analog thereof that can be detected within a targeted region in a mammal (e.g., a human patient).
[0017] By �recombinant human alpha-fetoprotein� is meant a polypeptide having substantially the same amino acid sequence as the protein encoded by the human alpha-fetoprotein gene as described by Morinaga et al., Proc. Natl. Acad. Sci., USA 80: 4604 (1983). The method of producing recombinant human alpha-fetoprotein in a prokaryotic cell is described in U.S. Ser. No. 08/133,773 to issue as U.S. Pat. No. 5,384,250.
[0018] The use of recombinant human alpha-fetoprotein for the treatment and diagnosis of cancer offers a number of advantages. For example, rHuAFP can be administered directly to a tumor site. Recombinant HuAFP can also be chemically defined and synthesized, and prepared in large quantities using the techniques of recombinant DNA. Moreover, unlike conventional cancer chemotherapies and radiotherapies, recombinant human alpha-fetoprotein causes minimal side effects such as nausea, vomiting, and neurotoxicity. Accordingly, relatively high doses of rHuAFP can be safely administered.
[0019] The diagnostic methods of the invention are advantageous since in that they allow for rapid and convenient diagnosis of a neoplasm. For example, the use of rHuAFP as a diagnostic agent (e.g., by radioimaging using scintigraphy) is especially advantageous for real time imaging of cancer in both pre-surgical or intraoperative localization and staging of a cancer, e.g., breast cancer, as well as during post-surgical examinations. Using such diagnostic procedures permits non-invasive determination of the presence, location, or absence of a neoplasm which is advantageous for monitoring the condition of a patient. other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
[0020] The drawings will first be described.
[0021]FIG. 1 is the nucleotide sequence (SEQ ID NO:______) and deduced amino acid sequence (SEQ ID NO:______) of the cDNA encoding human alpha-fetoprotein.
[0022]FIG. 2 is the SDS-PAGE analysis of rHuAFP Fragment I (SEQ ID NO:______) (Lane A, MW marker; Lane B, natural human alpha-fetoprotein (AFP); Lane C, unpurified rHuAFP; Lane D, rHuAFP Fragment I, and Lane E, rHuAFP (amino acids 1-590 of FIG. 1, SEQ ID NO:______).
[0023]FIG. 3 is a bar graph showing the effect of rHuAFP on estrogen-stimulated post-confluent growth of MCF-7 breast cancer cells.
[0024] There now follows a detailed description of methods and compounds of the invention.
[0025] As summarized above, the invention provides compositions for therapies and diagnostic methods for the prevention and treatment of a neoplasm, involving the use of rHuAFP or anti-neoplasm fragments or analogs thereof. Methods for producing such rHuAFP in a prokaryotic cell are described in U.S. Ser. No. 08/133,773 to issue as U.S. Pat. No. 5,384,250. The invention further provides such rHuAFP (or a fragment or analog thereof) linked to a toxin or a detectable label for use as anti-cancer or diagnostic agents, respectively. Production of such fragments and analogs of rHuAFP, as well as anti-cancer and diagnostic agents of rHuAFP, will now be discussed in greater detail.
[0026] The invention includes biologically active fragments of rHuAFP. A biologically active fragment of rHuAFP is one that possesses at least one of the following activities: (a) directs a specific interaction with a target cell, e.g., binds to a cell expressing a receptor which is recognized by rHuAFP (e.g., the membrane of a cancer cell such as an MCF-7); or (b) halts, reduces, or inhibits the growth of a neoplasm (e.g., binds to a cell surface receptor and imparts an anti-proliferative signal). The ability of rHuAFP fragments or analogs to bind to a receptor which is recognized rHuAFP can be tested using any standard binding assay known in the art.
[0027] Accordingly, a rHuAFP fragment, like the full-length rHuAFP molecule, can be used as a targeting agent for a toxin (for therapy) or a detectable compound (for diagnosis). Recombinant HuAFP fragments that effectively target a toxin or act as detectable compounds to label a cell expressing a receptor which is recognized by rHuAFP or a fragment or analog thereof are useful in the invention.
[0028] In general, fragments of rHuAFP are produced according to the techniques of polypeptide expression and purification described in U.S. Ser. No. 08/133,773 (U.S. Pat. No. 5,384,250). For example, suitable fragments of rHuAFP can be produced by transformation of a suitable host bacterial cell with part of an HuAFP-encoding cDNA fragment (e.g., the cDNA described above) in a suitable expression vehicle. Alternatively, such fragments can be generated by standard techniques of PCR and cloned into the expression vectors (supra). Accordingly, once a fragment of rHuAFP is expressed, it may be isolated by various chromatographic and/or immunological methods known in the art. Lysis and fractionation of rHuAFP-containing cells prior to affinity chromatography may be performed by standard methods. Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry And Molecular Biology, Work and Burdon, eds., Elsevier, 1980).
[0029] A rHuAFP fragment may also be expressed as a fusion protein with maltose binding protein produced in E. coli. Using the maltose binding protein fusion and purification system (New England Biolabs, Beverly, Mass.), the cloned human cDNA sequence can be inserted downstream and in frame of the gene encoding maltose binding protein (malE), and the malE fusion protein can then be overexpressed. In the absence of convenient restriction sites in the human cDNA sequence, PCR can be used to introduce restriction sites compatible with the vector at the 5′ and 3′ end of the cDNA fragment to facilitate insertion of the cDNA fragment into the vector.
[0030] Following expression of the fusion protein, it can be purified by affinity chromatography. For example, the fusion protein can be purified by virtue of the ability of the maltose binding protein portion of the fusion protein to bind to amylose immobilized on a column.
[0031] To facilitate protein purification, the pMalE plasmid contains a factor Xa cleavage site upstream of the site into which the cDNA is inserted into the vector. Thus, the fusion protein purified as described above can then be cleaved with factor Xa to separate the maltose binding protein from a fragment of the recombinant human cDNA gene product. The cleavage products can be subjected to further chromatography to purify rHuAFP from the maltose binding protein. Alternatively, a fragment of rHuAFP may be expressed as a fusion protein containing a polyhistidine tag can be produced. Such an alpha-fetoprotein fusion protein may then be isolated by binding of the polyhistidine tag to an affinity column having a nickel moiety which binds the polyhistidine region with high affinity. The fusion protein may then be eluted by shifting the pH within the affinity column. The rHuAFP can be released from the polyhistidine sequences present in the resultant fusion protein by cleavage of the fusion protein with specific proteases.
[0032] Recombinant HuAFP fragment expression products (e.g., produced by any of the prokaryotic systems described in U.S. Ser. No. 08/133,773, issuing as U.S. Pat. No. 5,384,250) may be assayed by immunological procedures, such as Western blot, immunoprecipitation analysis of recombinant cell extracts, or immunofluorescence (using, e.g., the methods described in Ausubel et al., Current Protocols In Molecular Biology, Greene Publishing Associates and Wiley Interscience (John Wiley & Sons), New York, 1994).
[0033] Once a fragment of rHuAFP is expressed, it is isolated using the methods described supra. Once isolated, the fragment of rHuAFP can, if desired, be further purified by using the techniques described supra. Fragments can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, Ill.). The ability of a candidate rHuAFP fragment to exhibit a biological activity of alpha-fetoprotein is assessed by methods known to those skilled in the art (e.g., those described herein).
[0034] The purified recombinant gene product or fragment thereof can then be used to raise polyclonal or monoclonal antibodies against the human recombinant alpha-fetoprotein using well-known methods (see Coligan et al., eds., Current Protocols in Immunology, 1992, Greene Publishing Associates and Wiley-Interscience). To generate monoclonal antibodies, a mouse can be immunized with the recombinant protein, and antibody-secreting B cells isolated and immortalized with a non-secretory myeloma cell fusion partner. Hybridomas are then screened for production of recombinant human alpha-fetoprotein (or a fragment or analog thereof)-specific antibodies and cloned to obtain a homogenous cell population which produces monoclonal antibodies.
[0035] As used herein, the term �fragment,� as applied to a rHuAFP polypeptide, is preferably at least 20 contiguous amino acids, preferably at least 50 contiguous amino acids, more preferably at least 100 contiguous amino acids, and most preferably at least 200 to 400 or more contiguous amino acids in length. Fragments of rHuAFP molecules can be generated by methods known to those skilled in the art, e.g., proteolytic cleavage or expression of recombinant peptides, or may result from normal protein processing (e.g., removal of amino acids from nascent polypeptide that are not required for biological activity).
[0036] Recombinant HuAFP fragments of interest include, but are not limited to, Domain I (amino acids 1 (Thr)-197 (Ser), see FIG. 1, SEQ ID NO:______), Domain II (amino acids 198 (Ser)-389 (Ser), see FIG. 1, SEQ ID NO:______), Domain III (amino acids 390 (Gln)-590 (Val), see FIG. 1, SEQ ID NO:______), Domain I+II (amino acids 1 (Thr)-389 (Ser), see FIG. 1, SEQ ID NO:______), Domain II+III (amino acids 198 (Ser)-590 (Val), see FIG. 1, SEQ ID NO:______), and rHuAFP Fragment I (amino acids 266 (Met)-590 (Val), see FIG. 1, SEQ ID NO:______). Activity of a fragment is evaluated experimentally using conventional techniques and assays, e.g., the assays described herein.
[0037] The invention further includes analogs of full-length rHuAFP or fragments thereof. Analogs can differ from rHuAFP by amino acid sequence differences, or by modifications (e.g., post-translational modifications) which do not affect sequence, or by both. Analogs of the invention will generally exhibit at least 80%, more preferably 85%, and most preferably 90% or even 99% amino acid identity with all or part of a rHuAFP amino acid sequence. Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally occurring rHuAFP by alterations in primary sequence, for example, substitution of one amino acid for another with similar characteristics (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish the polypeptide's biological activity. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989, or Ausubel et al., supra)). Also included are cyclized peptide molecules and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g., β or γ amino acids, or L-amino acids with non-natural side chains (see e.g., Noren et al., Science 244:182, 1989). Methods for site-specific incorporation of non-natural amino acids into the protein backbone of proteins is described, e.g., in Ellman et al., Science 255:197, 1992. Also included are chemically synthesized polypeptides or peptides with modified peptide bonds (e.g., non-peptide bonds as described in U.S. Pat. No. 4,897,445 and U.S. Pat. No. 5,059,653) or modified side chains to obtain the desired pharmaceutical properties as described herein. Useful mutants and analogs are identified using conventional methods, e.g., those described herein.
[0038] The cloning, expression, isolation and characterization of exemplary rHuAFP fragments now follows. These examples are provided to illustrate, not limit, the invention.
[0039] Plasmid constructs encoding fragments of human alpha-fetoprotein were prepared using polymerase chain reaction (PCR) techniques known to those skilled in the art of molecular biology, using oligonucleotide primers designed to amplify specific portions of the human alpha-fetoprotein gene (see e.g., PCR Technology, H. A. Erlich, ed., Stockton Press, New York, 1989; PCR Protocols: A Guide to Methods and Applications, M. A. Innis, David H. Gelfand, John J. Sninsky, and Thomas J. White, eds., Academic Press, Inc., New York, 1990, and Ausubel et. al., supra).
[0040] The following six rHuAFP fragments were prepared to evaluate their biological activity (e.g., according to the methods disclosed herein):
[0041] Amino acids sequences were deduced from those shown for human alpha-fetoprotein (1(Thr)-590 (Val); SEQ ID NO:______) in FIG. 1. Fragments of rHuAFP designated Domain I, Domain II, Domain III, Domain I+II, Domain II+III and rHuAFP Fragment I were synthesized using standard PCR reaction conditions in 100 μL reactions containing 34 μL H2O, 10 μL 10X reaction buffer, 20 μL 1 mM dNTP, 2 μL DNA template (HuAFP cloned in pI18), appropriate 5′ and 3′ oligonucleotide primers (10 μL 10 pmol/μL 5′ primer, 10 μL 10 pmol/μL 3′ primer), 1 μL glycerol, 10 μL DMSO, and 1 μL Pfu polymerase (Stratagene, LaJolla, Calif.). Primers used for PCR amplifications were:
[0042] Accordingly, primer pairs DomI25 and DomI3, DomII5 and DomII3, DomIII5 and DomIII3, 5′rHuAFP Fragment I and DomIII3, DomI25 and DomII3, and DomII5 and DomIII3 were used to isolate cDNA sequences of Domain I, Domain II, Domain III, rHuAFP Fragment I, Domain I+II, and Domain II+III, respectively, of rHuAFP. Annealing, extension, and denaturation temperatures were 50� C., 72� C., and 94� C., respectively, for 30 cycles. PCR products were purified according to standard methods. Purified PCR products encoding Domain I and Domain I+II were digested individually with KpnI and BamHI and cloned separately into KpnI/BamHI-treated pTrp4. Purified PCR products encoding Domain II, Domain III, Domain II+III, and rHuAFP Fragment I were digested individually with Bsp106I and BamHI and were cloned separately into Bsp106I/BamHI-treated pTrp4. Each plasmid construct was subsequently transformed into competent E. coli cells. Since the expression product will begin with the amino acid sequence encoded by the translation start signal methionine, it is expected that such signal will be removed, or in any event, not affect the bioactivity of the ultimate expression product.
[0043]E. coli containing the expression plasmid encoding rHuAFP Fragment I was cultured and purified. FIG. 2 (lane D) shows the SDS-PAGE profile of the purified rHuAFP Fragment I. N-terminal amino acid sequence analysis showed that rHuAFP Fragment I possessed the amino acid sequence Ser267-Tyr-Ile-Cys-Ser-Gln-Gln-Asp-Thr275 (SEQ ID NO:______) which corresponds to the expected N-terminal amino acid sequence of rHuAFP Fragment I (see FIG. 1, SEQ ID NO:______) where the initiating methionine is cleaved intracellularly.
[0044] A hybrid cytotoxin of rHuAFP is prepared by conjugating a full-length rHuAFP or a fragment or analog thereof to any number of known toxic entities using conventional techniques. Such toxins are useful for inhibiting the development of a neoplasm (as described infra). Useful cytotoxins are preferably significantly cytotoxic only when present intracellularly and are substantially excluded from any given cell in the absence of a targeting domain. As described below, peptide toxins fulfill both of these criteria and are readily incorporated into hybrid molecules. If desired, a mixed cytotoxin (i.e., a cytotoxin composed of all or part of two or more toxins) can also be used. Several useful toxins are described in more detail below.
[0045] Toxin molecules useful in the method of the invention are preferably toxins, such as peptide toxins, which are significantly cytotoxic only when present intracellularly. of course, under these circumstances the molecule must be able to enter a cell bearing the targeted receptor. This ability depends on the nature of the molecule and the nature of the cell receptor. For example, cell receptors which naturally allow uptake of a ligand are likely to provide a means for a molecule which includes a toxin to enter a cell bearing that receptor. As is discussed below, the peptide toxin useful in the methods of the invention is fused to a rHuAFP (or fragment or analog thereof) binding domain by producing a recombinant DNA molecule which encodes a hybrid protein molecule.
[0046] Many peptide toxins have a generalized eukaryotic receptor binding domain; in these instances the toxin must be modified to prevent intoxication of non-receptor bearing cells. Any such modifications must be made in a manner which preserves the cytotoxic functions of the molecule (see U.S. Department of Health and Human Services, U.S. Ser. No. 401,412). Potentially useful toxins include, but are not limited to: cholera toxin, ricin, 0-Shiga-like toxin (SLT-I, SLT-II, SLT IIV), LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, Pseudomonas exotoxin, saporin, modeccin, and gelanin.
[0047] The cytotoxic portion of some molecules useful in the invention, if desired, can be provided by a mixed toxin molecule. A mixed toxin molecule is a molecule derived from two different polypeptide toxins. Generally, as discussed above, polypeptide toxins have, in addition to the domain responsible for generalized eukaryotic cell binding, an enzymatically active domain and a translocation domain. The binding and translocation domains are required for cell recognition and toxin entry respectively. The enzymatically active domain is the domain responsible for cytotoxic activity once the molecule is inside a cell.
[0048] Naturally-occurring proteins which are known to have a translocation domain include diphtheria toxin, Pseudomonas exotoxin A, and possibly other peptide toxins. The translocation domains of diphtheria toxin and Pseudomonas exotoxin A are well characterized (see, e.g., Hoch et al., Proc. Natl. Acad. Sci. USA 82:1692, 1985; Colombatti et al., J. Biol. Chem. 261:3030, 1986; and Deleers et al., FEBS Lett. 160:82, 1983), and the existence and location of such a domain in other molecules may be determined by methods such as those employed by Hwang et al. Cell 48:129, 1987); and Gray et al. Proc. Natl. Acad. Sci. USA 81:2645, 1984).
[0049] For example, one useful rHuAFP/mixed toxin hybrid molecule is formed by fusing the enzymatically active A subunit of E. coli Shiga-like toxin (see, e.g., Calderwood et al., Proc. Natl. Acad. Sci. USA 84:4364, 1987) to the translocation domain (amino acid residues 202 through 460) of diphtheria toxin, and to rHuAFP. The rHuAFP portion of the three-part hybrid causes the molecule to attach specifically to cells bearing receptors which is recognized by rHuAFP, and the diphtheria toxin translocation portion acts to insert the enzymatically active A subunit of the Shiga-like toxin into the targeted cell. The enzymatically active portion of Shiga-like toxin, like diphtheria toxin, acts on the protein synthesis machinery of the cell to prevent protein synthesis, thus killing the cell.
[0050] Functional components of the hybrid cytotoxins of the invention are linked together via a non-covalent or covalent bond, or both. Non-covalent interactions can be ionic, hydrophobic, or hydrophilic, such as interactions involved in a leucine-zipper or antibody-protein G interaction (see, e.g., Derrick et al., Nature 359:752, 1992). An example of a covalent linkage is a disulfide bond.
[0051] A hybrid cytotoxin is prepared by chemically conjugating rHuAFP (or fragment or analog) to a any number of known toxic entities, e.g., those described above. Such reactions are carried out by standard techniques known to those skilled in the art. A typical way of conjugating a protein to a protein toxin (including,. e.g., bacterial toxins such as diphtheria toxin or Pseudomonas exotoxin A, or plant toxins such as ricin) is by crosslinking through a disulfide bond (see, e.g., Chang et al., J. Biol. Chem. 252:1515, 1977) or a heterobifunctional molecule (see, e.g., Cawley et al. Cell 22:563, 1980). See also Stevens et al., U.S. Pat. No. 4,894,227.
[0052] Alternatively, the hybrid cytotoxin is prepared by expression of a hybrid DNA engineered to encode both the rHuAFP (or a fragment or analog thereof) and the toxin (or a toxic portion thereof), using technology available to those of ordinary skill in the art of making such hybrids (see, e.g., Murphy, U.S. Pat. No. 4,675,382, and Chadhary et al., Proc. Natl. Acad. Sci. USA 84:4538, 1987). For example, a recombinant fusion protein of rHuAFP and a cytotoxic agent is made according to methods known in the art (see, e.g., Murphy supra and Huston et al., Meth. Enzymol. 203:46, 1991). If the hybrid cytotoxin is produced by expression of a fused gene, a peptide bond serves as the link between the cytotoxic agent and the targeting ligand. Another method useful for conjugating a protein or polypeptide to a protein toxin employs the polymer, monomethoxy-polyethylene glycol (mPEG), as described in Maiti et al., Int. J. Cancer Suppl. 3:17, 1988.
[0053] If desired, following its synthesis, the hybrid cytotoxin is affinity purified according to standard methods using antibodies against the targeting portion of the molecule, e.g., antibodies against human alpha-fetoprotein. Similarly, antibodies directed against the cytotoxic agent are also useful for purifying the hybrid cytotoxin molecule by standard immunological techniques. The resulting hybrid cytotoxin is then formulated for use as an agent against unwanted cells, e.g. cancer cells, following procedures standard in the field of pharmacology.
[0054] Molecules of the invention can be screened for the ability to decrease viability of cells bearing the targeted receptor by means of assays known in the art, e.g., those methods described herein.
[0055] Because hybrid cytotoxins of the invention are potent cytotoxic agents for cells bearing the a receptor which is recognized by rHuAFP, rHuAFP is useful in the treatment of diseases involving unwanted alpha-fetoprotein receptor-positive cells, e.g., cancer cells.
[0056] Recombinant rHuAFP or a fragment or analog thereof can be attached to a detectable label to produce an agent useful for detecting and localizing a neoplasm in vivo, in situ, or in vitro. Methods for attaching such labels to proteins are known in the art. For example, a detectable label is attached by chemical conjugation, but where the label is a polypeptide, it could alternatively be attached by genetic engineering techniques.
[0057] Detectable labels are generally selected from a variety of such labels known in the art, but are normally radioisotopes, flurophores, enzymes (e.g., horseradish peroxidase), or other moieties or compounds which emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate. Various detectable label/substrate pairs (e.g., horseradish peroxidase/diaminobenzidine, avidin/streptavidin, luciferase/luciferin, β-galactosidase/X-gal(5-Bromo-4-Chloro-3-Indoyl-D-Galactopyranoside), and methods for labelling proteins for such detection purposes are known in the art. The usefulness of such an agent can be assayed, for example, by implanting a tumor cell line, e.g, MCF-7, into a host, e.g., a mouse, and determining whether the agent of the invention detectably labels the tumor produced by such implanted cells, e.g., by radioimaging using scintigraphy. Such an agent can also be used to assay for the presence of any unwanted cell bearing an alpha-fetoprotein receptor, e.g., by using Western blot analysis or histochemical staining of a tissue sample, according to known methods.
[0058] Anti-cancer agents of the invention (e.g., rHuAFP or a fragment or analog thereof; or a hybrid cytotoxin of rHuAFP) are useful for inhibiting a neoplasm, e.g., breast or prostate carcinomas. Those skilled in the art will understand that any number of methods, both in vitro and in vivo, are used to determine the efficacy of anti-cancer agents useful in the methods of the invention. For example, the reduction of tumor growth can be monitored in a mouse or rat growing a prostate cancer (e.g., tumor xenografts of LNCaP androgen receptor-positive human prostate cancer cell line) following the administration of the test compound. In a working example, a human tumor cell lines (e.g., cell lines such as MCF-7(ATCC HTB 22), T-47D (ATCC HTB 133), MDA-MB-231 (ATCC HTB 26), BT-20 (ATCC HTB 19), NIH:OV-CAR-3 (ATCC HTB 161), LnCaP.FGC (ATCC CRL 1740), and Du-145 (ATCC HTB 81) growing in culture is released from monolayer by trypsinization, diluted into single-cell suspension and then solidified by centrifugation into a pellet which IS subsequently exposed to 15 μl fibrinogen (50 mg/ml) and 10 μl thrombin (50 units/ml) for 30 minutes at 37� C. Fibrin clots containing tumor are then cut into pieces approximately 1.5 mm in diameter. Each piece of tumor is subsequently implanted under the kidney capsule of a mouse according to standard methods. If desired, mice can be immunosuppressed by daily subcutaneous (s.c.) injection of 60 mg/kg cyclosporine A (Sandimmune IV) beginning immediately prior to tumor implantation according to conventional methods. If necessary, estrogen and androgen supplementation of mice is achieved by standard methods, e.g, implantation of silastic tubing containing estradiol or by injection of testosterone propionate. Typically, hormone supplementation is commenced on the day of tumor implantation. Generally, administration of the test molecule is initiated prior to tumor implantation and/or after tumor implantation. Control animals receive a placebo, e.g., human serum albumin or diluent, similarly administered as for rHuAFP or related molecules. The effect of the test molecule on tumor growth is monitored according to any standard method. For example, tumor growth is monitored by weekly measurement of tumor size by laparotomy using a dissecting microscope equipped with an ocular micrometer. A molecule shown experimentally to halt or reduce or inhibit the growth of such implanted tumors is considered useful in the invention.
[0059] Toxicity of test compounds towards cells bearing receptors that are recognized by rHuAFP can be tested in vitro according to any standard protocol. For example, a cultured cancer cell line, e.g., MCF-7 estrogen-receptor-positive human breast cancer cell line, is maintained in plastic tissue culture flasks (Costar) in DMEM with penicillin (100 U/ml), streptomycin (100 μg/ml), 5% fetal calf serum, insulin (10 ng/ml), L-glutamine (2 mM) and non-essential amino acids (1%). Cells are seeded in 96-well V-bottomed plates (Linbro-Flow Laboratories, McLean, Va.) at a concentration of 1�105 per well in complete medium. Putative toxins are added to varying concentrations (10−12M to 10−6M) and the cultures are incubated for 18 hrs. at 37� C. in a 5% CO2 atmosphere. Following incubation, the plates are centrifuged for 5 min. at 170�g, and the medium removed and replaced with 100 μl leucine-free medium (MEM, Gibco) containing 8 μCi/ml (3H-leucine; New England Nuclear, Boston, Mass.). After an additional 90 min. at 37� C., the plates are centrifuged for 5 min. at 170�g, the medium is removed, and the cells are collected on glass fiber filters using a cell harvester (Skatron, Sterling, Va.). Filters are washed, dried, and counted according to standard methods. Cells cultured with medium alone serve as the control. A test compound which reduces or halts or inhibits cell growth compared to untreated control cells, is detected as an indication of toxicity and is considered useful in the invention.
[0060] Evaluation of whether a test compound confers protection against the development of a neoplasm (e.g., breast or prostate cancers) generally involves using an animal known to develop a neoplasm (e.g., the transgenic mouse described in U.S. Pat. No. 4,736,866). An appropriate animal is treated with the test compound according to standard methods, and a reduced incidence of neoplasm development, compared to untreated control animals, is detected as an indication of protection.
[0061] As is discussed below, I have discovered that unglycosylated rHuAFP produced in a prokaryotic expression system is effective in treating cancer. For example, rHuAFP has been found to be a potent inhibitor of breast carcinoma growth in vitro.
[0062] The experimental examples described below demonstrate the efficacy of rHuAFP as an anti-cancer agent. These examples are provided to illustrate, not limit, the invention.
[0063] Dulbecco's modified Eagle's medium (DMEM), RPMI 1640, fetal calf serum, glutamine, non-essential amino acids and penicillin-streptomycin mixture were obtained from GIBCO (BRL). Donor calf serum was obtained from Hyclone, Logan, Utah, and porcine insulin was obtained from Squibb, Inc., Princeton, N.J.
[0064] The MCF-7 estrogen-receptor-positive human breast cancer cell line was obtained from Dr. Alberto C. Baldi, Institute of Experimental Biology and Medicine, Buenos Aires, Argentina. Stock cultures were maintained in plastic tissue culture flasks (Costar) in DMEM with penicillin (100 U/ml), streptomycin (100 μg/ml), 5% fetal calf serum, insulin (10 ng/ml), L-glutamine (2 mM) and non-essential amino acids (1%).
[0065] Recombinant HuAFP was synthesized and purified using the methods described in U.S. Ser. No. 08/133,773 issuing as U.S. Pat. No. 5,384,250. rHuAFP can be obtained from Immtek, Inc. (Boston, Mass.).
[0066] This assay is based on the finding that MCF-7 cells in estrogen-containing medium grow past confluence and accumulate into foci; but, in the absence of estrogen, cell proliferation stops after the cultures establish cell-cell contact, and no foci are formed (see, e.g., Gierthy et al., Breast Cancer Res. Treat. 12:227, 1988). 1�107 MCF-7 breast cancer cells were seeded in 16-mm wells contained in 24-well tissue culture plates. Culture medium was phenol red-free DMEM supplemented with 5% donor calf serum (prescreened for absence of detectable estrogens), L-glutamine (2 mM), non-essential amino acids (1X, GIBCO), insulin (10 ng/ml), penicillin-streptomycin (1X, GIBCO) and estradiol diluted to a final concentration of 1.8�10−9 M. Cultures were refed at 24 hr and every 4 days thereafter with 2 ml of culture medium containing rHuAFP and human serum albumin to yield a final protein concentration of 100 μg/ml per well. Cells reached confluence within 5 days, and a substantial number of foci were apparent within 10 days in wells containing estrogen alone. Cells were fixed with buffered formalin and stained with 1% Rhodamine B. The stained foci were quantitated using an Artek 870 Macro-Micro Automated Colony Counter. Data are presented as mean number of foci per treatment group.
[0067] The results shown in FIG. 3 demonstrate that rHuAFP inhibits estrogen-stimulated postconfluent growth of MCF-7 breast cancer cells in vitro. Control experiments using human albumin or no protein had no effect on MCF-7 foci formation. These data indicate that rHuAFP has a direct inhibitory effect on the growth of carcinoma cell cultures.
[0068] As demonstrated above, rHuAFP is effective in inhibiting a neoplasm, e.g., a breast cell carcinoma. Accordingly, compounds of the invention can be formulated according to known methods to prepare pharmaceutically useful compositions. Treatment of human patients will be carried out using a therapeutically effective amount of an anti-cancer agent of rHuAFP in a physiologically acceptable carrier. Suitable carriers and their formulation are described for example in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the anti-cancer agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the type of disease, extensiveness of the disease, and size of the patient suffering from the disease. Generally amounts will be in the range of those used for other agents used in the treatment of cancer, although in certain instances lower amounts will be needed because of the increased specificity of the compound. For example, rHuAFP is administered systemically, as described below, at a dosage that inhibits malignant cell proliferation, typically in the range of 0.1 ng-10 g/kg body weight.
[0069] Furthermore, the method of the invention can also employ combination therapy in which rHuAFP is administered either simultaneously or sequentially with a chemotherapeutic agent. Typically, a chemotherapeutic agent is administered according to standard methods or, alternatively, in a dose which is lower than the standard dose when the chemotherapeutic agent is used by itself. Examples of chemotherapeutic agents include, without limitation, mechlorethamine, cyclophosphamide, ifosfamide, L-sarcolysin, chlorambucil, hexamethylmelamine, thiotepa, busulfan, carmustine, lomustine, semustine, streptozocin, dacarbazine, methotrexate, fluorouracil, cytarabine, mercaptopurine, thioguanine, pentostatin, vinblastine, vincristine, etoposide, teniposide, actinomycin D, daunomycin, doxorubicin, bleomycin, plicamycin, mitomycin, cisplatin, mitoxantrone, hydroxyurea, procarbozine, mitotane, aminoglutethimide, prednisone, hydroxyprogesterone, diethylstilbestrol, tamoxifen, flutamide, or leuprolide.
[0070] Treatment is started generally with the diagnosis or suspicion of a neoplasm and is generally repeated on a daily basis. Protection from the development of neoplasm is also achieved by administration of rHuAFP on a daily basis. If desired, the efficacy of the treatment or protection regimens is assessed with the methods of monitoring or diagnosing patients for cancer.
[0071] Furthermore, the compounds of the invention can also be used to treat mammals to destroy any unwanted cells bearing alpha-fetoprotein receptors associated with a pathological condition. The method(s) of the invention can also be used to treat non-human mammals, for example, domestic pets, or livestock. As described below, the anti-cancer agents of the invention can be administered systemically or locally.
[0072] For use as an anti-cancer agent, the compounds of the invention can be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections which provide continuous, sustained levels of the drug in the patient. In other preferred routes of administration, the compounds of the invention can be given to a patient by injection of a slow release preparation, for example, in a slowly dissociating polymeric or crystalline form; this sort of sustained administration can follow an initial delivery of the drug by more conventional routes (for example, those described above). Alternatively, the compounds can be administered using an infusion pump, thus allowing a precise degree of control over the rate of drug release, or through installation of the compounds in the nasal passages in a similar fashion to that used to promote absorption of insulin. As an alternative to nasal transmucosal absorption, the compounds can be delivered by aerosol deposition of the powder or solution into the lungs.
[0073] The anti-cancer agents of the invention also can be administered locally to treat cancer. Since the desired action of the agent is generally upon a circumscribed mass of tissue, for example a tumor, delivery of the drug by means which result in high local concentrations in the vicinity of the tumor is especially desirable.
[0074] Recombinant HuAFP (or fragment or analog thereof) linked to a detectable label finds diagnostic use in the detection or monitoring or assaying for the presence of a neoplasm (e.g., breast or prostate cancers).
[0075] For example, in vivo studies can be conducted on human patients to determine the presence of a neoplasm using a detectably labelled rHuAFP (e.g., Tc-99m-labelled rHuAFP). In general, the detectably labelled rHuAFP is administered intravenously and imaging can be performed using scanners by methods known to those skilled in the art, e.g., by radioimaging using scintigraphy.
[0076] In another working example, a neoplasm or any cell bearing a receptor which is recognized by rHuAFP may be detected in a tissue sample, e.g., a biopsy, a bodily fluid, by using rHuAFP (or fragment or analog thereof) linked to a detectable label. After determining that a patient should be tested for the presence of such cells, a tissue sample, a biopsy, or a sample of bodily fluid, preferably lymph, blood, serum, or urine, is collected from the patient. Accordingly, the subcellular location or presence of a receptor which is recognized by rHuAFP is determined either in situ or in vitro using fractionated cells by any standard biochemical or histochemical procedure (see e.g., Ausubel et al., supra; Bancroft and Stevens, Theory and Practice of Histological Techniques, Churchill Livingstone, 1982). Appropriate control samples for the assay include a tissue sample or a bodily fluid collected from individuals who do not have cells bearing alpha-fetoprotein (negative control), or samples which contain a known, predetermined amount of alpha-fetoprotein receptor (positive control).
[0077] The diagnostic assay may be performed in solution or may use a solid (insoluble) support (e.g. polystyrene, nitrocellulose, or beads) or in a tissue sample prepared for histological examination, using any standard methods. For example, to determine whether the patient from whom the test sample was collected has cells bearing receptors which is recognized by rHuAFP, the level of binding of the detectably labelled rHuAFP in the test sample is compared to the level of binding in the negative and/or positive control samples. A level of binding in the test sample greater than the level of binding in the negative control sample, or at least equal to the level of binding in the positive control sample, indicates that the patient has cells bearing alpha-fetoprotein receptors.
[0078] Materials for performing the diagnostic assays according to the methods of the invention may be provided as a kit having instructions for use. In general, the kit is composed in part of a rHuAFP (or fragment or analog thereof). This kit may further include a second reagent, e.g., a detectable label, which is used to label rHuAFP (or a fragment or analog thereof). The kits exemplified above are useful in, for example, detecting the presence of a tumor in a sample of human tissue in vitro, or for in vivo examination purposes.
[0079] The experimental examples described below demonstrate the efficacy of rHuAFP diagnosing a neoplasm. These examples are provided to illustrate, not limit, the invention.
[0080] MCF-7 human breast cancer cells implanted in the lateral thorax region of CB-17 SCID mice were grown to a size of 1 cm diameter (approx. 5 gm) under estrogen stimulation according to methods known in the art.
[0081] 99mTc-recombinant labelled alpha-fetoprotein was prepared from an AFP aliquot mixed with 0.5 ml 0.9% sodium chloride injection solution (Baxter Healthcare Corporation, Deerfield, Ill.). The solution is added to an UltraTag RBC Reaction Vial (Mallinckrodt Medical Inc., St. Louis, Mo. 63134 Lot No. 0683040), containing stannous chloride dihydrate, sodium citrate dihydrate, and dextrose anhydrous, in a lyophilized form stored under argon. The contents of the vial are mixed by gentle swirling, and incubated at room temperature for 5 minutes. At the completion of the incubation, 0.8-1.2 Gbq Technetium 99mTc Sodium Pertechnetate Injection is added (99mTc Generator Mallincrokt Medical, Inc. St. Louis, Mo.) in a volume of 1-2 ml. The contents of the vial are mixed by gentle swirling and incubated for 15 minutes. Dose aliquots were assayed at 0, 3, and 6 hours after preparation. Thin-layer chromatography performed on preparations using ITLC-SG (Gelman Instrument Co., Ann Arbor, Mich.) with 0.9% NaCl showed 95-99% of the 99Tc was bound to the recombinant alpha-fetoprotein.
[0082] Experimental animals are sedated with Medafane. A 24 gauge, � inch catheter (Surflo IV catheter, Terumo Medical) is then secured in a lateral tail vein. The animal is then further anesthetized with a slow infusion of 20-25 mg/kg body weight of pentobarbital intravenously. Anesthesia is maintained for restraint as required with injections of 5 mg of additional pentobarbital.
[0083] Isotope biodistribution data is collected using a Elscint Dymax 409 gamma camera. This data is subsequently analyzed by a computer (Siemans Gammasonics Microdelta). Animals are imaged in triples, being placed in the dorsal recumbent position on a thin polyethylene panel. To eliminate motion during imaging, the animals are restrained as necessary, on these panels by strips of tape over their extremities so as not to restrict respiration. Dynamic images obtained over 60 minutes are used to determine the biodistribution of the labeled protein. Typically, twelve sequential, five minute images are obtained with low energy general purpose collimation, and 1.5 hardware zoom into the computer matrices having 128 by 128 picture elements. Study animals are typically injected with 37MBq of Tc-99m labelled protein.
[0084] Following administration of 37MBq (approx. 4-6 μg Tc-99m recombinant human alpha-fetoprotein) in the tail vein, tracer biodistribution kinetics were measured during the initial hour after injection and at 24 hours. Tissue uptake kinetics were measured in % injected activity/per 100 ROI (Region of Interest) pixels (% IA). During the first hour there is rapid renal clearance, mild localization in the liver and little evident activity in other tissues. At 1 hour, tumor uptake was (mean�SEM: 1.9�0.3% IA) and the tumor to heart (T/H) region ratio was 0.84�0.23. By 24 hours, tumor uptake was (0.8+/−0.1% IA) and T/A and tumor to background (T/B upper chest) region ratios were 1.43�0.41 and 2.66�0.54, respectively. Studies comparing 99mTc-labelled rHuAFP to 99mTc-labelled human serum albumin (used as a non-specific protein control) repeated in the same animals showed that T/B image ROI activity ration was 2.7 and 5.8 for 99mTc-labelled rHuAFP at 1 and 24 hr, respectively and at 24 hours was 40% greater for 99mTc-labelled rHuAFP compared to Tc-99m human serum albumin. These results show that rHuAFP can be labelled with Tc-99m and that this labelled agent has low non-specific tissue uptake and rapid renal clearance from the blood. Localization in human breast cancer xenografts is initially rapid, increases with time, and is due to specific tumor uptake. These results demonstrate that rHuAFP labelled with Tc-99m is useful as a diagnostic agent for breast carcinoma.
[0085] As discussed above, rHuAFP (or fragment or analog thereof) linked to a detectable label finds diagnostic use in the detection or monitoring or assaying of a neoplasm (e.g., a breast cell carcinoma). Accordingly, patients who present with the classical symptoms of cancer, e.g., breast cancer or prostate cancer, or have a medical history which indicates susceptibility to such cancer may be tested with the methods of the invention. Other appropriate patients for such testing include those who have a family history of breast or prostate carcinomas. Patients who are receiving drugs or have been exposed to toxins implicated in the induction of a cancer should also be tested.
[0086] The diagnostic methods employing detectably labelled rHuAFP (or a fragment or analog thereof) of the invention may be used to detect the presence of a cancer prior to, or after the onset of, clinical symptoms associated with the cancer.
[0087] The method of the invention facilitates diagnosis of a neoplasm prior to or coincident with the onset of clinical symptoms (e.g., a palpable tumorous mass). For example, the method of the subject invention may provide a diagnosis of breast cancer prior to onset of clinical symptoms. Furthermore, the method of the invention allows the clinician to provide an accurate diagnosis of a neoplasm such as breast or prostate cancer.
[0088] Diagnostic imaging methods of the invention will be carried out using a diagnostically effective amount of a diagnostic agent of rHuAFP in a physiologically acceptable carrier. Suitable carriers and their formulation are described for example in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the diagnostic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and with the type of disease, extensiveness of the disease, and size of the patient suffering from the disease. Generally, however, amounts will be in the range of those used for other agents used in the diagnosis of cancer, although in certain instances lower amounts will be needed because of the increased specificity of the compound. For example, a detectably labelled rHuAFP is administered intravenously to a patient, as is described above, at a dosage that allows imaging of a neoplasm, e.g., by radioimaging using scintigraphy. Typically, a dosage is in the range of 0.1 ng-10 g/kg body weight.
[0089] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Legal Events
Original Image