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650 of 669 people found the following review helpful
4.0 out of 5 stars Charming, touching and engaging story
This is a delightful, poignant tale of a retired couple, Harold and Maureen, living out their days in Devon, when something happens that will change their future. And it is such a small occurrence on the face of it - a letter arrives for Harold from a former colleague of his at the brewery, Queenie Hennessy. Harold writes a reply, and he sets off down the road to post it...
Published on 5 Jan. 2012 by L. H. Healy
15 of 16 people found the following review helpful
2.0 out of 5 stars Self-consciously quirky
Gosh, this book irritated me. Clearly I have no heart, but I simply don't understand the rave reviews, the Booker Prize nomination, nor the recommendations from a friend who told me it was one of her favourite books from last year. I. Don't. Get. It. It's saccharine sweet, knowingly quirky and then it heaps on a surprise reveal for a tearjerker ending that felt...
Published 18 months ago by Julia Flyte
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650 of 669 people found the following review helpful
4.0 out of 5 stars Charming, touching and engaging story,
L. H. Healy "Books are life, beauty and truth." (Cambridgeshire, UK) - See all my reviews
This is a delightful, poignant tale of a retired couple, Harold and Maureen, living out their days in Devon, when something happens that will change their future. And it is such a small occurrence on the face of it - a letter arrives for Harold from a former colleague of his at the brewery, Queenie Hennessy. Harold writes a reply, and he sets off down the road to post it. But then he continues walking. And carries on walking, and it becomes his purpose to get to Queenie, to save her, all the hundreds of miles away up in a Hospice in Berwick-upon-Tweed, on foot, in just his yachting shoes.
Beautifully understated, the story plays out so well, there is sadness, some very touching moments, and there is some very well-observed gentle humour too. For Harold, and for Maureen, there is the time and space to take stock and think about their lives together, their son David, and about the events in the past that have brought them to where they are now. Can things be different for them; can they heal the divide that has grown? The reader is not party to the full story until close to the end of the novel. So we can only guess at the reason behind Harold's determination, whatever the odds, to get to Queenie, though we know it's not romantic love.
There is hope despite the difficult times. There is some lovely prose as Harold recognises and admires the nature all around him. His journey becomes more than just one that concerns himself and Queenie; it grows to involve the people he meets on his way, such a variety, by and large he is enriched by his encounters and buoyed by them. He is taken into strangers' confidences, and realises that so many people, despite appearances, have this inner torment that they carry with them. There are beautiful, simple but striking insights into humans, made through Harold.
This is a gentle, touching and rewarding tale, and what a promising, engaging first novel; it's a real accomplishment. I feel sure very many readers will enjoy this.
28 of 29 people found the following review helpful
5.0 out of 5 stars Beautifully written and emotional,
Rachel Joyce's The Unlikely Pilgrimage of Harold Fry is beautiful and simple, yet at the same time it's is packed with emotion, self-doubt and heartache that's sure to hit a nerve with every reader.
It begins when Harold Fry receives a letter from an old colleague and friend informing him that she has been diagnosed with terminal cancer. What starts as a trip to the nearest post-box to send a letter expressing his condolences turns into an epic journey across the country. Without a map, a change of clothes or a mobile phone Harold's walk is dependent completely on his instincts, the kindness of strangers and the strength of his belief in the fact that if he keeps on walking, Queenie will keep on living.
Harold's walk seems so simple on the outside. As he describes it, it's just putting one foot in front of the other. But as he attempts to walks from Cornwall to Berwick-upon-Tweed, Harold is forced to confront the tragedies of his past, his estrangement from his wife and growing detachment from the world.
Left behind, his wife Maureen is also battling her own demons as she struggles with feelings of repressed loss and anger. Harold's walk and the space left by his absence, prompts her own personal journey.
The bit I loved most about this book was how the two central characters somehow, against the backdrop of everything that's happened in their lives, managed to find a way to rediscover their love for each other. It's an intense and emotional read and it really drives home the fact that it's never too late to change.
237 of 249 people found the following review helpful
5.0 out of 5 stars Overwhelming,
Jade66 (Bournemouth) - See all my reviews
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I glanced at a few of the reviews before buying this book, none of them do it justice and I doubt if mine will either. This is a wonderful story that entertains and disquiets in equal measure. Ostensibly it is about a man, just retired, who sets out to walk from Devon to Berwick on Tweed after receiving a letter from an old work colleague. The colleague, Queenie, is dying of cancer. Harold pens a quick reply and sets off to post it, but somehow the posting of this letter seems inadequate. He decides instead to walk the 500 odd miles to Berwick, taking us with him.
It is clear very early on that Harold's life has been a disappointment. An inability to connect with his son, (stemming from his own neglectful childhood) has driven a stake between him and his wife, Maureen, and what was once a good marriage has deteriorated into a hopeless desert of non communication.
It is during his long walk that we discover all about Harold, and Maureen, and their son David, and all about the long held grievances and misunderstandings that have culminated in their isolation and loneliness. Sometimes these memories are extremely painful and I found myself moved beyond belief at this fictional tale.
One of the 2 star reviews on this page unbelievably states "nothing much happens". Nothing could be further from the truth. Everything happens as this endearing man struggles to make sense of his life, struggles to find hope and optimism when doors have been closed resolutely in his face, and struggles to assert his humanity on an indifferent world.
This is a story about all those things we leave unsaid, of all those regrets we fight daily to forget. Wonderful writing, clear recognizable characters, a story that won't leave you, and an examination in depth of all those weird and wonderful contradictions that make us what we are.
326 of 348 people found the following review helpful
5.0 out of 5 stars A wonderful book,
Maggie (UK) - See all my reviews
Be prepared to confront your personal demons when you read this book. For the mature reader in particular, I challenge you not to find something in this novel to make you at the least uneasy. It is ultimately an uplifting story, but along the way there is a great deal of pain and for many there will be uncomfortable home truths about things that we could have handled better, regrets about slipping into complacency and about the loss of passion. There is the terrible pain caused by the loss of a child; the guilt engendered by failure to appreciate and help a friend; the estrangement of a once-loving couple - and the knowledge that many of these things cannot be put right however much you want to.
Harold's walk is the vehicle for exploring these ideas and more. A very ordinary and unassuming man, not in any sense a hero, Harold's whim to walk to Berwick on Tweed to see a dying friend and by so doing to save her from cancer, provides the author with the opportunity to weave in the stories of many other people who, in the words of Henry David Thoreau, are living "lives of quiet desperation". The people he meets on his journey are often kind and generous but many are battling their own demons. At one point the simple pilgrimage that Harold has unwittingly created clashes with our modern world of celebrity - represented by the PR men and tabloid journalists - and you pray for Harold to escape all this and revert to his simple goal.
In the end Harold and his wife Maureen - a somewhat stereotypical middle-aged woman with net curtains and a clipped way of speaking that discourages anything unconventional - do find resolution and redemption. To say more would spoil the story. Do read it - it will make you laugh and cry but it will not leave you untouched.
15 of 16 people found the following review helpful
2.0 out of 5 stars Self-consciously quirky,
Julia Flyte - See all my reviews
Gosh, this book irritated me. Clearly I have no heart, but I simply don't understand the rave reviews, the Booker Prize nomination, nor the recommendations from a friend who told me it was one of her favourite books from last year. I. Don't. Get. It. It's saccharine sweet, knowingly quirky and then it heaps on a surprise reveal for a tearjerker ending that felt manipulative in the extreme.
Harold Fry is 65 and retired. He lives in Devon in England's south-west with Maureen, his wife of 45 years. The marriage is not terribly happy; they co-exist, but that's about it. One day he gets a letter from a former colleague whom he has not seen or talked to in many, many years. Queenie is writing to tell him that she has terminal cancer. Harold writes a letter in reply, goes out to post it and then spontaneously decides to walk to Queenie, (a journey of 627 miles). Between numerous references to blisters and his inappropriate boat shoes (which he will stubbornly refuse to replace), there are numerous encounters with an array of quirky characters who it would seem are desperate to unburden themselves on a stranger. Along the way he thinks a great deal about his upbringing, his relationship with his wife and his troubled relationship with his son. I wanted to shout at him to stop being such a martyr and I also wanted to slap almost every person that he met along the way.
This reminds me of books like Major Pettigrew's Last Stand, with its conviction that England is populated with nothing but endearing eccentrics. Even the whimsical chapter names "Harold and the Hiking Man and the Woman who Loved Jane Austen" or "Harold and the Barman and the Woman with Food" had me wanting to beat the book against a wall, screaming. To ram home the cutesy factor, each jolly little chapter title is accompanied by a playfully quaint illustration of a hedgehog or a pansy or a crow. So lovable!
In case you have written me off by now as a cold-hearted monster, I must point out that the central message didn't escape my notice - how could it, being laid on like a trowel? Harold realises that "in walking to atone for the mistakes he had made, it was also his journey to accept the strangeness of others" - meaning that he doggedly thinks the best of every moron who crosses his path. And yes, I was moved by the revelation near the book's conclusion. Hence my generosity in doubling my star rating. Actually that's a little unfair - it's not totally awful. It's an easy read, mercifully short and if you're less hard-hearted that me, you may even like it.
126 of 138 people found the following review helpful
3.0 out of 5 stars An interesting journey,
bomble "bomble" (Cambridge, UK) - See all my reviews
One of the oldest kinds of story is 'the journey' - the road trip - the odyssey. Most try hard to be epic in some sense, looking for superlatives of ardour, distance or grand purpose. What makes Harold Fry's pilgrimage a worthwhile one therefore is its humble and na�ve intent that gives Rachel Joyce ample space to explore characters and the emotions that they experience.
The bare bones of the story wouldn't fill more than a couple of pages, but Joyce has given life to her characters both in their past and present circumstances, making this book a lot more substantial. That said it comes as no surprise that she has honed her skills for the medium of Radio 4 afternoon plays as you could assume that she is writing for much the same cross-section of the population. There are some more 'sensitive' passages and the occasional profanity but you basically get the feeling that this is a book for the middle ground and unashamedly so.
Joyce's style is highly readable and it didn't take long to finish. She builds her characters in layers from the outside in as you gradually come to know their deeper thoughts, their joys and their sorrows. And it would be fair to say that I enjoyed the walk in Harold's shoes.
73 of 80 people found the following review helpful
5.0 out of 5 stars a wonderful book,
C. Bones "surreyman" - See all my reviews
I think this amazing debut novel could be as successful in 2012 as David Nicholls' One Day was in 2011. I don't mean that they are similar novels but they both offer that same blend of humour, wisdom and heart tugging pathos. And yet for about 50 pages I wasn't sure. Harold had set off on his journey and I started to wonder whether he was just a bit simple minded and unlikely to be an interesting companion over 300 pages. How wrong I was ! By the end I was in tears.
This is a story that develops in unexpected ways. It is by no means just the simple and charming tale that you may expect. Rachel Joyce starts by writing about her characters in a stripped down way that makes them very ordinary indeed. It appears that there are no heroes here. Just very ordinary people who on the whole fail to deal very well with life's tough knocks. And from this unpromising beginning she slowly start to weave her magic.
So what is it about ? Well, everything really, the whole of life. Its certainly about what love can achieve and its most definitely about what a lack of love can achieve. Its also about faith but not faith as you know it. This is not a story where anyone has any blazing convictions or sudden revelations. This is the sort of faith which starts off in misguided ways, humans stumbling along (literally in Harold's case) in a generally confused way and from this mixture of good and daft intentions a small light may start to flicker. And did I say that Ms Joyce writes like a dream? Well she does. How on earth can you produce a first book that is this well written ?
But my tip to would-be readers is to try and avoid too many reviews. As I said earlier this is a story that unfolds in unexpected ways and the last thing you want is learn anything too soon. Not only is the author a great writer but she doesn't put a foot wrong in the pacing and structure of her story. Just buy it. Just read it !
22 of 24 people found the following review helpful
3.0 out of 5 stars Sweet and charming but not without faults,
R. A. Davison (UK) - See all my reviews
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The Unlikely Pilgrimage Of Harold Fry by Rachel Joyce was originally born out of a BBC Radio Play, and now finds itself on the Booker Prize longlist for 2012, something which created some media surprise when it was announced.
Harold Fry is a sweet portrait of a man who has lived by all appearances an ordinary and mundane existence, who receives a letter from a dying friend; setting out to post his response he finds he cannot stop walking and eventually resolves to walk all the way to visit his old colleague Queenie Hennessy. It is not however, a short journey for Harold lives in Dorset and Queenie in Berwick-upon-Tweed.
Quite why Queenie is so important is revealed slowly over the course of the novel, which is by turns heartwarming and heartbreaking. The style of prose has a fable like quality to it, and though the story packs a emotional punch with its terribly human psychology and identifiable feelings, the actual writing is fundamentally simplistic, making me wonder whether it is worthy of the Booker accolade, and certainly of winning it, though it is a nice nod to a sweet book.
I found the section where other people join in Harold's mission and hijack it for their own purposes really quite annoying, I have seen it done as a device in other novels which involve a mission or a cause and find it irritating. Essentially it's like the section in Forrest Gump when he runs on his own for ages. And that's the problem, this phenomena is quite un-British, and quite cheesily American, which is something that Harold Fry overall is not, Harold and his wife Maureen being quite quintessentially British which is the novel's central charm.
Though Harold's eventual reunion with Queenie is what I had expected all along, the novel is as with life about the journey not the destination. The end of the novel is incredibly touching, and you must have a heart of stone if you are not moved by the revelation towards the novel's close. 7/10
3 of 3 people found the following review helpful
2.0 out of 5 stars I got so bored. This is a road trip in the ilk ...,
I am sorry to say that I left Harold to reach journey's end without me, I got so bored.
This is a road trip in the ilk of "The One Hundred Year Old Man who Climbed out of the Window and Disappeared". I dare say that if you like the latter and indeed any story of sweet little old men running around the countryside, then you will probably enjoy Harold Fry. To my mind any book title that reads like a Tweet is to be avoided.
This is a twee little story about a retiree who decides to walk from Devon to Berwick upon Tweed to help a Friend (who once did him a Good Deed) in Urgent Need. Despite the fact that the friend is at death's door, Harold eschews the car and even avoids the bus, opting instead to go on foot (in his plimsolls). Cue a series of Little Adventures as Harold meets Many Interesting People Along The Way. This handy device saves the author from worrying about any form of character development as each character has A Story To Tell.
This is "The Incredible Journey" meets "Worzel Gummidge". Some people will like its simple style but for me it was just simplistic.
11 of 12 people found the following review helpful
5.0 out of 5 stars Careful catalogue of kindnesses,
Katharine Kirby "Kate" (HELSTON, Cornwall United Kingdom) - See all my reviews
Without doubt this is the most refreshing, uplifting and exciting new novel. So far Rachel Joyce been busy writing plays for Radio 4. You know the ones that stop you in your daily task, that have you weeping with understanding and poignant sadness, in the early afternoon. The Unlikely Pilgrimage sprang from such a source.
I travelled companionably with courteous, self effacing, resourceful Harold Fry, more in hope than expectation; up through England from Devon, aiming for north of the border. There lies poor Queenie, a friend from his working days, a lady who he once had to drive from place to place. She was a good friend and looked out for Harold, eventually to her cost. Her cancer is terminal, her letter short but gripping, Harold feels drawn to visit her, to try to save her. All on a whim but one you feel anyone could be caught up by. The idea that somehow you can thwart fate by believing in a goal and stubbornly sticking to it. No careless or selfish hero is Harold, he maintains his links with home and gives himself no quarter or relief. His forty year old marriage has sadly turned sour, his son David appears an enigma, his wife closed in to her world of cleaning, tidying and shutting him out of her heart. She can't stop herself behaving this way although she recognises what she is doing. In the spirit of something must change the book takes off when, 'We all sit down far more than are meant to' Harold decides, and so along the road we go, gathering skills, experience and memories along the way.
Not an active gentleman, only used to driving about the countryside; being on foot shows Harold, a wholly different environment. One that can heal, help and encourage. He realises how little he needs and wants even. If you can be touched by the humanity that bubbles beneath the surface of everyday life, rarely tested but still stoically standing ready to offer the best of human nature, this will reinforce your confidence in people in general. The descriptions are fresh, the pace perfect, the denouement surprising and immensely satisfying.
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The Unlikely Pilgrimage Of Harold Fry by Rachel Joyce


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subset: commoncrawl


text:
The preparation and characterization of antibodies that bind to antigens on CLL or other cancer cells, especially to antigens upregulated in the cancer cells, and the identification and characterization of antigens present on or upregulated by cancer cells are useful in studying and treating cancer.
1. A method of treating chronic lymphocytic leukemia (CLL) comprising administering to a patient suffering from CLL an antibody or antigen-binding fragment thereof that specifically binds to OX-2/CD200, wherein said antibody or antigen-binding fragment thereof comprises a light chain CDR1 having the sequence set forth in SEQ ID NO: 5; a light chain CDR2 having the sequence set forth in SEQ ID NO: 21; a light chain CDR3 having the sequence set forth in SEQ ID NO: 29; a heavy chain CDR1 having the sequence set forth in SEQ ID NO: 50; a heavy chain CDR2 having the sequence set forth in SEQ ID NO: 69; and a heavy chain CDR3 having the sequence set forth in SEQ ID NO: 88.
2. The method of
3. The method of
4. The method of
5. The method of
6. A method of treating chronic lymphocytic leukemia (CLL) comprising administering to a patient suffering from CLL an antibody or antigen-binding fragment thereof that specifically binds to OX-2/CD200, wherein said antibody or antigen-binding fragment thereof comprises a light chain CDR1 having the sequence set forth in SEQ ID NO: 13; a light chain CDR2 having the sequence set forth in SEQ ID NO: 23; a light chain CDR3 having the sequence set forth in SEQ ID NO: 38; a heavy chain CDR1 having the sequence set forth in SEQ ID NO: 56; a heavy chain CDR2 having the sequence set forth in SEQ ID NO: 75; and a heavy chain CDR3 having the sequence set forth in SEQ ID NO: 94.
7. The method of
8. The method of
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10. The method of
11. A method of treating chronic lymphocytic leukemia (CLL) comprising administering to a patient suffering from CLL an antibody or antigen-binding fragment thereof that specifically binds to OX-2/CD200, wherein said antibody or antigen-binding fragment thereof comprises a light chain CDR1 having the sequence set forth in SEQ ID NO: 12; a light chain CDR2 having the sequence set forth in SEQ ID NO: 23; a light chain CDR3 having the sequence set forth in SEQ ID NO: 37; a heavy chain CDR1 having the sequence set forth in SEQ ID NO: 55; a heavy chain CDR2 having the sequence set forth in SEQ ID NO: 74; and a heavy chain CDR3 having the sequence set forth in SEQ ID NO: 93.
12. The method of
13. The method of
14. The method of
15. The method of
This application is a continuation in part of PCT/US01/47931 filed on Dec. 10, 2001 which is an international application that claims priority to U.S. Provisional Application No. 60/254,113 filed Dec. 8, 2000. The entire disclosures of both the aforementioned international and provisional applications are incorporated herein by reference.
Cell lines derived from chronic lymphocytic leukemia (CLL) cells and the uses thereof in the study and treatment of CLL disease are disclosed. In particular, this disclosure relates to a CLL cell line designated "CLL-AAT", deposited on December 11, 2001 with the American Type Culture Collection (Manassas, Va., USA) in accordance with the terms of the Budapest Treaty under ATCC accession no. PTA-3920.
Chronic Lymphocytic Leukemia (CLL) is a disease of the white blood cells and is the most common form of leukemia in the Western Hemisphere. CLL represents a diverse group of diseases relating to the growth of malignant lymphocytes that grow slowly but have an extended life span. CLL is classified in various categories that include, for example, B-cell chronic lymphocytic leukemia (B-CLL) of classical and mixed types, B-cell and T-cell prolymphocyic leukemia, hairy cell leukemia, and large granular lymphocytic leukemia.
Of all the different types of CLL, B-CLL accounts for approximately 30 percent of all leukemias. Although it occurs more frequently in individuals over 50 years of age it is increasingly seen in younger people. B-CLL is characterized by accumulation of B-lymphocytes that are morphologically normal but biologically immature, leading to a loss of function. Lymphocytes normally function to fight infection. In B-CLL, however, lymphocytes accumulate in the blood and bone marrow and cause swelling of the lymph nodes. The production of normal bone marrow and blood cells is reduced and patients often experience severe anemia as well as low platelet counts. This can pose the risk of life-threatening bleeding and the development of serious infections because of reduced numbers of white blood cells.
To further understand diseases such as leukemia it is important to have suitable cell lines that can be used as tools for research on their etiology, pathogenesis and biology. Examples of malignant human B-lymphoid cell lines include pre-B acute lymphoblasticleukemia (Reh), diffuse large cell lymphoma (WSU-DLCL2), and Waldenstrom's macroglobulinemia (WSU-WM). Unfortunately, many of the existing cell lines do not represent the clinically most common types of leukemia and lymphoma.
The use of Epstein Barr Virus (EBV) infection in vitro has resulted in some CLL derived cell lines, in particular B-CLL cells lines, that are representative of the malignant cells. The phenotype of these cell lines is different than that of the in vivo tumors and instead the features of B-CLL lines tend to be similar to those of Lymphoblastoid cell lines. Attempts to immortalize B-CLL cells with the aid of EBV infection have had little success. The reasons for this are unclear but it is known that it is not due a lack of EBV receptor expression, binding or uptake. Wells et al. found that B-CLL cells were arrested in the G 1/S phase of the cell cycle and that transformation associated EBV DNA was not expressed. This suggests that the interaction of EBV with B-CLL cells is different from that with normal B cells. EBV-transformed CLL cell lines moreover appear to differentiate, possessing a morphology more similar to lymphoblastoid cell lines (LCL) immortalized by EBV.
An EBV-negative CLL cell line, WSU-CLL, has been established previously (Mohammad et al., (1996) Leukemia 10(1):130-7). However, no other such cell lines are known.
There remains a need in the art, therefore, for a CLL cell line which has not been established by transformation with EBV, and which expresses surface markers characteristic of primary CLL cells.
In one embodiment an CLL cell line of malignant origin is provided that is not established by immortalisation with EBV. The cell line, which was derived from primary CLL cells, and is deposited under ATCC accession no. PTA-3920. In a preferred embodiment, the cell line is CLL-AAT. CLL-MT is B-CLL cell line, derived from a B-CLL primary cell.
In a further aspect, the CLL-AAT cell line is used to generate monoclonal antibodies useful in the diagnosis and/or treatment of CLL. Antibodies may be generated by using the cells as disclosed herein as immunogens, thus raising an immune response in animals from which monoclonal antibodies may be isolated. The sequence of such antibodies may be determined and the antibodies or variants thereof produced by recombinant techniques. In this aspect, "variants" includes chimeric, CDR-grafted, humanized and fully human antibodies based on the sequence of the monoclonal antibodies.
Moreover, antibodies derived from recombinant libraries ("phage antibodies") may be selected using the cells described herein, or polypeptides derived therefrom, as bait to isolate the antibodies on the basis of target specificity.
In a still further aspect, antibodies may be generated by panning antibody libraries using primary CLL cells, or antigens derived therefrom, and further screened and/or characterized using a CLL cell line, such as, for example, the CLL cell line described herein. Accordingly, a method for characterizing an antibody specific for CLL is provided, which includes assessing the binding of the antibody to a CLL cell line.
In a further aspect, there is provided a method for identifying proteins uniquely expressed in CLL cells employing the CLL-AAT cell line, by methods well known to those, skilled with art, such as by immunoprecipitation followed by mass spectroscopy analyses. Such proteins may be uniquely expressed in the CLL-AAT cell line, or in primary cells derived from CLL patients.
Small molecule libraries (many available commercially) may be screened using the CLL-AAT cell line in a cell-based assay to identify agents capable of modulating the growth characteristics of the cells. For example, the agents may be identified which modulate apoptosis in the CLL-AAT cell line, or which inhibit growth and/or proliferation thereof. Such agents are candidates for the development of therapeutic compounds.
Nucleic acids isolated from CLL-AAT cell lines may be used in subtractive hybridization experiments to identify CLL-specific genes or in micro array analyses (e.g., gene chip experiments). Genes whose transcription is modulated in CLL cells may be identified. Polypeptide or nucleic acid gene products identified in this manner are useful as leads for the development of antibody or small molecule therapies for CLL.
In a preferred aspect, the CLL-AAT cell line may be used to identify internalizing antibodies, which bind to cell surface components which are internalized by the cell. Such antibodies are candidates for therapeutic use. In particular, single-chain antibodies, which remain stable in the cytoplasm and which retain intracellular binding activity, may be screened in this manner.
In yet another aspect, a therapeutic treatment is described in which a patient is screened for the presence of a polypeptide that is upregulated by a malignant cancer cell and an antibody that interferes with the metabolic pathway of the upregulated polypeptide is administered to the patient.
"CLL", as used herein, refers to chronic lymphocytic leukemia involving any lymphocyte, including but not limited to various developmental stages of B cells and T cells, including but not limited to B cell CLL. B-CLL, as used herein, refers to leukemia with a mature B cell phenotype which is CD5+, CD23+, CD20dim+, sIgdim+ and arrested in G0/G1 of the cell cycle.
"Malignant origin" refers to the derivation of the cell line from malignant CLL primary cells, as opposed to non-proliferating cells which are transformed, for example, with EBV. Cell lines according to this disclosure may be themselves malignant in phenotype, or not. A CLL cell having a "malignant" phenotype encompasses cell growth unattached from substrate media characterized by repeated cycles of cell growth and exhibits resistance to apoptosis.
Preparation of Cell Lines
Cell lines may be produced according to established methodologies known to those skilled in the art. In general, cell lines are produced by culturing primary cells derived from a patient until immortalized cells are spontaneously generated in culture. These cells are then isolated and further cultured, to produce clonal cell populations or cells exhibiting resistance to apoptosis.
For example, CLL cells may be isolated from peripheral blood drawn from a patient suffering from CLL. The cells may be washed, and optionally immunotyped in order to determine the type(s) of cells present. Subsequently, the cells may be cultured in a medium, such as a medium containing IL-4. Advantageously, all or part of the medium is replaced one or more times during the culture process. Cell lines may be isolated thereby, and will be identified by increased growth in culture.
Preparation of Monoclonal Antibodies
Antibodies, as used herein, refers to complete antibodies or antibody fragments capable of binding to a selected target. Included are Fv, ScFv, Fab′ and F(ab′)2, monoclonal and polyclonal antibodies, engineered antibodies (including chimeric, CDR-grafted and humanized, fully human antibodies, and artificially selected antibodies), and synthetic or semi synthetic antibodies produced using phage display or alternative techniques. Small fragments, such Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution.
The antibodies are especially indicated for diagnostic and therapeutic applications. Accordingly, they may be altered antibodies comprising an effector protein such as a toxin or a label. Especially preferred are labels which allow the imaging of the distribution of the antibody in vivo. Such labels may be radioactive labels or radiopaque labels, such as metal particles, which are readily visualisable within the body of a patient. Moreover, the labels may be fluorescent labels or other labels which are visualisable on tissue samples removed from patients.
Recombinant DNA technology may be used to improve the antibodies produced in accordance with this disclosure. Thus, chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications. Moreover, immunogenicity may be minimized by humanizing the antibodies by CDR grafting and, optionally, framework modification. See, U.S. Pat. No. 5,225,539, the contents of which are incorporated herein by reference.
Antibodies may be obtained from animal serum, or, in the case of monoclonal antibodies or fragments thereof produced in cell culture. Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial or preferably mammalian cell culture. The selected cell culture system preferably secretes the antibody product.
In another embodiment, a process for the production of an antibody disclosed herein includes culturing a host, e.g. E. coli or a mammalian cell, which has been transformed with a hybrid vector. The vector includes one or more expression cassettes containing a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding the antibody protein. The antibody protein is then collected and isolated. Optionally, the expression cassette may include a promoter operably linked to polycistronic, for example bicistronic, DNA sequences encoding antibody proteins each individually operably linked to a signal peptide in the proper reading frame.
Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which include the customary standard culture media (such as, for example Dulbecco's Modified Eagle Medium (DMEM) or RPMI 1640 medium), optionally replenished by a mammalian serum (e.g. fetal calf serum), or trace elements and growth sustaining supplements (e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like). Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art. For example, for bacteria suitable culture media include medium LE, NZCYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2�YT, or M9 Minimal Medium. For yeast, suitable culture media include medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.
In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies. Techniques for bacterial cell, yeast, plant, or mammalian cell cultivation are known in the art and include homogeneous suspension culture (e.g. in an airlift reactor or in a continuous stirrer reactor), and immobilized or entrapped cell culture (e.g. in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges).
Large quantities of the desired antibodies can also be obtained by multiplying mammalian cells in vivo. For this purpose, hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody-producing tumors. Optionally, the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection. After one to three weeks, the antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristine. After one to two weeks, ascitic fluid is taken from the animals.
The foregoing, and other, techniques are discussed in, for example, Kohler and Milstein, (1975) Nature 256:495-497; U.S. Pat. No. 4,376,110; Harlow and Lane, Antibodies: a Laboratory Manual, (1988) Cold Spring Harbor, the disclosures of which are all incorporated herein by reference. Techniques for the preparation of recombinant antibody molecules is described in the above references and also in, for example WO97/08320; U.S. Pat. No. 5,427,908; U.S. Pat. No. 5,508,717; Smith, 1985, Science, Vol. 225, pp 1315-1317; Parmley and Smith 1988, Gene 73, pp 305-318; De La Cruz et al, 1988, Journal of Biological Chemistry, 263 pp 4318-4322; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,223,409; WO88/06630; WO92/15679; U.S. Pat. No. 5,780,279; U.S. Pat. No. 5,571,698; U.S. Pat. No. 6,040,136; Davis et al., Cancer Metastasis Rev.,1999;18(4):421-5; Taylor, et al., Nucleic Acids Research 20 (1992): 6287-6295; Tomizuka et al., Proc. Nat. Academy of Sciences USA 97(2) (2000): 722-727. The contents of all these references are incorporated herein by reference.
The cell culture supernatants are screened for the desired antibodies, preferentially by immunofluorescent staining of CLL cells, by immunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radioimmunoassay
For isolation of the antibodies, the immunoglobulins in the culture supernatants or in the ascitic fluid may be concentrated, e.g. by precipitation with ammonium sulfate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like. If necessary and/or desired, the antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno-) affinity chromatography, e.g. affinity chromatography with a one or more surface polypeptides derived from a CLL cell line according to this disclosure, or with Protein-A or G.
Another embodiment provides a process for the preparation of a bacterial cell line secreting antibodies directed against the cell line characterized in that a suitable mammal, for example a rabbit, is immunized with pooled CLL patient samples. A phage display library produced from the immunized rabbit is constructed and panned for the desired antibodies in accordance with methods well known in the art (such as, for example, the methods disclosed in the various references incorporated herein by reference).
Hybridoma cells secreting the monoclonal antibodies are also contemplated. The preferred hybridoma cells are genetically stable, secrete monoclonal antibodies described herein of the desired specificity and can be activated from deep-frozen cultures by thawing and reckoning.
In another embodiment, a process is provided for the preparation of a hybridoma cell line secreting monoclonal antibodies directed to the CLL cell line is described herein. In that process, a suitable mammal, for example a Balb/c mouse, is immunized with a one or more polypeptides or antigenic fragments thereof derived from a cell described in this disclosure, the cell line itself, or an antigenic carrier containing a purified polypeptide as described. Antibody-producing cells of the immunized mammal are grown briefly in culture or fused with cells of a suitable myeloma cell line. The hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example, spleen cells of Balb/c mice immunized with the present cell line are fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag 14, the obtained hybrid cells are screened for secretion of the desired antibodies, and positive hybridoma cells are cloned.
Preferred is a process for the preparation of a hybridoma cell line, characterized in that Balb/c mice are immunized by injecting subcutaneously and/or intraperitoneally between 106 and 107 cells of a cell line in accordance with this disclosure several times, e.g. four to six times, over several months, e.g. between two and four months. Spleen cells from the immunized mice are taken two to four days after the last injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, preferably polyethylene glycol. Preferably, the myeloma cells are fused with a three- to twenty-fold excess of spleen cells from the immunized mice in a solution containing about 30% to about 50% polyethylene glycol of a molecular weight around 4000. After the fusion, the cells are expanded in suitable culture media as described hereinbefore, supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells.
In a further embodiment, recombinant DNA comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to the cell line described hereinbefore are produced. The term DNA includes coding single stranded DNAs, double stranded DNAs consisting of said coding DNAs and of complementary DNAs thereto, or these complementary (single stranded) DNAs themselves.
Furthermore, DNA encoding a heavy chain variable domain and/or a light chain variable domain of antibodies directed to the cell line disclosed herein can be enzymatically or chemically synthesized DNA having the authentic DNA sequence coding for a heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof. A mutant of the authentic DNA is a DNA encoding a heavy chain variable domain and/or a light chain variable domain of the above-mentioned antibodies in which one or more amino acids are deleted or exchanged with one or more other amino acids. Preferably said modification(s) are outside the CDRs of the heavy chain variable domain and/or of the light chain variable domain of the antibody in humanization and expression optimization applications. The term mutant DNA also embraces silent mutants wherein one or more nucleotides are replaced by other nucleotides with the new codons coding for the same amino acid(s). The term mutant sequence also includes a degenerated sequence. Degenerated sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded. Such degenerated sequences may be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host, particularly E. coli, to obtain an optimal expression of the heavy chain murine variable domain and/or a light chain murine variable domain.
The term mutant is intended to include a DNA mutant obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.
For the assembly of complete tetrameric immunoglobulin molecules and the expression of chimeric antibodies, the recombinant DNA inserts coding for heavy and light chain variable domains are fused with the corresponding DNAs coding for heavy and light chain constant domains, then transferred into appropriate host cells, for example after incorporation into hybrid vectors.
Recombinant DNAs including an insert coding for a heavy chain murine variable domain of an antibody directed to the cell line disclosed herein fused to a human constant domain g, for example γ1, γ2, γ3 or γ4, preferably γ1 or γ4 are also provided. Recombinant DNAs including an insert coding for a light chain murine variable domain of an antibody directed to the cell line disclosed herein fused to a human constant domain κor λ, preferably κ are also provided
Another embodiment pertains to recombinant DNAs coding for a recombinant polypeptide wherein the heavy chain variable domain and the light chain variable domain are linked by way of a spacer group, optionally comprising a signal sequence facilitating the processing of the antibody in the host cell and/or a DNA coding for a peptide facilitating the purification of the antibody and/or a cleavage site and/or a peptide spacer and/or an effector molecule.
The DNA coding for an effector molecule is intended to be a DNA coding for the effector molecules useful in diagnostic or therapeutic applications. Thus, effector molecules which are toxins or enzymes, especially enzymes capable of catalyzing the activation of prodrugs, are particularly indicated. The DNA encoding such an effector molecule has the sequence of a naturally occurring enzyme or toxin encoding DNA, or a mutant thereof, and can be prepared by methods well known in the art.
Antibodies and antibody fragments disclosed herein are useful in diagnosis and therapy. Accordingly, a composition for therapy or diagnosis comprising an antibody disclosed herein is provided.
In the case of a diagnostic composition, the antibody is preferably provided together with means for detecting the antibody, which may be enzymatic, fluorescent, radioisotopic or other means. The antibody and the detection means may be provided for simultaneous, separate or sequential use, in a diagnostic kit intended for diagnosis.
Uses of the CLL Cell Line
There are many advantages to the development of a CLL cell line, as it provides an important tool for the development of diagnostics and treatments for CLL.
A cell line according to this disclosure may be used for in vitro studies on the etiology, pathogenesis and biology of CLL. This assists in the identification of suitable agents that are useful in the therapy of CLL disease.
The cell line may also be used to produce monoclonal antibodies for in vitro and in vivo diagnosis of CLL, as referred to above, and for the screening and/or characterization of antibodies produced by other methods, such as by panning antibody libraries with primary cells and/or antigens derived from CLL patients.
The cell line may be used as such, or antigens may be derived therefrom. Advantageously, such antigens are cell-surface antigens specific for CLL. They may be isolated directly from cell lines according to this disclosure. Alternatively, a cDNA expression library made from a cell line described herein may be used to express CLL-specific antigens, useful for the selection and characterization of anti-CLL antibodies and the identification of novel CLL-specific antigens.
Treatment of CLL using monoclonal antibody therapy has been proposed in the art. Recently, Hainsworth (Oncologist 5 (5) (2000) 376-384) has described the current therapies derived from monoclonal antibodies. Lymphocytic leukemia in particular is considered to be a good candidate for this therapeutic approach due to the presence of multiple lymphocyte-specific antigens on lymphocyte tumors.
Existing antibody therapies (such as Rituximab™, directed against the CD20-antigen, which is expressed on the surface of B-lymphocytes) have been used successfully against certain lymphocytic disease. However, a lower density CD20 antigen is expressed on the surface of B-lymphocytes in CLL (Almasri et al., Am. J. Hematol., 40 (4) (1992) 259-263).
The CLL cell line described herein thus permits the development of novel anti-CLL antibodies having specificity for one or more antigenic determinants of the present CLL cell line, and their use in the therapy and diagnosis of CLL.
In a particularly useful embodiment, the antibody binds to or otherwise interferes with the metabolic pathway of a polypeptide that is upregulated by a malignant cancer cell. For instance, the antibody can bind to the upregulated polypeptide and in this manner prevent or inhibit the polypeptide from interacting with other molecules or receptors. Alternatively, the antibody may bind to a receptor with which the upregulated polypeptide normally interacts, thereby preventing or inhibiting the polypeptide from binding to the receptor. As yet another alternative, the antibody can bind to an antigen that modulates expression of the polypeptide, thereby preventing or inhibiting normal or increased expression of the polypeptide. For example, the peptide OX-2/CD200 is upregulated in a portion of CLL patients. Because the presence of OX-2/CD200 has been associated with reduced immune response, it would be desirable to interfere with the metabolic pathway of OX-2/CD200 so that the patient's immune system can defend against the cancer more effectively.
Thus, in another embodiment, a method for treating a cancer patient in accordance with this disclosure includes the steps of screening a cancer patient for the presence of a polypeptide that is upregulated by a malignant cancer cell and administering an antibody that interferes with the metabolic pathway of the upregulated polypeptide. In a particularly useful embodiment, a CLL patient is screened for overexpression of OX-2/CD200 and an antibody that interferes with the metabolic pathway of OX-2/CD200 is administered to the patient. As described in detail below, one such antibody is scFv9 (see
In order that those skilled in the art may be better able to practice the compositions and methods described herein, the following examples are given for illustration purposes.
Isolation of Cell Line CLL-MT
Establishment of the Cell Line
Peripheral blood from a patient diagnosed with CLL was obtained. The WBC count was 1.6�108/ml. Mononuclear cells were isolated by Histopaque-1077 density gradient centrifugation (Sigma Diagnostics, St. Louis, Mo.). Cells were washed twice with Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), and resuspended in 5 ml of ice-cold IMDM/10% FBS. Viable cells were counted by staining with trypan blue. Cells were mixed with an equal volume of 85% FBS/15% DMSO and frozen in 1 ml aliquots for storage in liquid nitrogen.
Immunophenotyping showed that >90% of the CD45+lymphocyte population expressed IgD, kappa light chain, CD5, CD19, and CD23. This population also expressed low levels of IgM and CD20. Approximately 50% of the cells expressed high levels of CD38. The cells were negative for lambda light chain, CD10 and CD138
An aliquot of the cells was thawed, washed, and resuspended at a density of 107/mL in IMDM supplemented with 20% heat-inactivated FBS, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, 50 μM 2-mercaptoethanol, and 5 ng/ml recombinant human IL-4 (R & D Systems, Minneapolis, Minn.). The cells were cultured at 37� C. in a humidified 5% CO2 atmosphere. The medium was partially replaced every 4 days until steady growth was observed. After 5 weeks, the number of cells in the culture began to double approximately every 4 days. This cell line was designated CLL-MT.
Characterization of the Cell Line
Immunophenotyping of the cell line by flow cytometry showed high expression of IgM, kappa light chain, CD23, CD38, and CD138, moderate expression of CD19 and CD20, and weak expression of IgD and CD5. The cell line was negative for lambda light chain, CD4, CD8, and CD10.
Immunophenotyping of the cell line was also done by whole cell ELISA using a panel of rabbit scFv antibodies that had been selected for specific binding to primary B-CLL cells. All of these CLL-specific scFv antibodies also recognized the CLL-AAT cell line. In contrast, the majority of the scFvs did not bind to two cell lines derived from B cell lymphomas: Ramos, a Burkitt's lymphoma cell line, and RL, a non-Hodgkin's lymphoma cell line.
Selection of scFv Antibodies for B-CLL-specific Cell Surface Antigens using Antibody Phase Display and Cell Surface Panning
Immunizations and scFv Antibody Library Construction
Peripheral blood mononuclear cells (PBMC) were isolated from blood drawn from CLL patients at the Scripps Clinic (La Jolla, Calif.). Two rabbits were immunized with 2�107 PBMC pooled from 10 different donors with CLL. Three immunizations, two sub-cutaneous injections followed by one intravenous injection, were done at three week intervals. Serum titers were checked by measuring binding of serum IgG to primary CLL cells using flow cytometry. Five days after the final immunization, spleen, bone marrow, and PBMC were harvested from the animals. Total RNA was isolated from these tissues using Tri-Reagent (Molecular Research Center, Inc). Single-chain Fv (scFv) antibody phage display libraries were constructed as previously described (Barbas et al., (2001) Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). For cell surface panning, phagemid particles from the reamplified library were precipitated with polyethylene glycol (PEG), resuspended in phosphate-buffered saline(PBS) containing 1% bovine serum albumin (BSA), and dialysed overnight against PBS.
Antibody Selection by Cell Surface Panning
The libraries were enriched for CLL cell surface-specific antibodies by positive-negative selection with a magnetically-activated cell sorter (MACS) as described by Siegel et al. (1997, J. Immunol. Methods 206:73-85). Briefly, phagemid particles from the scFv antibody library were preincubated in MPBS (2% nonfat dry milk, 0.02% sodium azide in PBS, pH 7.4) for 1 hour at 25� C. to block nonspecific binding sites. Approximately 107 primary CLL cells were labeled with mouse anti-CD5 IgG and mouse anti-CD19 IgG conjugated to paramagnetic microbeads (Miltenyi Biotec, Sunnyvale, Calif.). Unbound microbeads were removed by washing. The labeled CLL cells ("target cells") were mixed with an excess of "antigen-negative absorber cells", pelleted, and resuspended in 50 μl (1010−1011 cfu) of phage particles. The absorber cells serve to soak up phage that stick non-specifically to cell surfaces as well as phage specific for "common" antigens present on both the target and absorber cells. The absorber cells used were either TF-1 cells (a human erythroleukemia cell line) or normal human B cells isolated from peripheral blood by immunomagnetic negative selection (StemSep system, StemCell Technologies, Vancouver, Canada). The ratio of absorber cells to target cells was approximately 10 fold by volume. After a 30 minute incubation at 25� C., the cell/phage mixture was transferred to a MiniMACS MS+ separation column. The column was washed twice with 0.5 ml of MPBS, and once with 0.5 ml of PBS to remove the unbound phage and absorber cells. The target cells were eluted from the column in 1 ml of PBS and pelleted in a microcentrifuge at maximum speed for 15 seconds. The captured phage particles were eluted by resuspending the target cells in 200 μl of acid elution buffer (0.1 N HCl, pH adjusted to 2.2 with glycine, plus 1 μg/ml BSA). After a 10 minute incubation at 25� C., the buffer was neutralized with 12 μL of 2M Tris base, pH10.5, and the eluted phage were amplified in E. coli for the next round of panning. For each round of panning, the input and output phage titers were determined. The input titer is the number of reamplified phage particles added to the target cell/absorber cell mixture and the output titer is the number of captured phage eluted from the target cells. An enrichment factor (E) is calculated using the formula E=(Rn output/Rn input)/(R1 output/R1 input). In most cases, an enrichment factor of 102−103 fold should be attained by the third or fourth round.
Analysis of Enriched Antibody Pools Following Panning
After 3-5 rounds of panning, the pools of captured phage were assayed for binding to CLL cells by flow cytometry and/or whole cell ELISA:
To screen individual scFv clones following panning, TOP10F′cells were infected with phage pools as described above, spread onto LB plates containing carbenicillin and tetracycline, and incubated overnight at 37� C. Individual colonies were inoculated into deep 96-well plates containing 0.6-1.0 ml of SB-carbenicillin medium per well. The cultures were grown for 6-8 hours in a HiGro shaking incubator (GeneMachines, San Carlos, Calif.) at 520 rpm and 37� C. At this point, a 90 μl aliquot from each well was transferred to a deep 96-well plate containing 10 μL of DMSO. This replica plate was stored at −80� C. IPTG was added to the original plate to a final concentration of 1 mM and shaking was continued for 3 hours. The plates were centrifuged at 3000 xg for 15 minutes. The supernatants containing soluble scFv antibodies were transferred to another deep 96-well plate and stored at −20� C.
A sensitive whole-cell ELISA method for screening HA-tagged scFv antibodies was developed:
Primary screening of the scFv clones was done by ELISA on primary CLL cells versus normal human PBMC. Clones which were positive on CLL cells and negative on normal PBMC were rescreened by ELISA on normal human B cells, human B cell lines, TF-1 cells, and the CLL-AAT cell line. The clones were also rescreened by ELISA on CLL cells isolated from three different patients to eliminate clones that recognized patient-specific or blood type-specific antigens. Results from representative ELISAs are shown in
The number of unique scFv antibody clones obtained was determined by DNA fingerprinting and sequencing. The scFv DNA inserts were amplified from the plasmids by PCR and digested with the restriction enzyme BstNI. The resulting fragments were separated on a 4% agarose gel and stained with ethidium bromide. Clones with different restriction fragment patterns must have different amino acid sequences. Clones with identical patterns probably have similar or identical sequences. Clones with unique BstNI fingerprints were further analyzed by DNA sequencing. Twenty-five different sequences were found, which could be clustered into 16 groups of antibodies with closely related complementarity determining regions (
Characterization of scfv Antibodies by Flow Cytometry
The binding specificities of several scFv antibodies were analyzed by 3-color flow cytometry (
To determine if the antigens recognized by the scFv antibodies were overexpressed on primary CLL cells, PBMC from five CLL patients and five normal donors were stained with scFv and compared by flow cytometry (
CLL patients can be divided into two roughly equal groups: those with a poor prognosis (median survival time of 8 years) and those with a favorable prognosis (median survival time of 26 years). Several unfavorable prognostic indicators have been identified for CLL, most notably the presence of VH genes lacking somatic mutations and the presence of a high percentage of CD38+B cells. Since scFv-9 recognizes an antigen overexpressed in only a subset of CLL patients, we sought to determine if scFv-9 antigen overexpression correlated with the percentage of CD38+cells in blood samples from ten CLL patients (
Identification of Antigens Recognized by scFv Antibodies by Immunoprecipitation (IP) and Mass Spectrometry (MS)
To identify the antigens for these antibodies, scFvs were used to immunoprecipitate the antigens from lysates prepared from the microsomal fraction of cell-surface biotinylated CLL-AAT cells (
The following references are incorporated herein by reference to more fully describe the state of the art to which the present invention pertains. Any inconsistency between these publications below or those incorporated by reference above and the present disclosure shall be resolved in favor of the present disclosure.
It will be understood that various modifications may be made to the embodiments disclosed herein. For example, as those skilled in the art will appreciate, the specific sequences described herein can be altered slightly without necessarily adversely affecting the functionality of the antibody or antibody fragment. For instance, substitutions of single or multiple amino acids in the antibody sequence can frequently be made without destroying the functionality of the antibody or fragment. Thus, it should be understood that antibodies having a degree of homology greater than 70% to the specific antibodies described herein are within the scope of this disclosure. In particularly useful embodiments, antibodies having a homology greater than about 80% to the specific antibodies described herein are contemplated. In other useful embodiments, antibodies having a homology greater than about 90% to the specific antibodies described herein are contemplated. Therefore, the above description should not be construed as limiting, but merely as exemplifications of preferred embodiments. Those skilled in the art will envision other modifications within the scope and spirit of this disclosure.
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Originally written on Celtics Life | Last updated 2/13/13
The Celtics host the Bulls tonight at the Garden (7:30 tip), in the 4th and final meeting between the clubs this season. So far this year the Bulls have won 2 of the 3 match-ups, including the only game in Boston. Here's what rounds 1-3 have looked like. Nov. 12th: Celtics 101, Bulls 95 - Celtics win in Chicago behind Rajon Rondo's near triple-double (20 points, 10 assists, 9 boards). Luol Deng goes for 27 and 10 in a losing effort. Dec. 18th: Bulls 100, Celtics 89 - Rondo again puts up big numbers (26 points, 8 assists), but this time the Bulls win easy behind Deng and Carlos Boozer (21 points each), and a huge performance by their bench (outscored the C's reserves 37-16) Jan. 18th: Bulls 100, Celtics 99 - Ugh. Almost a month later this one still hurts. The Celtics fell to the Bulls in overtime on a last second Marco Belinelli floater, only after a terrible jump ball call allowed the Bulls to send the game to OT. This was the 2nd loss in a row for the C's during a losing streak ...
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List-it is a fast, simple, 100% Free+Open Source (MIT License) note-taking tool for Firefox. Designed by a research group at MIT, List-it lets you quickly take short notes to yourself + quickly access them whenever and wherever you need 'em.
Troubleshooting Facebook Games in Firefox
Having trouble playing your favorite Facebook game in your favorite browser? Thankfully, there is an easy way you can troubleshoot and fix this type of problem. I will walk you through the steps, step by step and hopefully you will get back to playing your favorite Facebook game in no time!
Keep Java Active
Having problems with having to re-activate Java for a website you use a lot in Firefox? There is a really quick fix for this that some people may or may not have seen. With that said, I thought I might share it with you all real quick right here.
Abduction – Easier Website Screenshots
The Firefox extension Abduction makes it easy to do webpage screenshots. This extension for Firefox has the potential to be a website designer's best friend. Being able to do quick and easy screenshots can make any type of website design work easier. You could cut minutes out of building that online portfolio or whatever other projects you might have.
The fantastic Firefox add-on betterFox claims that it can make Firefox 15% faster. Is it true? Well, it does contain more than a few little tweaks here and there that will make browsing your favorite websites using Firefox a little faster.
SEO Doctor
Need another SEO-inspired add-on for Firefox? SEO Doctor is made with both beginners and experienced SEOs in mind and it's scoring system and recommendations are based on official SEO documents, namely Google Webmaster guidelines, Google Image guidelines and Google SEO starter guide as well as my own experience.


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UPrinting Business Card Giveaways 205
This Friday giveaway is sponsored by UPrinting, a shop that provides a wide range of online printing services. We are giving away 3 sets of 250 die-cut UPrinting business card printing to 3 winners. Each winner will receive a set of 250 business card printing with free shipping. To enter this contest, all you have to do is write a comment in this post. Contest ends Dec 29, 2011.


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Shameful Vienna Antics Contrived to Change Course of Celts' History; Parkhead Coach Grant Recalls the Utter Chaos of His Club's Last Clash with Rapid and Insists That the Ramifications Were Long-Lasting
Byline: Jim Black
PETER GRANT can think of two reasons as to why Celtic lost so much ground on their archrivals and fell so spectacularly from grace for the best part of a decade - Mo Johnston decided to sign for Rangers and, more crucially, Rapid Vienna were given a second chance in the European Cup Winners' Cup.
As far as historic moments go, Grant insists the significance of that shambolic meeting with the Austrians in late 1984 simply cannot be underplayed. In fact, the former Parkhead captain, now assistant coach, claims the events surrounding the Cup Winners' Cup tie destroyed the ambitions of a generation of players and set the club back years.
As preparations begin to host Rapid in European competition again this week, 25 years on from an unforgettable episode, focus has temporarily switched to the Scottish football archives of infamy.
To recap, Celtic had lost the first leg of the tie in Austria 3-1 but looked to be heading through when they went 3-0 up in the return meeting in Glasgow. However, Rapid were incensed when the third goal, scored by the late Tommy Burns, was allowed to stand.
This resulted in Burns suffering two assaults, the second of which saw Roman Kienast sent off.
The award of a penalty to Celtic sparked further bedlam, during which Rapid substitute Rudolph Weinhofer feigned injury as missiles were hurled from the stands.
Grant missed the penalty, but Celtic still won 3-0 and, seemingly, secured their passage. However, Rapid appealed against the result and, remarkably, UEFA decreed the second leg should be replayed 100 miles from Celtic Park.
Old Trafford was named as the venue for Celtic's subsequent 1-0 defeat and they were punished again after Celtic fans attacked Rapid goalkeeper Herbert Fuhrer and goalscorer Peter Pacult (now Rapid's coach) at the end of the match.
The home leg of the following season's meeting with Atletico Madrid had to be played behind closed doors as a result. Celtic lost and, according to Grant, the price of defeat was heavy.
'What happened set Celtic back five to 10 years, in my opinion, because the better you did in Europe, the more players wanted to come to the club,' he said.
'Celtic were, and still are, a massive name, but to attract the best players you have to be progressing in Europe and that is still the case to this day.
'I felt that the Rapid affair and the Mo Johnston incident were two massive moments in the club's history.
'I honestly don't believe we would have gone so long without winning the title or that Rangers would have achieved nine-in-a-row if Mo Johnston had returned to Celtic.
'Rangers had the money to buy the England goalkeeper, captain and right-back, as well as other top-quality players.
'Meantime, we were financing a new stadium and the club couldn't pay top dollar to bring players of a similar quality to Celtic Park.
'So these events had a huge effect on the club and the supporters and they also impacted on players' careers, including my own.
'The effects of the Rapid Vienna affair were felt far beyond the tie when we had to play Atletico Madrid behind closed doors the following season.
'Having done exceptionally well to draw 1-1 in the first leg in Spain, the complete lack of atmosphere in the return match definitely affected our performance and we lost 2-1. 'That period was the most disappointing of my career because we had a good mix of young and experienced players and we proved that by going to Madrid and getting a result.
'I really do feel we could have gone on to achieve something in Europe with the players we had at that time, guys like Danny McGrain, Paul McStay, Roy Aitken and Tommy Burns.
'So the Rapid business had a definite snowball effect, both financially for the club and on the players because we felt we had beaten them fair and square and had done nothing wrong as a team. …


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You're not the boss of me
Joined: Nov 2006
Location: Downtown
Oddometer: 156
Crazy warm day here on the east coast
DR Rock and I decide to take advantage of the crazy weather we are having here on the east coast.
So...at 11 am, when the threat of rain had subsided, and after vetoing his planned route of interstate 80 to the Delaware Water Gap route because of my fear of speed, we set out for Northwest CT via the Saw Mill Parkway.
Gently building up to the parkway speed we drove through Harlem.
Notice the truck with Florida plates driving in reverse; he almost takes out the gentleman on the bicycle...got to love it....dude, this in NYC not Oxford Florida.
Then as my stomach began to turn in anticipation of heading onto the Sawmill parkway to the cross county parkway to ....the Hutchinson parkway ..merging onto the Merrit parkway...
I can finally begin to relax on the country roads of Connecticut:
Unknown to me, DR Rock had a hidden agenda......
Time to learn how to pick up the bike once you put it down. You see in NYC we have been having a run of bad luck where either his bike or mine has been found down on the ground by either cage strikes or gusts of wind propelling our bike covers like windsurfers, to the ground. So after his demonstration I give it a try........
and decide...........
I would rather bask in the sun....or RIDE (if anyone has any suggestions for lifting techniques I am all ears ).
Stopped for gas and a quick bite to eat at an old mom and pop grocery store.
Where else but in Greenwich Connecticut (..."Greenwich is one of the wealthiest towns per capita in the world...") do you have the option to leave a penny, take a penny or... leave a dollar / take a dollar????
Eating our snacks on the porch, this 86 year old man told us two jokes ...
The first of which we didn't get..
and the second one we can't remember.
From there we headed on
to familiar territory..
Where I still feel more comfortable riding down second avenue amidst Manhattan traffic than I do travelling a speed anywhere above 60 mph. Will I ever make it to I-80????? I will keep working on it. Thanks for a great ride DR Rock....got to love that global warming.....
Mod Squad
Joined: Aug 2002
Location: Toronto, ON
Oddometer: 57,139
Beautiful!! The weather is crazy for January in the east, it was 11 Celsius today in Toronto! thanks for the ride report and the great pics.. maybe you can do your next report with larger pics! More to look at!
keep the rubber side down
Joined: Jun 2006
Location: NYC
Oddometer: 142
hey ldf,
If your bike has taken a nap (even if it was just a dry run), you're not a n00b anymore! Get rid of that n00bness :-)
I made a slightly larger loop than you guys did, going into MA and back down. It got up to 70F! Crazy.
Part of the problem
Joined: Aug 2006
Location: NYfC, yff
Oddometer: 5,536
Ride to support global warming
This is all Al Gore's fault. I saw that movie... Didn't he invent global warming?
I guess it's my turn for the next ride report.
I still can't believe people leave a dollar / take a dollar in Greenwich, CT. I wouldn't believe it if I didn't see it.
One thing I learned on this ride is that roads that show up as dotted lines on GPS in this area are not dirt / undeveloped roads. They are PRIVATE. With a guard house. And a guard. .
This was our track:
Just riding around. Fun.
"I came into this game for the action, the excitement; go anywhere, travel light... get in, get out... wherever there's trouble, a man alone... Now they got the whole country sectioned off; you can't make a move without a form." --Robert De Niro as Archibald 'Harry' Tuttle in Brazil, 1985. The Mobius Trip index | Spot tracking live 4/18-5/4/13 | AdventureLoft Tent Space
loosely portrayed
Joined: Aug 2001
Location: Brooklyn
Oddometer: 31,764
Great ride report Donna, but why you trying to lift that bike.....
(That's just plain cruel Doc....)
That's what yer hubby's for
Looks like fun
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text: My husband planned this 11th anniversary weekend to take in the local parks and fall colors. We stayed in the Tamarack log cabin that was set up on a hill away from the other units. Lovely & clean accommodations. A beautiful park setting with wildlife, pond & fire pit available to us to each evening. It was far enough away... More


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Top StoryBeadlock Basics: Ultra Wheels Xtreme X103 Beadlock Wheels
While these specialty wheels might not be for everyone, there's no better time than now to take a close look at the Xtreme X103 beadlock wheel and how it work.
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In the diesel performance realm, it's not uncommon for general repair items to morph into high-horsepower builds. Case in point, this '09 GMC Sierra came into Flynn's Shop in Alexander, Illinois.


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Zimbabwe Professional Hunters and Guides Association
This is an association dedicated to the perusal of ethical and sportsman like hunting and guiding in Zimbabwe. We aim to maintain the highest standards of professionalism amongst our members, and are committed to the long term management and utilisation of wildlife.
We are committed to the training and education of future hunters and guides in the industry both in Zimbabwe and Africa as a whole.
With-in this site you will find news and views, lists of members,firearms regulations and other useful information regards hunting in Zimbabwe. We hope you find this site informative and helpful.


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Cost of Baja 500
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Beastly Adventurer
Joined: Oct 2007
Location: North of Jack Daniel's, South of Country Music
Oddometer: 2,997
Originally Posted by
Strong Bad
How do you chase both sides (Pacific and Gulf) with one chase truck? Using last year's course as an example, half way was around San Matais. There is no way you could do a rider change at San Matias/Mike's Sky Rancho Road and see the rider again before the final hwy 3 road crossing the other side of Ojos.
Here is how you do it: Send Chase 1 to where they cross the hwy at Ojos. Chase 2 goes to where they come out to the road @ Cerro Colorado. Chase 1 goes from Ojos on down to the base of the summit at Santa Catarina. Chase 2 turns around at Cerro Colorado and goes all the way around to Llano Colorado on the Pacific side. After Llano Colorado, Chase 2 catches the rider again at Santo Thos. The Chase 1 goes on to Borrego and follows back towards Trinidad doing the rider change somewhere between the two. After the rider change, Chase 1 goes to the Hwy 3 road crossing the other side of Ojos.
Adding another chase adds so much more money to the equation... in that case I'll just do it solo...
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Awesome. Give puddycat the Bengals since they're his team, and switch my 2nd team to the Patriots or Dolphins. Again only if someone doesn't want them for their first teams. Thanks.
Awesome. Give puddycat the Bengals since they're his team, and switch my 2nd team to the Patriots or Dolphins. Again only if someone doesn't want them for their first teams. Thanks.
I'll take Buffalo as my second team, unless someone wants them as their first choice..
I'll take the Pats + 1 if needed.
I'm tryin' to explain to you that Ronald Reagan was the devil. Ronald Wilson Reagan? Each of his names has six letters? 6-6-6? Man, doesn't that offend you?
-Huey Freeman
Cool. Give Mavfan the Patriots, and I'll take the Dolphins and/or Ravens as needed. The more the merrier.
Cool. We have commitments for up to 16 teams with the following 11 teams taken (as first choices): Jets, Patriots, Bengals, Browns, Colts, Broncos, Eagles, Giants, Packers, Vikings, Seahawks
Anyone who conducts an argument by appealing to authority is not using his intelligence; he is just using his memory. - Leonardo da Vinci
I'll take the Ravens like in years past.
I can also grab another team if needed, doesn't matter which.
Don't know what happened to femurov, but I'll try to open a new thread tomorrow. We'll have 3 deadlines for proxies each day and try to finish it up in a week. Send proxy lists to the draftcommish account. Make sure they have enough names on them or a pick will be made for you. Trades will be limited to before your block. It sucks to try to project that, but it's the best I can do on short notice.
We'll start the draft on Wednesday morning. I'll block it all out as best I can. Pm me if you have any questions. Thanks guys. I might need a couple of gms to take on one more team if you have the time.
Bills - Roadblock
Dolphins - Ellis269
Jets - Roadblock
Patriots - Mavfan2390
Bengals - puddycat
Browns - Ellis269
Ravens - NormanBates
Steelers - NormanBates
Colts - Natjaxbree
Jaguars - List
Texans -
Titans - Maroon Typhoon
Broncos - Femurov
Chargers -
Chiefs -
Raiders - puddycat
Cowboys - Femurov
Eagles - Wings
Giants - TeddyB
Redskins - femurov
Bears - Mavfan2390
Lions -
Packers - MaroonTyphoon
Vikings - TeddyB
Buccaneers - Natjaxbree
Falcons -
Panthers - Roadblock
Saints -
Cardinals - McGruffHawk
49ers - Ellis269
Rams - MaroonTyphoon
Seahawks - McGruffhawk
Last edited by Draftcommish; 04-18-2012 at 08:25 PM.
These are the open teams, if you have any prefrence. . . pick them now. Steelers, Jaguars, Texans, Titans, Chargers, Chiefs, Cowboys, Redskins, Bears, Lions, Falcons, Panthers, Saints. Anyone who wants a 3rd team may have it. If you said you can do two, but don't pick a 2nd team. . . I'll assign another one to you. Any teams that are not represented will use a value chart for their proxy lists. I've got several to choose from, so I'm not too worried about filling up all the teams.
GMs who've said that they can represent a 2nd team, but haven't picked or assigned one yet include Mavfan2390, Wings and NormanBates. Haven't heard back from femurov or jammer yet, but those two usually take two teams in this one as well. That should put us pretty near to filling up.
Last edited by Draftcommish; 04-17-2012 at 07:37 PM.
Let's give the Cardinals to McGruffHawk and the Raiders to puddycat. Those were more or less their requests earlier.
I'll take the Titans for my third team.
I'd be glad to pick up the Panthers as a third team, unless someone wold like them as a #2. If that is the case, I'd like to have Houston.
Time to assign some teams to GMs so that the top half of the draft is represented well.
Maroon Typhon can have the Titans.
Roadblock can have the Panthers.
Femurov can have the Cowboys.
Mavfan2390 can have the Bears.
Anybody else want another team?
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can someone post pictures please!
I will tonight I promise this time. I just took some new pictures 5 min ago , I will be home at 7 and post them. And there was no public soft openings just as I was told on Monday and today
---------- Post added 06-14-2012 at 02:40 PM ----------
I have an iPhone and the pictures are 16-20 times to big to post with mc limitations
Im here for fun and info not be a bully! Carsland here I come


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2013-12-23 - Swedish high-energy Heavy/Speed Metal outlet ENFORCER are proud to announce their first ever European headlining tour which will see the Stockholm based foursome hit the road in 2014. After presenting their current record »Death By Fire« the audiences of the "Hunting Across Europe" Tour alongside label- and country mates GRAND MAGUS and British Metal veterans ANGEL WITCH in early 2013, the aspiring act has moved up to the next level. Joining ENFORCER on the road will be Canadian Metalheads SKULL FIST and Dutch Hard Rock assault VANDERBUYST.
Stated ENFORCER vocalist/guitarist Olof Wikstrand: "It's our first headline tour ever and it's gonna be great to finally give the fans a proper long set including songs from all our three albums. This time we also have the chance to bring much of our stage props, lightning rigs and other things to make the show complete. You can expect hell on earth! Expect the ultimate heavy metal show of the new millennium."
Don't miss your chance to be part of the high voltage Metal package at the following dates:
30.01. NL Amsterdam - Melkweg
31.01. DE Saarbrücken - Garage
01.02. DE Essen - TuRock
02.02. NL Drachten - Iduna
03.02. UK London - The Underworld
04.02. FR Paris - Divan Du Monde
05.02. DE Köln - MTC
06.02. DE Pforzheim - Kupferdächle
07.02. CH Aarau - Kiff
08.02. DE Lichtenfels - Paunchy Cats
09.02 IT Turin - Nightbreaker Club
10.02. IT Bologna - Freak Out Club
11.02. AT Graz - Explosiv
12.02. AT Dornbirn - Conrad Sohm
13.02. DE München - Backstage
14.02. DE Aalen - Rock It
15.02. BE Kortjijk - JC Den Ast
16.02 DE Darmstadt - Steinbruch Theater
18.02. HU Budapest - Dürer Kert
19.02. CZ Prague - Club Kain
20.02. PL Krakow - Klub Kwadrat
21.02. DE Leipzig - Bandhouse
22.02. AT Wien - Escape
23.02. DE Berlin - K 17
24.02. DE Hamburg - Marx
25.02. DK Copenhagen - TBC
26.02. NO Oslo - Vulcan arena / Pokal Room
27.02 SE Stockholm - Pussyagogo
28.02. SE Umea - House Of Metal
01.03. SE Göteborg - Truckstop Alaska
The current ENFORCER record »Death By Fire« is out now. Order your copy here: http://smarturl.it/Enforcer
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ENFORCER - »From Beyond« out now!
2015-02-27 11:30 - The wait is finally over! Swedish Heavy Metal foursome ENFORCER release their brand new masterpiece »From Beyond« today. Details
ENFORCER – video clip released
2015-02-20 16:30 - Today ENFORCER presents their video clip for the album opening track 'Destroyer'. The video was shot in Arvika/Sweden and edited by Gustav Öhman Spjuth. Details
ENFORCER – new album trailer released!
2015-02-18 14:15 - Swedish Heavy Metal foursome ENFORCER will release their upcoming new album »From Beyond«. Enjoy the new album trailer, featuring statements and some new song excerpts. Details
ENFORCER – lyric video for released!
2015-01-16 12:30 - Today, ENFORCER reveals the official lyric video for 'Undying Evil'. The clip perfectly underlines the gloomy and blasphemous mood of the album track. Details
ENFORCER – new album »From Beyond« revealed!
2015-01-05 13:00 - Swedish Heavy Metal act ENFORCER has finished the recordings of their upcoming new album, called »From Beyond«. Details
ENFORCER – additional headliner shows ...
2014-10-21 13:30 - Swedish speed metal maniacs ENFORCER have just announced their return to the European roads for November. Following the success of their latest album »Death By Fire«, the ... Details
ENFORCER: "Roll The Dice" - Diamonds On ...
2013-02-05 01:00 - As 'Death By Fire World Tour' draws near, Enforcer would like to thank everyone who came out to see us on the previous tour. Watch Enforcer performing 'Roll the Dice' live ... Details
ENFORCER: "Death By Fire" – out now!
2013-02-01 03:00 - Today is the release of the new ENFORCER record "Death By Fire". If you wanna take listen, click on the exclusive stream on the homepage of UK's Terrorizer magazine ... Details
ENFORCER: "Death By Fire" albumstream on ...
2013-01-30 02:00 - UK's Terrorizer Magazine gives you an exclusive listen to the new album by Swedish heavy metal upstarts ENFORCER. Titled 'Death By Fire', the record marks their debut for ... Details
ENFORCER: Videoclip Online Premiere on ...
2013-01-21 01:00 - From 15.00 CET on German Rock hard magazine premiers the new ENFORCER Video Clip for "Mesmerized By Fire" on their homepage: Details


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This (ultimately unused) logo was created for a company that deals with compressing oil and gas as its pumped out of the earth. I aimed to communicate the pipes within pipes that it takes to get efficient flow. Also, I wanted to make a unique typeface.
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