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I was hoping shortly after Labour Day as well.
Check out NVCR in premarket. It is not just the premarket share price, which can be manipulated with a fake bid or ask and removed quickly before the open, BUT THE PREMARKET VOLUME THAT IS JUST SHORT OF A MILLION SHARES. Something very real is happening. This is no preopen mirage!
I am fully on board with the law suit and following it as closely as I can. Hoping for great results. The only question is whether it will settle or will go to trial and how long that might take.
DD, I am with you for the most part. I now recognize that I do not know enough about what is going on behind the curtain to really know whether the silence from management or the delay is really warranted. I would prefer a little more real news (not bread crumbs) even at the risk of shorts twisting it some what. As far as the lengthy delay, I simply do not know the real reason why it is happening, so I will reserve my judgement on this until I do know more. But I will not wait forever - dangling the carrot only goes so far. We do need an element of trust in the management - otherwise you might as well sell your shares.
OK, that is great and clear. BUT, I would suggest in your future posts that can be read as bashing, be explicit that you are only bashing the management and not the stock. It will be easier to understand your position. It usually comes across as a total bash of everything in consort with the shorts - not in your favor if you still hold a large position.
Mike, I do not usually respond to those I consider bashers. I do not have enough hours in the day as it is. I will make an exception to that here. @FeMike, @PoorMan
Here is one thing both you and PM can do to help us believe you. In an post simply state the following.
1) I believe management is terrible and the main cause for the NWBO decline.
2) NWBO science works as shown in the trials and JAMA article and Bosch ASCO presentation and can save lives.
3) DCVAX-L works and with the right management is worth a lot of money
4) NWBO patent assets are very valuable.
5) MAA will be applied for and gain UK approval even if delayed due to management
Are you Peter Davis? PgsD - mid initials gs? You are always retweeting him? What are his credentials?
More on the Kessev Tov case from the Memo:
Amazing how sloppy the defendants were! See note 4 below!:
This was my response to Bear on this matter:
Bear, I do not agree. They may have had an agreement for use of poly in the trials. But I doubt they had one tied up for all future commercial use after approval. While ordinary contracts in the course of doing business do not require disclosure, any such far reaching contract (deal - however it is structured) IMO would be considered material and require disclosure. We have had no such disclosure, therefore I conclude that no such deal has been made as of yet.
I hadn't noticed before. We had an LL and an LP, now we have an LL and 2 X LP. Cute. Like in the Superman comics, all women in Supe's life were LL.
Bear, here is what I posted back in May 2023 about BP or NWBO buying Oncovir:
hankmanhub
Re: meirluc post# 595498
Tuesday, May 23, 2023 10:57:18 PM
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I might voice a possible concern. As this thread seems to assume that Oncovir on its own is going nowhere with the patent due to expire shortly, but would be worth alot in the hands of NWBO as part of the dcvax platform. It seems to me that with LP seemingly demanding a top dollar from BP (whichever one) the thing to do for a BP is to acquire Oncovir at a multiple of its current value, but this would still be a dirt cheap entree to NWBO for a necessary ingredient for DCVAX. Right now would the time to do this as the BP could easily outbid NWBO in its bid for Oncovir at this time - this may not be true further down the road..
hankmanhub
Re: meirluc post# 595503
Tuesday, May 23, 2023 11:18:23 PM
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My point was not for BP to buy Oncovir for itself, but to use it as an entree to make a deal with LP and NWBO for something they (NWBO) may need more than just a large checkbook. This would be chips on the table which they can use to make a good deal with NWBO.
hankmanhub
Re: skitahoe post# 595504
Tuesday, May 23, 2023 11:24:14 PM
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Skit, I do not think what the owners of Oncovir are looking for is guaranteed continued employment. that is small potatoes. They will want a cut of the pie, and a seat at the table when Oncovir is taken over by BP (now) or by NWBO (later) to merge the DCVAX platform with iclc and thus also extend the patent that is near expiration.
hankmanhub
Re: meirluc post# 595514
Tuesday, May 23, 2023 11:50:53 PM
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While you are right, the cost of iclc after the patent expires will not be terribly high, but thejr lost opportunity costs of not owning Oncovir and being able to control iclc by extending its patent will be a significant bargaining chip and worth alot to NWBO. The whole (merged DCVAX and iclc) is much greater than the sum of its parts.
On July 20 & 25 I posted the following:
hankmanhub
Re: Doc logic post# 613686
Tuesday, July 25, 2023 4:46:07 PM
Post#
613710
of 623388
On the 19th I posted several thoughts on the possibilities for the additional modules (pages) which I copied below. I have inserted into your post the correspondence to my item numbers from my post, Seems like the ideas are pretty similar except I offered more choices.
.
To tell you the truth there are a few things that 700,000 extra pages could be focused on. ((4)) First of all this trial started as de novo GBM then turned to GBM and rGBM. As you know GBM was reclassified based on IDH status too before results were published so plenty of things this extra paperwork could represent. ((2)) The possibilities also includes supporting evidence of tissue agnostic status due to the natural in vivo way DCs and, therefore, DCVax works through the imitation of the process of DC maturation/activation and subsequent immune response in almost ALL DISEASES. ((2.1)) Then there is the use of Poly ICLC as an activator of increased immune response ((3)) and digitization of Edens which will be the answer, at least on an early interim basis, for getting a personalized vaccine treatment to the masses of patients wanting and needing them. Take your pick, they are all good choices.
hankmanhub
07/20/23 10:00 AM
#611917 RE: hankmanhub #611478
Addition to my post:
I would add one more possibility that I missed in the original post;
2.1) to account for Poly iclc?
Thus the post now reads:
At 27 modules and 1.7 million total pages,
I assume that as 1.7 M pages is much larger the the usual submission, then it follows that 27 modules is much larger than usual as well. Anyone know how many modules the typical submission contains? If for example the answer id say 20 modules, then we have 7 extra modules. (my math is very good).
My question is, what would be the substance of these say 7 extra modules?
1) due to the diff requirements of the several RAs?
2) to account for tissue agnostic?
2.1) to account for Poly iclc?
3) to account for EDEN?
4) to account for the addition of rGBM?
5) to account for solid tumors?
6) to account for Direct?
7) to account for the many possible combos, in particular those trials already underway?
8) to treat DCVAX as a platform?
9) to account for ECA?
10) to help lead up to any of the above to ease future trials and lighten their submissions?
11) For any combination of the above?
Clearly something is happening here, even if we are only actually applying for GBM in the UK.
Any ideas?
Whatever the answer to the above we really seem to be on the one yard line this time. Can't be long now!
True, even so, that is still far short of 1300
And most of the 1300 were never treated with DCVAX-L.
Who is peter davis?
This article totally ignores DCVAX-L in its vaccine section. It only mentions 20 mRNA vaccines. Nothing about dendritic cell vaccines at all. Like they do not exist! Lousy research on their part!
What makes you say that?
Of course the main difference between now and then is that approval is around the corner (I know, you've heard that before) and they will be in a position to actual carry out those combo trials.
Ex, so which is it now? Are you arguing that they truly have good reason for the delay - so everything is OK, but there will be a delay to be expected. Or is the delay really a bad thing because there is no good reason for it and they only want to delay the bad news PR as long as they can?
My guess, is they could - but will they. The past record is not one to inspire much confidence that they will. Personally, I lack the expertise to determine the viability of a criminal case or its likelihood, but certainly hope one is possible and likely.
I think that this is the old interview from a couple of months back – nothing new. At the start of the interview LG is introduced as just having come back from ASCO, so that places this interview back in early to mid June 2023. Nevertheless, there are a couple of points that I now noticed that I did not pick up on the last time I heard it.
LG said that it is being double checked now…. So that tells us it is being checked for at least 2 ½ months or longer at this time – so they must be very close to finished now.
LG also said that they “would be filing there…” (not clear where “there” is -- US?) then he says “we will apply in the other countries … in each of the 4 countries”, then he lists off 3 of them, UK, Canada and Germany (doesn’t mention the 4th, and then continues to say that “we will be filing in each of those places IN ORDER, to obtain commercial approval…” -- So it seems that they are going to file for approval serially – not what I had hoped to hear.
Idle speculation and any response I might make would be idle speculation as well.
UNLESS THEY ARE SETTLED!
Danish, Robotdroid knows nothing is happening in the case at this time until both the final response on the MTD is in on Sept 27 and some more time for the judge to rule on the MTD, so lets guess sometime in mid to late October. So In the meantime he keeps asking the safe question, why nothing is happening in the case, as he knows nothing will happen until sometime in October or so. Once the judges denies the MTD, that question will not be as safe, because as we approach discovery, the defendants will throw in the ball and seek to arrange a settlement. But in the meantime until about October - nothing big will happen in this case. The coming steps will be:
1) FINAL RESPONSE BY DEFENSE ON THE MTD ON SEPT 27
2) RULING BY THE JUDGE - SOMETIME IN OCTOBER MOST PROBABLY
3 MOVES TO START OF DISCOVERY - SHORTLY THEREAFTER
So do not expect anything newsworthy to happen before say end of Oct or Nov.
In the old days of my early investment career, a retail investor's game was to try to spot the promising new companies, with sound science and promising ideas to develop and get in early while it was still cheap. The normal expectation was that as the good news kept coming, the stock would continue to climb based on these expectations. The risk was in making the wrong choice with a company whose expectations did not pan out. Good news then continue to climb, bad news - well that's the risk you took with these risky investments. But going DOWN on good news (especially in a big way like May 10) was not expected, and retail investors had confidence in the market and felt they had a shot at winning if their research was good.
This was important for these young developing companies. It meant they had a dependable source of funding from this retail investor market. They just had to convince the investors they had the real deal and were not selling snake oil - which if they really had sound science and good research was not hard for them to do. This was the foundation of the ability of young companies, with new ideas and technologies to get funded and eventually bring their products to market. This used to be investing 101. This is what made the US the number one source of new development and technology in the WORLD.
The SEC is now allowing this system to be hijacked by the MMs and hedge funds and shorts etc, and turning this whole market on its head. It is now a very uneven playing field. The rules and regulations are all being played by the MMs et al. And those regs that are on the books are not being adequately enforced. And when they do catch someone with egregious offenses they get off with a mere slap on the wrists, and pay the fines as part of the cost of doing business. The fact pattern of an NWBO should not be able to exist. NWBO is the poster boy for this situation. Imagine if the MMs were to win and force NWBO (can't happen anymore - but imagine) to close its doors. Imagine the tremendous loss to the millions suffering from cancer - all to the gain of a few who are able to manipulate the market to their benefit and due to the weak and lackadaisical oversight. Imagine the loss to the world of all the small companies with promising technology that were never allowed to make it to the market and of which you never even heard!
Congress needs to revamp the laws to maintain the US position as the foremost place to develop new and promising technology. This is seriously at risk without prompt serious action with teeth by congress - not just a band aid change to a couple of regs just for show. The SEC needs to be an effective regulator who has the tools it needs AND THE WILL to really regulate!
NWBO gives us basis to assert strongly that something is very wrong with the state of the market system in the US (and Canada) and its supposed regulation which has totally failed to do its job. the pervasive control of the MMs and hedge funds and dark money over the market without serious fear of significant retribution tells small retailer investors - the game is rigged against you - you are not playing on a fair, level playfield.
It goes against all logic, that a small bio, with a completed, successful PIII, against GBM, the most aggressive of cancers, that has resisted treatment to the present, without ANY bad news should be trading so low, and constantly be going down - EVERYDAY. In particular, with more announcement expected imminently such as MAA etc, delayed as it may be.
Furthermore, this treatment promises to be a platform for other solid cancers, and perhaps other diseases as well. People spend more for a lottery ticket where the odds of winning are far, far worse than a lottery ticket here on NWBO. and people do not resell their 50 cent lottery ticket for 48 cents the next day. Given that the odds of success for DCVAX-L are very high, and the pay-back very large, it is a major failing of the US markets that investment in promising companies isn't rewarded with the positive expectations. Rather the structure of the market allows punishing such promising companies until they actually have real revenue to show. Very good expectations are no longer rewarded, therefore the US market will not be a good place to invest in young promising stocks with great expectations but no current real revenue to show at their startup! Retail investors will no longer be willing to invest in them, and will wait until they are almost ready to show real revenue. This source of funding for these promising young companies will disappear, and these companies will be left to the mercies (such as they are) of the predatory lenders as their only source of funding that is very highly dilutive. Shame on the SEC and the greedy MMs for ruining this market.
An interesting bit of history and two of many comments of interest:
Profile photo for Ken Saladin
Ken Saladin
·
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Former professor of histology (microscopic anatomy)Updated 3y
What would happen if a few cancer cells from another person were injected into your bloodstream?
Originally Answered: What would happen if few cancer cells from other person are injected into your bloodstream?
Something like this has already been done in a horribly unethical experiment. In the 1950s, a virologist and cancer researcher at Sloan Kettering named Chester Southam was working with the famous HeLa strain of cancer cells (named for the source patient Henrietta Lacks). He wanted to know if there was a danger of these giving cancer to one of the researchers by accidental exposure.
In 1954, he injected HeLa cells into a dozen hospitalized cancer patients without telling them what he was doing or why. He lied and said he was testing their immune systems, but he was really experimenting to see if the cancer from Henrietta Lacks would grow in another person. Several of these grew to tumors about 2 cm in size and one metastasized to the patient’s lymph nodes.
Since these patients already had other cancers, Southam next wanted to see if the HeLa tumors would take in healthy persons. One hundred fifty state prisoners volunteered for various reasons (such as feeling it would help atone for their crimes) and he injected HeLa into 65 of them in 1956. Tumors grew in the prisoners too, on the arms at the injection site. All of the prisoners’ bodies fought them off and the tumors vanished.
Southam injected HeLa into more than 600 other people in the years following that, incuding every OB-GYN surgery patient at Sloan Kettering, lying to these patients about what he was doing. “We’re just testing for cancer,” he told them.
When all of this came to light in the 1960s, a reporter from Science asked Southam why he didn’t inject the cells into himself. His reply was basically that he was more important than these patients; his life less dispensable. “Let’s face it,” he said to the reporter, “there are relatively few skilled cancer researchers, and it seemed stupid to take even a little risk.”
Stupid to risk his life for research; not too stupid (in his opinion) to risk the lives of hundreds of other people.
The ethical codes in place today would prohibit such a horrific experiment. Southam’s career would have been at an end if he had done such experiments in disregard to any such code of medical ethics, and he likely would have been stripped of his medical license and perhaps imprisoned. But such codes didn’t exist in the 1950s.
(Reference: Rebecca Skloot, The Immortal Life of Henrietta Lacks, pp. 127–136)
The comments:
Ray Schilling
· 6y
It is unfortunate that the researcher never asked the question why the tumors grew in the cancer patients, but not in the healthy patients. Had he pursued this further he could have solved the immunosuppression problem that exists in cancer patients. Out of this could have emerged a general cancer vaccine or learn about the mechanism how to stimulate the immune system in cancer patients.
Zeynep Bilgi
· 5y
actually he did ask that question and laid substantial groundwork in cancer immunology. he even tried to cure cancer using viruses and also investigated antibodies against transplanted tumor tissue. he later used newborn mice for experimental work.
A good summary on the generation of antibody diversity.
You may have to read it a couple of times.
Sorry, the images for the fids did not transfer properly.
Generation of Antibody Diversity
WRITTEN BY
Oliver Backhaus
Submitted: October 9th, 2016 Reviewed: November 29th, 2017 Published: February 21st, 2018
FROM THE EDITED VOLUME
Antibody Engineering
Edited by Thomas Böldicke
CHAPTER METRICS OVERVIEW
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Abstract
Because of the huge diversity, the immunoglobulin repertoire cannot be encoded by static genes, which would explode the genomic capacity comprising about 20,000–25,000 human genes. The immunoglobulin repertoire is provided by the process of somatic germ line recombination, which is the only controlled alteration of the genomic DNA after meiosis. It takes place in mammalian B lymphocyte (B cells) precursors in the bone marrow. The genome germ line sequence of undeveloped B cells is organized in gene segments and compromise V (variable), D (diversity), and J (joining) gene segments constituting the variable domain of the heavy chain and only V and J genes for building up the variable domain of the light chain. The rearrangement of the variable region follows a strict order. The following processes that participate in the generation of antibody diversity were summarized—allelic, combinational, and junctional diversity, pairing of IgH and IgL, and receptor editing—which all together produce the primary antigen repertoire (pre-antigen stimulation). When a B cell encounters a foreign antigen, affinity maturation and class switch are induced. Thereby the antibody repertoire increases. The resulting secondary immunoglobulin repertoire reveals in humans at least 1011 specificities for different antigens.
Keywords
• antibody diversity
• somatic recombination
• somatic hypermutation
• class-switch recombination
• allelic exclusion
• B-cell receptor editing
• pairing of VH and VL
• germinal center
Author Information
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Chapter sections
1. Introduction
The immune system is a complex system, comprising different organs and many specialized cell types, which are carrying out their development, maturation, and pathogen recognition at various sides in the body. The immune system has two major approaches to recognize and attack pathogens. The first is the innate immunity followed by the delayed adaptive immune response, based on specific antigen recognition receptors. The innate immune system is nonspecific and uses general pathogen recognition mechanisms, through pathogen-associated molecular patterns (PAMPs) recognized by cell surface or intracellular pattern recognition receptors (PRRs), such as toll-like receptors or NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs) [1]. Cell types of the innate immunity are monocytes/macrophages, dendritic cells, mast cells, natural killer cells, granulocytes, B1 cells, and innate lymphoid cells (ILCs). Although it lacks specificity, it can react immediately on the invading pathogens and activates the adaptive immune system by presentation of the foreign antigen peptides.
The adaptive immune system needs to be activated and primed by the antigen and therefore acts delayed from the initial pathogen attack. It is based mainly on two cell types, the B cell and the T cell. Both cell types express specific receptors on their cell surface for pathogen recognition. Many different B- and T-cell clones exist in parallel inside the body, and each has a different receptor specificity to the antigen. These receptors were called B-cell receptor (BCR) and T-cell receptor (TCR). It is remarkable that despite the relatively small genome size of approximately 20,000–25,000 human genes [2, 3], the human body can produce an antibody repertoire which can recognize almost every possible antigenic structure. Of course, this cannot be achieved by encoding the antigen receptor specificity directly in the genome sequence.
The huge B-cell diversity is generated by a complex multistep process, starting in the bone marrow and ending up in the peripheral lymphoid tissues, such as lymph nodes, spleen, or mucosal lymphoid tissue. In the maturation of functional BCR or TCR, the antigen receptor genes were rearranged from many different possible gene segments to form a full receptor. In each step, the receptor is tested for functionality and excluded when it reveals self-antigen reactivity in order to prevent autoimmunity and making the immune system self-tolerant.
The B-cell maturation occurs inside the bone marrow before the B cells migrate to peripheral lymphoid tissues. On the contrary to B cells, T-cell progenitors migrate to the thymus to differentiate and to mature. After their maturation, B and T cells meet again in lymph nodes. In the germinal centers of the lymph node, antigens were presented to the B cells through antigen-presenting cells, particularly through follicular dendritic cells (FDC). In response to a foreign pathogen, B cells with the highest antigen affinity were selected from a pool of different BCR clones. This process is organized in a form of a repetitive cycle inside of the dark and light zone of a germinal center of the lymph node and is known as the cyclic reentry model (Figure 4).
An essential part of the cycle is the BCR affinity maturation of the B cells. It begins with the tight controlled somatic hypermutation (SHM), particularly in the variable regions of the light and heavy chain of the antigen receptor and is only active in the dark zone of the germinal center. This process creates BCRs with higher affinity, whereby the mutations which produced very low or nonfunctional receptors were excluded. Finally, high-affinity B cells differentiate either into plasma cells, which start to produce secreted antibodies with the same specificity as the BCR, or they differentiate into memory cells, conferring lifelong immunity.
This chapter will discuss in detail the different steps and processes, which contribute to the high diversity of B cells. Many steps are similar for the generation of T-cell receptor diversity and were not covered by this article.
2. The primary antibody repertoire
2.1. Combinatorial diversity of immunoglobulins
Before immature naive B cells encounter a foreign antigen, their genomic sequence is rearranged by a well-controlled process, called somatic DNA recombination. This process is unique in lymphocytes, and except of the meiosis in the gametes, this is the only DNA recombination of somatic cells [4]. Before B cells leave the bone marrow to the secondary lymphatic organs, somatic DNA recombination takes place. The sum of all B lymphocytes in an individuum, producing different antibodies with different specificities and affinities, is designated as the antibody repertoire. In humans, the antibody repertoire consists of at least 1011 specificities [4]. The number varies and is limited by the total number of B cells and encountered antigens of an individuum. The immunoglobulin loci contain gene fragments to build up all immunoglobulin variable domains of the heavy and light chain. The different immunoglobulin loci are located on different chromosomes (Chr), the heavy chain on Chr14, the kappa light chain on Chr2, and the lambda light chain on Chr22. In contrast to the light chain loci, the heavy chain locus has several constant regions; each represents a different immunoglobulin isotype, e.g., IgM, IgD, IgG1, Ig2a, IgG2b, IgG3, IgE, and IgA in mice. The gene segments consist of different germ line sequences. For example, the variable gene locus of the heavy chain comprises 38–46 genes, which varies between individuals.
Besides different germ line segments, there exist a relative large number of pseudogenes of which some can undergo recombination leading to a nonfunctional variable region. An overview of the number of gene segments in the respective gene locus is given in Table 1 (slightly modified from IMGT [5]).
Immunoglobulin (Ig) gene segments
Gene locus Ig chain Chromosomal location Locus size (kb) Variable (V) Diversity (D) Joining (J) Constant (C)
IGH Heavy chain 14q32.33 1250 38–46 23 6 9
IGK ? Light chain 2p11.2 18201
34–38 0 5 1
IGL ? Light chain 22q11.2 1050 29–33 0 4–5 4–5
Table 1.
Number of functional human immunoglobulin gene segments in the heavy and light chain locus.
1
In one known haplotype, the locus size is reduced to 500 kb comprising only 17–19 IG?V genes.
The light chain loci have only variable (V) and joining (J) gene segments, whereby the heavy chain locus additionally has a diversity (D) gene segment, which lay between the V and J genes of the heavy chain variable region. One of each gene segment is randomly selected by the RAG1/RAG2 recombinase and joined together to form the variable region (Figure 2c) as shown as example with the variable region of the ? light chain. The recombination steps of the V region follow a strict order. The variable light chain recombines first with the V-J segments. Afterward the constant (C) domain is joined through RNA splicing of the primary RNA to the variable region. The construction of the V region of the heavy chain begins with the recombination of the D and the J gene; then the V gene is joined to the DJ segment. Finally, the C domain is joined through RNA splicing of the primary RNA. Figure 1 gives an overview of the respective steps of the V(D)J recombination for construction of the V region of the heavy chain immunoglobulin.
Figure 1.
V(D)J recombination of the heavy chain immunoglobulin (IgH) from germ line gene segments. The immunoglobulin locus is organized in gene segments: the variable (V), diversity (D), and joining (J) and constant (C) gene segment. The variable (V) region comprising the V, D, and J gene segments is generated by random recombination of these sequences. L = leader sequence.
The figure illustrates the somatic recombination event of the antibody heavy chain in the bone marrow of developing B cells. At first, one of the D and J segments is randomly chosen and rearranged. In the following step, one of the variable gene segments is joined to form the V-D-J variable region. This process is catalyzed by the recombination activating gene 1/recombination activating gene 2 (RAG1/RAG2) recombinase. In the immature B cells in the bone marrow, the variable region is transcribed with the constant mu (Cµ) and the constant gamma (Cd) chain, which produces two different mRNAs through alternative splicing which are finally translated into either IgM or IgD immunoglobulin.
The guided fashion of the recombination is mediated by recombinase signaling sequences (RSSs). The RSS is always directly adjacent to the coding region of the gene segments (Figure 2A). The nucleotide structure of the RSS is well defined and conserved (Figure 2B). A heptamer of seven conserved nucleotides is linked with a non-conserved linker sequence to a conserved nine-nucleotide nonamer [6, 7, 8]. The linker sequence is either 12 or 23 nucleotides long, and only a RSS with a 12 bp linker sequence can recombine with a 23 bp linker RSS, which is called the 12/23 rule. With the 12/23 rule, only corresponding gene segments can recombine. For instance, the V gene segments of the lambda light chain are always flanked downstream by a 23 bp RSS, and the genes of the J segments of the lambda light chain are always flanked upstream by a 12 bp RSS to the coding sequence. For the kappa light chain, it is the other way around, with the 12 bp RSS at the end of the V gene and the 23 bp RSS upstream of the coding sequence of the J gene. The heavy chain diversity gene segment is flanked by a 12 bp linker RSS from both sides and the V gene and the J gene segments with a 23 bp linker RSS upstream of the coding sequence, respectively. This allows only recombination in the desired V-D-J orientation, whereby during the recombination, the sequence between the chosen genes is excised and discarded. Figure 2 shows the position and structure of recombinase signal sequences (RSSs) at the V, J, and D gene segments and RSS-guided RAG-dependent V-J rearrangement of the variable domain of the ? chain.
Figure 2.
Position and structure of recombinase signal sequences (RSSs) at the V, D, and J gene segments and RSS-guided RAG-dependent V-J ? rearrangement (A). Schematic representation of the position and orientation of the different recombinase signal sequences (RSSs) at the V (variable), D (diversity), and J (joining) gene segments of the heavy (H) and of V?, V?, J?, and J? of the lambda (?) and kappa (?) locus. (B) Conserved nucleotide sequences of the two different RSSs. In each case, a conserved heptamer sequence and a conserved nonamer sequence encompass a non-conserved spacer sequence. Two different RSSs exist, which have either a 23 base pair (bp) spacer or a 12 base pair spacer. (C) The RAG1/RAG2 (recombination activating gene)-dependent rearrangement of the V region (here demonstrated with the variable domain of the ? chain) is mediated through a guided fashion by recombinase signal sequences (RSSs). The RAG1/RAG2 complex always binds a 23 bp spacer RSS together with a 12 bp spacer RSS and then mediates DNA cleavage between each gene segment (here V? and J?) and its heptamer. The sequence between the chosen genes are excised and discarded. The process described is called deletional joining and occurs when the two gene segments to be fused are in the same transcriptional orientation. However, in some instances, the two segments to be fused are in opposite transcriptional orientations in the germ line (inversional joining).
2.2. Junctional diversity of immunoglobulins
During V(D)J recombination the diversity of immunoglobulins is further increased by incorporation of additional nucleotides between the junctions of the V, D, and J gene segment of the heavy and V and J gene segment of the light chain. Especially the diversity of the CDR3 (complementarity-determining region), which has a huge influence on the antigen binding [9, 10], is affected with high frequency by this process, because of its position between the V and J gene segments in the heavy chain and between the V and J gene segments in the light chain. The CDR1 and CDR2 loops are not affected by junctional diversity, because of their position in the V gene segment of the heavy and light chain.
When two gene segments guided by the recombinase signaling sequences (RSSs) and the RAG1/RAG2 complex were brought together, the RAG complex excises the intervening DNA and produces short hairpins on both sides of the immunoglobulin gene segments (Figure 3). Then the Artemis/DNA-dependent protein kinase (DNA-PK) complex is recruited and cuts the DNA strand randomly at the site of the hairpin of both ends of the DNA strands [11, 12, 13]. This can produce palindromic DNA sequences at the side of the gene segment joint, and these nucleotides are called P nucleotides, because of its palindrome nature. Next, the terminal deoxynucleotidyl-transferase (TdT) adds further nucleotides at the single-stranded P nucleotide stretch [14]. The nucleotides were added randomly without any DNA template; hence they are called N nucleotides (non-template). After addition of a couple of N nucleotides, some base pairs between both single-stranded DNA stretches and the mismatched nucleotides were removed by an exonuclease; in this process the Artemis might be involved. The remaining gaps were filled by a DNA polymerase, and finally both DNA strands were joined together by the DNA ligase IV/X-ray repair cross-complementing protein 4 (XRCC4) complex.
Figure 3.
Junctional diversity is produced by incorporation of additional nucleotides between the junctions of immunoglobulin germ line gene segments of the variable region. After the RAG1/RAG2 (recombination activating gene) complex has removed the intervening DNA between two gene segments, in this case the V? and J? gene segment of the light chain, short hairpins were formed at both DNA blunted ends. Next, the Artemis is recruited and catalyzes a random single-stranded break at both DNA strands. This can produce in many cases a palindromic DNA sequence. These nucleotides are designated as P nucleotides. The single-stranded DNA is further extended by the addition of random nucleotides by the enzyme terminal deoxynucleotidyl-transferase (TdT). These nucleotides were named N nucleotides, because they are added without a DNA template. Some nucleotides at both single-stranded stretches match and can form hydrogen bonds (black lines). The mismatched DNA bases were removed by an exonuclease, and the remaining gaps were filled by a DNA polymerase and both DNA strands ligated. Underlined is the inserted sequence between the V? and J? gene segment.
The presence of N nucleotides is not equally distributed in the light and heavy chain [4]. The light chain has a remarkable lower appearance of N nucleotides in comparison to the heavy chain. The reason for this difference is the expression pattern of the terminal deoxynucleotidyl-transferase, which is much higher when the heavy chain is rearranged and already lower when subsequently the light chain is rearranged. The incorporation of additional nucleotides has not only beneficial effects, of cause the affinity of the antibody can be changed dramatically, but also missense mutations can be produced by violating the 3 bp codon structure, which can produce a frameshift in the coding sequence (non-productive rearrangements, see Figure 3).
2.3. Antibody diversity is further expanded by allelic exclusion, B-cell receptor editing, and pairing of VH and VL
In most cases, only one functional allele of an immunoglobulin gene is expressed. The other gene is transcripted in parallel, but usually only one of them can assemble into a functional B-cell receptor (BCR) [15]. Allelic exclusion means that only clonally identical BCRs were expressed on the B-cell surface and not two different versions from two different alleles. In diploid organisms, such as mammals, two different copies of a gene are on a chromosome. For the immunoglobulin gene loci, only one allele is expressed on the B-cell surface. When V(D)J rearrangement did not produce a functional BCR, the second allele will be activated and tested. When this will also fail, the B cell will die by apoptosis; this process is called clonal deletion. The choice of two different immunoglobulin alleles further increases the antibody diversity [16].
The exact mechanism of allelic exclusion is not completely understood by now, but in general some important steps are known. During pre-B-cell development when the heavy and light chain rearrangement takes place in the bone marrow; only one allele is chosen for recombination, whereas the other will be silenced. When a functional heavy chain is produced, RAG1/RAG2 recombinase expression will be decreased, and RAG1/RAG2 is targeted for degradation [4]. Furthermore, the RAG1/RAG2 recombinase access to the heavy chain loci will be decreased. Later, when the light chain is rearranged, the prevented access to the heavy chain loci is sustained, and no further rearrangement or change of allele activity can occur.
Although some essential steps in the mechanism are known, the precise mechanism is still unknown and under controversial discussion [16].
When the production of a functional B-cell receptor fails, another immunoglobulin allele is tested, or the BCR could undergo additional rounds of V(D)J recombination, until a functional receptor will be produced or no further V, D, and J genes for recombination were available. Usually, V(D)J recombination ends when a functional BCR is produced. When a functional BCR exhibits reactivity against antigens of the own body (self-reactivity), a specialized mechanism attempts to rescue the functional BCR and tries to edit the self-reactive B-cell receptor. This mechanism is called receptor editing and is one of the key checkpoints and rescue mechanisms to ensure self-tolerance and to escape clonal deletion.
The idea of rendering self-reactive B cells by editing the BCR through continued recombination of the antibody genes was investigated by several groups between the late 1980s and early 1990s [17, 18, 19, 20]. In one experiment, an H-2Kb MHC class I and an anti-H-2Kb antibody was expressed ectopically in transgenic mice [20]. They found that the anti-H-2Kb B cells were absent in the periphery, but B cells with the anti-H-2Kb were still in the bone marrow, trying to edit the BCR by high levels of RAG1/RAG2 recombinase [21]. About 25% of the functional antibodies are produced by receptor editing [22].
But there are reports that about 50% of B cells are initially self-reactive, and it is suggested that receptor editing is the main mechanism to confer self-tolerance [21], beside the clonal deletion of self-reactive B cells in the bone marrow and anergy of self-reactive B cells in the periphery. Anergy and deletion inactivate or remove self-reactive clones. Receptor editing is based on secondary V? ? J? light chain rearrangements or, more rarely, by altering the variable region of heavy chains by the replacement of a VH gene segment in an established VHDJH rearrangement.
In conclusion, the modification of the V region by receptor editing extents the antibody diversity and rescue some B cells from apoptosis especially when self-reactivity was observed.
The pairing of heavy and light chains is considered to contribute not to the same extent to the antibody diversity as the processes of somatic recombination and junctional diversity mentioned before. By combination of different variable regions of the light (VL) and the heavy chain (VH), the antibody repertoire is further expanded. Previous studies suggested that the combination of different VH and VL is completely by chance and no preference of V gene pairing was observed [23, 24]. But more recent publications, unveiled some preferred VH and VL gene pairings in human and mouse antibodies, by searching a newer and larger antibody database set (KabatMan dataset [25]) not available in the previous studies before [26]. The results revealed that pairing preference do exists but only for a small proportion of germ line immunoglobulin gene sequences.
3. The secondary antibody repertoire
3.1. Somatic hypermutation
After the assembly of the V region of the heavy and light chain and cell surface expression of a functional BCR, naive B cells migrate to the secondary lymphatic organs, for example, to the lymph nodes. In the germinal center of the lymph node, the primary antibody repertoire is further diversified by introducing mutations in the V domains of the heavy and light chain mediated by the activation-induced cytidine deaminase (AID) [27, 28, 29]. This enzyme is only expressed and active in germinal center-activated mature B cells and is the key enzyme for the somatic hypermutation (SHM). The anatomical structure of the germinal center in the lymph node is divided macroscopically in two parts, the light zone and the dark zone. Somatic hypermutation mediated by AID activity takes place in the dark zone. Cells which produce a nonfunctional B-cell receptor (BCR) upon mutation are dying by apoptosis, whereby the B cells with a functional BCR will migrate into the light zone. In the light zone, positive selected B cells with a low-affinity B-cell receptor were stimulated for survival, proliferation, and reentry to the dark zone for a next round of affinity maturation. At this point, after several rounds of affinity maturation, B cells can leave the germinal center and differentiate into antibody producing plasma cells or B memory cells. B cells with very low affinity are suffering for survival signals, and before they can reenter the dark zone, they die by apoptosis [30].
During the migration, B cells change their expression pattern depending on their location in the light or dark zone of the germinal center. The C-X-C chemokine receptor type 4 (CXCR4) is one of the classical markers, which changes the expression level in response of the migration to the other germinal zone. In the dark zone, the B-cell CXCR4 expression is strong and is reduced in the light zone. CXCL12 is a ligand of CXCR4 and expressed on the cell surface of reticular cells in the dark zone. The CXCL12/CXCR4 signaling of B cells in the dark zone is regarded as a homing signal to keep B cells in the dark zone, if CXCR4 expression is high [31, 32]. CXCR4 deficiency in germinal B cells restricted the B cells to the light zone, but the deletion is not sufficient alone for functional transition of dark zone B cells (centroblasts) to light zone B cells (centrocytes) [31].
The process of affinity maturation is also known as the cyclic reentry model (Figure 4). It starts with the introduction of mutations in the V region initiated by AID. The induced mutation rate is about one nucleotide per 10,000 nucleotides after each cell cycle division [4]. This is much higher than the normal mutation rate of about 1010 mutations per cell cycle. Only a slight change in one or a few amino acids in the CDRs or frameworks of the V region can change dramatically the antigen affinity and specificity. Mutations can have detrimental effects and produce lower affinity B-cell receptors, especially when the complementarity-determining regions (CDRs) are affected with mutations leading to antibodies which cannot anymore recognize the antigen-binding site. At this stage, negative selection of B cells occurs. When B cells are affected by negative changes and were not able to produce a functional receptor presented on the B cell surface or lost antigen affinity, cell death by apoptosis is initiated. Subsequently, phagocytic clearance of apoptotic B cells is executed by tingible body macrophages (TBM).
Figure 4.
Positive selection of high-affinity B cells in the lymph node germinal center. B cells progress from the dark to the light zone. B cells in the dark zone highly express the chemokine receptor C-X-C chemokine receptor type 4 (CXCR4) and are undergoing somatic hypermutation of their variable-region genes. When they enter the light zone, CXCR4 expression is reduced. In the light zone, follicular dendritic cells (FDCs) present foreign antigens on their cell surface. B-cell clones, which have B-cell receptors (BCRs) with affinity to the antigen, can bind to it. The antigen/BCR complex is processed, and antigen peptides were presented through the MHC II for T-cell recognition. TFH bind to the antigen presented on MHC II via its T-cell receptor and the B cell receives survival and proliferation signals through CD40-CD40L interaction and cytokines secreted by the corresponding T cells. B cell clones with low affinity die by apoptosis. B-cell clones which receive TFH stimulation reenter the dark zone and upregulate CXCR4 expression. The affinity of the BCR is further increased by additional rounds of SHM, whereby B cells producing nonfunctional BCRs initiate apoptosis. B cells can repeat the cyclic affinity maturation until they express high-affinity BCR and finally leave the light zone for differentiation into antibody producing plasma cells. When B cells differentiate into plasma cells, they also switch their immunoglobulin class. Some B cells with lower affinity as plasma cells preferentially differentiate into memory B cells.
On the other hand, B-cell clones with a BCR of high affinity toward the antigen receive growth signals, for example, from the follicular T helper cells, and are expended. This principle is called positive selection. Selected B cells which have undergone affinity maturation are showing more mutations in the critical regions for the antigen binding, namely, the CDRs. A mutation in the CDR, which produces an amino acid change, very likely alters the antigen affinity.
B cells with sufficient affinity to the antigen which is presented by follicular dendritic cells (FDCs) in the light zone can capture it, process it, and present the antigen peptide via the major histocompatibility complex II (MHC II) to the T cells. Then, B-cell clones get survival and mitogenic signals through the T-cell receptor (TCR) recognition, CD40-CD40L interaction, and cytokine stimulation of T cells (Figure 4). As a consequence, B-cell receptors and CD40 cluster together and promote thereby positive selection signaling. Follicular dendritic cells present foreign antigens on their dendritic surface in form of iccosomes (immune complex-coated bodies) [33, 34, 35]. Iccosomes are antigen/antibody/complement complexes bound to Fc and complement receptors on FDCs. When B cells recognize antigens presented by iccosomes, they can take them up and process them for MHC II-mediated T-cell presentation. The efficiency and amount of iccosome uptake can also influence the fate of the B cell. B-cell clones with higher affinity for the antigen can capture more from the iccosome-presented antigen, which resulted in more representation of the processed antigen peptide on the B-cell surface, complexed in the MHC II molecule. Therefore, these clones get more surviving and proliferation signals in the light zone from the recognizing follicular T helper cell (TFH).
The process of somatic hypermutation (SHM) has not only a cellular dimension; it also has a molecular dimension, which can be characterized by the details of the mechanism of SHM and affinity maturation. The central enzyme in SHM is the activation-induced cytidine deaminase (AID). AID catalyzes the deamination of the DNA nucleotide cytosine to uracil, which is usually only present in RNA molecules.
The expression of AID is tightly restricted to germinal center B cells; this protects other cells from somatic hypermutation. Furthermore, it cannot act on predominantly double-stranded genomic DNA. To protect the majority of the genomic DNA from mutation, AID has developed a clever mechanism [36, 37, 38]. AID can act specifically only on single-stranded DNA molecules. The genomic DNA is released during transcription as a single strand by the RNA polymerase, which granted access of the AID for deamination. The immunoglobulin V region genes are actively transcribed in germinal center B cells, and somatic hypermutation can occur. Beside of the immunoglobulin V region, also some other transcribed genes can be affected by AID, fortunately by a lower frequency. AID has not only the function of somatic hypermutation by acting on the immunoglobulin V region loci; it can also activate the immunoglobulin class-switching process by acting on the residues in switch regions.
The deamination of cytidine to uracil by AID is the initiation step of SHM or class-switch recombination (CSR). Further mutation of the DNA around the initial deamination is executed by two different DNA repair pathways [39, 40, 41]. For example, the DNA mismatch repair process recognizes the wrong base pairing of uracil (U) to guanosine (G). Mismatch repair proteins MSH2 and MSH6 (mutS homolog 2/6) detect the wrong U/G base pairing, which then recruits DNA nucleases to remove the uracil and the adjacent nucleotides. The following DNA polymerase Pol? has no exonuclease activity and is error prone in B cells. The polymerase preferentially misincorporates thymidine (T), regardless of the template sequence, which leads to a preference of adenosine (A)-thymidine (T) mutations at the original targeted cytosine and the adjacent nucleotides by the mismatch repair pathway.
Alternatively, in the base excision repair pathway, the uracil DNA glycosylase (UNG) cleaves the uracil nucleobase from the uridine and leaves an abasic site in the DNA strand. During the following DNA replication, a random DNA base will be inserted in the opposite DNA strand of the abasic nucleotide. This is mediated by an error-prone DNA polymerase used in translesion DNA synthesis for damaged DNA caused by UV radiation.
As mentioned before, AID can also initiate class-switch recombination, by acting of apurinic/apyrimidinic endonuclease 1 (APE1) upon UNG-mediated introduction of an abasic nucleotide in the switch region. APE1 cleaves the DNA strand at the abasic site and produces a single-strand nick. In the switch regions, upstream of the constant region genes, the DNA nick is further cleaved which produces a double-strand break (DSB). This leads to a joint of another constant region gene to the V region, produced by the double-strand break repair machinery.
3.2. Class-switch recombination
In naive B cells, which had already rearranged their V region by somatic DNA recombination, two antibody isotypes are co-expressed at the same time. The V region and the µ chain (IgM) together with the d chain (IgD) were transcribed on the same RNA transcript. By alternative splicing, either the µ chain or the d chain is chosen, which produces two different messenger RNAs (Figure 1). Upon antigen contact and B-cell activation, B cells switch their antibody isotypes from IgM/IgD to IgG, IgA, or IgE. This is achieved by a process called class-switch recombination (CSR) or isotype switching. The antibody isotype is changed by an exchange of the constant region of the heavy chain locus. Only the constant region is replaced by CSR, which means the V region stays the same, but class switch confers the antibody the ability to interact with different effector molecules by their fragment crystallizable (Fc) region (Figure 5).
Figure 5.
Mechanism of switch recombination from IgM to IgG2b.
Unlike in the parallel expression of IgM and IgD, the class switch is a chromosomal DNA rearrangement, leading to only one ultimate antibody isotype in the affected B cell. The process is guided by conserved switch region (S) upstream of the heavy chain constant genes, coding for the respective constant domains. The switch regions are repetitive stretches of DNA placed in introns upstream to the C region genes [28, 42]. The initial activation of CSR is done by the enzyme activation-induced cytidine deaminase (AID), which has also an essential role in the somatic hypermutation process. This produces a single-stranded DNA break (nick) at two switch regions, and the DNA between both switch sites were irreversible excised. The removal includes always the µ and d chain constant region. Both DNA strands were brought together by the non-homologous end joining (NHEJ) mechanism; this rearranges the variable region with the constant region of the chosen immunoglobulin isotype. The decision which isotype will be produced is influenced by different cytokines, secreted by T cells [43, 44].
CSR is induced by the enzyme activation-induced cytidine deaminase (AID) acting on the switch regions (S) of the respective constant region gene. The non-homologous end joining (NHEJ) machinery joins the chosen constant gene segment to the V region (here Cy2b). The constant region gene, which is next to the V region, is then expressed together with the V(D)J gene sequence.
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Oliver Backhaus
Submitted: October 9th, 2016 Reviewed: November 29th, 2017 Published: February 21st, 2018
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Personally, I find the standard terminology "going concern" to be poorly chosen. Out of context it could mean either "on going enterprise" or "on going worry"
It's mostly a function of who has more leverage at that time, the investor or the company.
If they are in actual talks with NWBO on combos, I imagine I should rethink my position on who is behind the manipulation of NWBO stock. First, this leads me to think that the BPs are not a monolithic block. Some may still be behind the manipulation and others may be willing (trying) to get behind NWBO and set up combo trials. Look at who has more to lose and who has more to gain. I hope that discovery in the spoofing case will soon tell us more about who is who.
I would also highlight the following in the comparison that I have bolded below:
I do not have the time or know how to check this out about the history and future of some website.
I just reported what I saw when I went to this site. so I have no basis upon which to agree. You could be right, but I have no way to know that from what I personally saw at the website.