NorthWest Biotherapeutics Inc (NWBO)

[Pyrrhonian]

Gnawkz and beachlife,


Hey Pyrr, questions:

1. Are the DCs for DCVax-L not already matured when injected into the body? I thought only DCVax-D is partially matured upon injection.


They start out with immature DC to make DCVax-L. Immature DC are much more favorable than mature for picking up antigens. So it is useful to load immature DC. However, immature DC do not migrate well, and even if they did they would be tolerogenic. So a dedicated maturation step is necessary before injection. As for DCVax-L, the theory was that because DC mature when they encounter necrosed cells that therefore mixing immature DC with necrosed tumor lysate would cause these DC to find and pick up useful antigens (TAA) and then subsequently mature. An “all-in-one” step so to speak. Therefore no dedicated maturation step is used in making DCVax-L.

However this tech was developed in the late 90s (IND # 10206), and studies have since shown that necrosed tumor lysate (unlike other necrosed cells) is full of inhibitory cytokines that inhibit DC maturation. Other studies have also shown the method used to make DCVax-L as inferior to other methods of loading DC. And certain studies suggest it is not effective at all.

Immune responses seen from the UCLA studies may be confounded by other causes, such as ending SOC and natural immune rebound concurrent with vaccine initiation. Also injecting any foreign cellular substance into any person will cause some changes in immune functioning. Increases in CD4/CD8, etc.

The problem is mixing DC with freeze-thaw necrosed lysate inhibits their maturation, which hinders migratory capacity and also deadens t-cell response. Those that designed the vaccine weren’t aware of that issue at the time. And nothing has been done to remedy it.

This IND is old, and if they were to change the process or formula significantly such that the safety or efficacy of the product would be expected to be altered, then a new IND must be issued to advance it in human trials. That has not happened.

Here is a post explaining the three different INDs and their manufacturing processes:

http://investorshub.advfn.com/boards/read_msg.aspx?message_id=115344897


2. Is there a difference between partial maturity and immaturity? Previous communications from the company indicate there is a substantial difference. Have you found research to suggest otherwise?


Yes, there is, and there may be some maturation of the DC in DCVax-L, but I doubt it’s very much (due to inhibitory cytokines). Partially mature DC make some sense to inject lesions with because they may encounter naturally existing antigens within the tumor (some apoptotic cells, etc). Apparently in murine studies DCVax-Direct was injected into engrafted tumors with the DC either immature, partially mature, or fully mature, and the partially mature evoked the most effective outcome. However I think this has more to do with cytokine release and DC state than migration. The "mature" DC were probably just exhausted before injection, and the "partially mature" ones as NWBO calls them had probably just become mature. Full maturation precludes cytokine release.

When mixing DC with BCG + INF-y they will secrete IL-12 etc. due to "danger signals." If you wait too long they will become exhausted. 24 hours is too long. 6 hours is just about right, as these DC should be mature in phenotype while just beginning to secrete IL-12, etc. So NWBO's "partially mature" DCVax-Direct DC are likely fully mature DC that had only just begun to secrete immunogenic cytokines.

But in any event, I was only talking about intradermally injected DC. Immature don’t migrate well and partially mature don’t as well as mature, but worse, immature and partially mature are tolerogenic:

Freeze/thawing of cancer cells, certain immunogenic live cancer cells, and certain therapy-induced non-immunogenic cell death routines can induce a “limbo” state in DCs called semi-mature DCs which are not fully mature and can be either devoid of phenotypic maturation ligands or functional maturation depending on the context. Both immature DCs and semi-mature DCs cause T cell anergy and facilitate tolerogenicity thereby compromising anticancer immunity.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858649/figure/F1/

Here is the greater article: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858649/

Very much recommend reading that one through.

So the DC in DCVax-L are truly immature or partially mature, not "mature but un-exhausted" like the DC in DCVax-Direct.


3. The immaturity referenced in the case studies that you've shared, are those immature DCs or already on their pathway to maturation?


In the studies I sited all DC were matured before vaccination. They were loaded with TAA in their immature state first, however.

The problem is once DC incubate for 24 hours with inhibitory cytokines (such as exist in freeze-thaw necrosed tumor lysate) like with DCVax-L they become somewhat “fixed.” Then after injection the body undergoes a natural defensive mechanism (as with say a spider bite), whereby the perceived toxin or pathogen is quarantined and systematically removed or destroyed first. Inflammation is the first real step. Very few (about 1%) even fully mature DC can migrate away in time before the clamp comes down, but immature DC won’t really go anywhere and neither immature or partially mature DC can elicit an immunogenic t-cell response anyway, so if they manage to escape the body's protective mechanisms they will only elicit a tolerogenic response in t-cells.

There is one study that showed some partially mature DC were able to subsequently mature in a primates study, but these were not loaded DC, and the majority remained partially mature. See the last study sited. It's important because some of those DC, being unloaded, likely picked up bits of non-inhibitory necrotic material along the way that matured them.


4. Is the type of cancer important here? GBM vs Melanoma?


I really don’t think so. The immune system responds to a perceived pathogen (the injected vaccine) the same, and all tumors secrete inhibitory cytokines.


5. Is the size of the tumor an important factor here? The studies you've shared are for Melanoma, a type of cancer that can grow to be of a substantial size. For GBM, since space is limited, it cannot grow beyond a certain size. Is it logical to correlate the size of the tumor with the effectiveness of the immunosuppressive environment? (Larger tumor = stronger immunosuppressive environment.)


The influence freeze-thaw necrosed lysate has on immature DC (a negative one) when combining them in vitro is a constant regardless of that patient’s indication or tumor burden. But as for GBM, space is limited yes but that’s also why it’s so lethal.


6. In addition, if the size of the tumor is minimal, would the number of DCs needed to migrate to the lymph node be smaller for there to be a clinical benefit?


I would think so yes--provided those DC are immunogenic (unlike the DC in DVax-L). Although it seems even in complete resection and after SOC there are often quite a few cells left. RK put some of this info out there on ivil: http://investorsvillage.com/smbd.asp?mb=6543&mn=3917&pt=msg&mid=15477153

DC vaccines really have an uphill battle due to 99% of them on average being destroyed after ID injection. At best with various methods some 3% may survive to migrate.

GL buddy. I'd drop NWBO before next fall. Over the summer is imo best. June-ish. Jmo though.


Beachlife,

As I already sited (from the study you then requoted), even with 2.5mil DC injected intradermally only 1.5% of DC were found to migrate to lymphs. But, these were also definitely mature DC, as the vaccine studied loaded those DC with TAA and then had a dedicated maturational step applied to them before injection. Those few mature DC from that group that migrated (again, it’s less than 2%--the other 98% are phagocitized) can and do interact with t-cells and elicit an immunogenic response. But it’s pretty small, and probably not enough to significantly thwart progression in a disease like GBM except perhaps in some subgroup.

However, the DC in DCVax-L are almost certainly not mature, but are mostly immature and partially mature. These DC, even if they do migrate, with have a tolerogenic effect on the t-cells they interact with. I don’t think any of the DC in DCVax-L ever escape the localized immune response to destroy them, and so I believe 0% migrate, but even if some do they will be useless.

Showing studies that state ID delivery is better than IN or IV or SC is moot because those studies used TAA loaded then matured DC.

Can you show a trafficking study that used as its vaccine DCs loaded with freeze-thaw necrosed lysate? Or compared methods of delivery with the same? The only one I found was in the presentation AVII posted on ivil of GBM-vax (also AV0113), where the CSO said they just didn’t move when injected intradermally (ID).

http://investorsvillage.com/smbd.asp?mb=6543&mn=3902&pt=msg&mid=15475220

They also show that for their vacc (which to me looks almost identical with DCVax-L), intranodal (IN) delivery showed good preclinical responses whereas ID delivery did nothing for tumor burden in mice. I don’t see any reason to think DCVax-L will be different. I doubt those DC go anywhere, not even 1.5% of them. At least IN delivery would have given those DC a chance (though a pointless one, being tolerogenic).

AV0113 group actually performed slightly worse than control in PFS. OS was essentially the same between groups. That’s probably the DCVax-L P3’s fate.

Here are some relevant graphs and links and quotes:

Unmodified freeze-thaw tumor cell lysates inhibited the toll-like receptor-induced maturation and function of bone marrow-derived DCs, preventing up-regulation of CD40, CD86, and major histocompatibility complex class II, and reducing secretion of inflammatory cytokines [interleukin (IL)-12 p70, tumor necrosis factor-a, and IL-6].

Lysate-induced DC suppression was partially reversed in vitro by induction of tumor cell stress before lysis, and only DCs loaded with stressed lysates afforded protection against tumor challenge in vivo. These data suggest that ex vivo freeze-thaw of tumor cells does not effectively mimic in vivo immunogenic necrosis.

The commonest strategy, however, has been to load DCs with ostensibly “necrotic” tumor cell lysates, generated by repeated cycles of freezing and thawing, with the solid debris removed by centrifugation. DCs loaded with such lysates have provided modest protection in various animal models,9-11 and stimulated limited responses in a range of clinical trials.

As the immune response generated by DCs is related to their maturation state, we hypothesized that DCs, which had taken up TAA in the form of necrotic lysates, and were then matured using a range of TLR agonists, would represent a potent anticancer vaccine. In this study, we show that freeze-thaw lysates in fact inhibit the maturation of DCs in response to a range of TLR ligands, interfere with DC/T-cell interactions, and selectively target particular signaling pathways. To some extent, these effects are shown to be reduced by stressing cells before lysis. These data have important implications for the optimal preparation of tumor cell lysate-based vaccines for clinical application.

And they even touch on what UCLA have been trying from 2003-2010, and are continuing with even today:

Recent studies have shown that combining TLR ligands can enhance cytokine release by DCs,36 with the combination of poly I:C (a TLR3 agonist) and CpG (a TLR9 agonist), particularly effective for IL-12 p70 production. To test whether this potent combination could overcome the inhibition of phenotypic maturation and IL-12 p70 release caused by lysates, further experiments were conducted using poly I:C and CpG in place of LPS. As with LPS, up-regulation of CD40, CD86, and I-A was reduced by lysates, whereas CD80 remained unaffected (Fig. 1E); production of IL-12 p70 was again inhibited to a similar degree.

And then conclude with:

Therefore, tumor cells freeze-thawed in vitro to generate ostensibly necrotic and hence, potentially immunogenic lysates, inhibit full DC phenotypic maturation and inflammatory cytokine release. These effects are broad, extending over a range of tumor models, mouse strains, and maturation stimuli. These data also show that DCs pulsed with freeze-thaw lysates are likely to skew CD4 responses away from an optimal TH1 profile, are unable to cross-present lysate-derived TAA, and are less effective at activating specific CD8 cells even when favorably loaded exogenously with specific peptide epitopes.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901408/

PURPOSE:

We have investigated the capacity of immature and mature monocyte-derived DCs pulsed with melanoma-associated peptides (gp100 and tyrosinase) to induce a primary cytotoxic T-lymphocyte response in vivo.

EXPERIMENTAL DESIGN:

Advanced HLA-A2.1(+) melanoma patients were vaccinated with peptide- and keyhole limpet hemocyanin (KLH)-pulsed DCs, either immature (9 patients) or matured by monocyte-conditioned medium/tumor necrosis factor alpha/prostaglandin E(2) (10 patients).

RESULTS:

All patients vaccinated with mature DCs showed a pronounced proliferative T-cell and humoral response against KLH. By contrast, KLH responses were absent in most of the patients vaccinated with immature DCs. Delayed-type hypersensitivity (DTH) reactions against antigen-pulsed DCs were only observed in patients vaccinated with mature DCs and not in patients vaccinated with immature DCs. MHC-peptide tetramer staining of DTH-derived T cells revealed the presence of specific T cells recognizing the melanoma-associated peptides in 1 patient. In a second patient, DTH-derived T cells showed specific lysis of tumor cells expressing the antigens used for DC pulsing. Only patients vaccinated with mature DCs showed objective clinical responses. Interestingly, both patients with long-term progression-free survival (22 and >40 months) were both vaccinated with mature DCs and demonstrated antigen-specific T-cell reactivity of DTH-derived T cells.

http://www.ncbi.nlm.nih.gov/pubmed?Db=pubmed&Cmd=ShowDetailView&TermToSearch=14613986

We demonstrated that mice treated with bone marrow-derived DCs pulsed with HOCl-oxidized whole tumor cell lysate of ID8 expressing ovalbumin (ID8-ova) had the best tumor control with >60% cure rate [41]. In contrast, mice that were treated with DCs pulsed with freeze-thawed (100%) and UVB-irradiated ID8-ova whole tumor cell lysate (70%), succumbed to tumor growth and ascites formation. The superiority of HOCl-oxidized whole tumor cell lysate preparation could be attributed to the induction of less Treg cells in peripheral blood and absence of serum IL-10 in the vaccinated mice and not in mice treated with other UVB-irradiated or freeze-thawed whole tumor lysate preparations. We translated these findings into a phase I trial by immunizing patients with autologous DCs loaded with autologous whole tumor cell lysate prepared with HOCl-oxidation [41].

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494356/

AV0113, although injected IN, showed no PFS diff between groups. Again, citing some small studies that show ID delivery had more effect than IN is moot in this regard because the DC in DCVax-L/AV0113 are a mix of immature and partially mature DC and not exclusively fully mature as the DC in those studies were. Activartis at least conducted trafficking studies and both methods in preclinical studies and determined IN delivery was superior for WTL loaded DC vaccine.

http://www.nyas.org/Publications/Media/VideoDetail.aspx?cid=f16e3d44-c758-483b-8c68-0d9d5d2208dd

Here is the unfortunate result (and also the DCVax-L P3's primary endpoint):





Looks similar to this one, which compared mice treated with WTL loaded DC vaccine, PD-1 inhibitor, both or nothing (control). WTL loaded group was comparable to control:





And here is another comparing WTL loaded DC vaccine with cancer stem cell loaded DC vacc (such as CLBS does) and control:





http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063547/

Good luck everyone, sincerely. Not interested in continuing dialogue on this site going forward, but you can always pm me on Seeking Alpha.