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Re: skitahoe post# 692202
Friday, May 17, 2024 10:38:11 AM
ski, the pre-approval inspection is performed to assure regulatory authorities that the facility listed in the marketing application can manufacture the product, and that the data submitted is accurate and complete. Regulators will look to confirm that:
* The site conforms to the application
* The site demonstrates manufacturing reliability
* The site can scale its operations
* The site uses suitable and adequate analytical methodologies
* The site is producing authentic and accurate data
* The site is in full CGMP compliance
I have full confidence that the Sawston facility will pass the inspection as they have previously, regardless whether it’s the same inspectors or not. Although it’s probably the most important, Sawston may not be the only site visited, since other manufacturing sites were used for the clinical trials. (Cognate in Memphis, and Fraunhofer IZI in Germany) Obviously those other sites haven’t manufactured a product for Northwest Bio in a decade, but scheduling these visits in a timely manner could be more of an issue in my mind, and it seems impossible to predict if they even will, or the timing.
hyperopia
Re: ATLnsider post# 692493
Sunday, May 19, 2024 2:00:26 PM
Right ATLnsider, as you indicate, in the UCLA combination studies with DCVax-L, the poly-ICLC is administered separately as an adjuvant. However, in the early studies with DCVax-Direct, it was used in the manufacturing process, which increased the potency. (poly I:C, or its derivative, poly-ICLC is included in the Direct patents, and the more recent hyperactive patents)
NW Bio Receives U.S. Patent On Broad Processes For Producing More Potent Dendritic Cells
Next Generation Technology Already In DCVax®-Direct; Will Be Applicable To All DCVax® Products
Building upon the pure immature dendritic cells, NW Bio's patented methods develop mature and activated dendritic cells that are far more potent than dendritic cells produced in the standard way. For example, NW Bio's dendritic cells produce as much as 10X or more the amount of signaling compounds which are key to mobilizing other active agents of the immune system, such as T cells (which infiltrate and attack tumors) and B cells (which produce antibodies).
NW Bio is already using these next generation methods for producing more potent dendritic cells in its production of DCVax-Direct. The same patented methods for activating dendritic cells were also used in the pre-clinical animal studies with DCVax-Direct. In those studies, injection of these potent dendritic cells into some of the tumors in each of the animals resulted in complete clearance of all tumors (both the tumors injected with DCVax-Direct and the tumors not injected) in 80-100% of the animals in the various studies, indicating a system-wide immune response.
?
Going forward, NW Bio's now patented methods of producing more potent dendritic cells will also enable development of the next generation of NW Bio's other two product lines: DCVax-L and DCVax-Prostate. The current DCVax-L and DCVax-Prostate products have already delivered striking results in clinical trials to date, extending the time to tumor recurrence and the patients' survival time by 1-1/2 to 2 years or more, with a substantial portion of patients going far beyond that. Incorporating NW Bio's patented methods for more potent dendritic cells will enable production of enhanced versions of these DCVax-L and DCVax-Prostate products.
https://www.prnewswire.com/news-releases/nw-bio-receives-us-patent-on-broad-processes-for-producing-more-potent-dendritic-cells-198760831.html
For DCVax-L, Northwest Bio uses the most common method of culturing PBMC’s to generate dendritic cells, which relies on the monocytes adhering to the bottom surface of the polystyrene culture vessel in a medium of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), which induces differentiation into dendritic cells, and activates them.
I believe this increased-potency knowledge was gained in the DCVax-Direct studies because they were attempting to overcome the tumor’s defenses by delaying the activation and maturation of the dendritic cells, which wasn’t possible using culture flasks that are used to produce DCVax-L, so they attempted culturing the monocytes in suspension, in a bagged system without IL-4, using only GM-CSF, and then various other mediums to optimize and activate the dendritic cells, including poly I:C.
Northwest Bio stated that they intended to use this higher potency method across all product lines for the next generation of DCVax, but the Flaskworks’ eden system uses a polystyrene culture cartridge (similar to the well plates used in manual culturing protocols) which the monocytes adhere to, so I’ve not seen evidence that they’ve actually changed the manufacturing method for the next generation of DCVax-L. I believe the method of combining poly-ICLC with DCVax-L requires separate (nearly simultaneous) injections to produce the more potent effect in vivo.
ATLnsider
Re: hyperopia post# 692015
Thursday, May 16, 2024 6:26:39 PM
hyperopia, I noticed that you referenced me in this post.
ATLnsider has speculated that Northwest Bio is using poly-ICLC as a maturation and activation agent in the manufacturing process of DCVax-L and therefore, thinks it will be included in the marketing application. While I believe this manufacturing method may have been studied, I believe it was with DCVax-Direct, and not DCVax-L.
I wanted to correct and clarify my beliefs about DCVax-L and poly-ICLC. Here are my beliefs and opinions:
(1) I do not know for sure if poly-ICLC was included in the MAA filed with the MHRA.
(2) I hope poly-ICLC was included in the MAA filed with the MHRA, because more cancer patients will live longer, and more cancer patients will be effectively cured (5 of more years overall survival).
(3) After the 2010 clinical trial at UCLA proved that poly-ICLC was the best TLR agonist to use with DCVax-L (sometime around 2014), all current and future DCVax-L clinical trials have included DCVax-L along with poly-ICLC as the new control. There are no current clinical trials or future planned clinical trials, that do not include DCVax-L along with poly-ICLC.
(4) I am not aware of any clinical trials that have studied poly-ICLC with DCVax Direct. All of the completed trials, and future planned trials that I am aware of, are with DCVax-L along with poly-ICLC.
(5) The recently published peer-reviewed Nature study was of DCVax-L (ATL-DC) along with poly-ICLC.
However, I do believe there will be future basket trials with DCVax-L and DCVax Direct, along with poly-ICLC, that will test the efficacy and safety in treating multiple solid tumor cancers.
@d_stock07734
Some old news. But it has connection with NWBO now.
NWBO has the commander of the immune system (DCVax-L) and LP owns the maestro of the immune system (CYT 107). Is it great if there is a merger between the two?
New way to fight sepsis: Rev up patients' immune systems
Clinical trial demonstrates significant, lasting increase in key immune cells
https://eurekalert.org/news-releases/597984
Interleukin-7 restores lymphocytes in septic shock: the IRIS-7 randomized clinical trial
https://insight.jci.org/articles/view/98960
Excellent post by hyperopia:
Right ATLnsider, as you indicate, in the UCLA combination studies with DCVax-L, the poly-ICLC is administered separately as an adjuvant. However, in the early studies with DCVax-Direct, it was used in the manufacturing process, which increased the potency. (poly I:C, or its derivative, poly-ICLC is included in the Direct patents, and the more recent hyperactive patents)
NW Bio Receives U.S. Patent On Broad Processes For Producing More Potent Dendritic Cells
Next Generation Technology Already In DCVax®-Direct; Will Be Applicable To All DCVax® Products
Building upon the pure immature dendritic cells, NW Bio's patented methods develop mature and activated dendritic cells that are far more potent than dendritic cells produced in the standard way. For example, NW Bio's dendritic cells produce as much as 10X or more the amount of signaling compounds which are key to mobilizing other active agents of the immune system, such as T cells (which infiltrate and attack tumors) and B cells (which produce antibodies).
NW Bio is already using these next generation methods for producing more potent dendritic cells in its production of DCVax-Direct. The same patented methods for activating dendritic cells were also used in the pre-clinical animal studies with DCVax-Direct. In those studies, injection of these potent dendritic cells into some of the tumors in each of the animals resulted in complete clearance of all tumors (both the tumors injected with DCVax-Direct and the tumors not injected) in 80-100% of the animals in the various studies, indicating a system-wide immune response.
?
Going forward, NW Bio's now patented methods of producing more potent dendritic cells will also enable development of the next generation of NW Bio's other two product lines: DCVax-L and DCVax-Prostate. The current DCVax-L and DCVax-Prostate products have already delivered striking results in clinical trials to date, extending the time to tumor recurrence and the patients' survival time by 1-1/2 to 2 years or more, with a substantial portion of patients going far beyond that. Incorporating NW Bio's patented methods for more potent dendritic cells will enable production of enhanced versions of these DCVax-L and DCVax-Prostate products.
https://prnewswire.com/news-releases/nw-bio-receives-us-patent-on-broad-processes-for-producing-more-potent-dendritic-cells-198760831.html
For DCVax-L, Northwest Bio uses the most common method of culturing PBMC’s to generate dendritic cells, which relies on the monocytes adhering to the bottom surface of the polystyrene culture vessel in a medium of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), which induces differentiation into dendritic cells, and activates them.
I believe this increased-potency knowledge was gained in the DCVax-Direct studies because they were attempting to overcome the tumor’s defenses by delaying the activation and maturation of the dendritic cells, which wasn’t possible using culture flasks that are used to produce DCVax-L, so they attempted culturing the monocytes in suspension, in a bagged system without IL-4, using only GM-CSF, and then various other mediums to optimize and activate the dendritic cells, including poly I:C.
Northwest Bio stated that they intended to use this higher potency method across all product lines for the next generation of DCVax, but the Flaskworks’ eden system uses a polystyrene culture cartridge (similar to the well plates used in manual culturing protocols) which the monocytes adhere to, so I’ve not seen evidence that they’ve actually changed the manufacturing method for the next generation of DCVax-L. I believe the method of combining poly-ICLC with DCVax-L requires separate (nearly simultaneous) injections to produce the more potent effect in vivo.
https://investorshub.advfn.com/boards/read_msg.aspx?message_id=174448995
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Views
hyperopia
Re: ATLnsider post# 692493
Sunday, May 19, 2024 2:00:26 PM
For DCVax-L, Northwest Bio uses the most common method of culturing PBMC’s to generate dendritic cells, which relies on the monocytes adhering to the bottom surface of the polystyrene culture vessel in a medium of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), which induces differentiation into dendritic cells, and activates them.
I believe this increased-potency knowledge was gained in the DCVax-Direct studies because they were attempting to overcome the tumor’s defenses by delaying the activation and maturation of the dendritic cells, which wasn’t possible using culture flasks that are used to produce DCVax-L, so they attempted culturing the monocytes in suspension, in a bagged system without IL-4, using only GM-CSF, and then various other mediums to optimize and activate the dendritic cells, including poly I:C.
Northwest Bio stated that they intended to use this higher potency method across all product lines for the next generation of DCVax, but the Flaskworks’ eden system uses a polystyrene culture cartridge (similar to the well plates used in manual culturing protocols) which the monocytes adhere to, so I’ve not seen evidence that they’ve actually changed the manufacturing method for the next generation of DCVax-L. I believe the method of combining poly-ICLC with DCVax-L requires separate (nearly simultaneous) injections to produce the more potent effect in vivo.Right ATLnsider, as you indicate, in the UCLA combination studies with DCVax-L, the poly-ICLC is administered separately as an adjuvant. However, in the early studies with DCVax-Direct, it was used in the manufacturing process, which increased the potency. (poly I:C, or its derivative, poly-ICLC is included in the Direct patents, and the more recent hyperactive patents)NW Bio Receives U.S. Patent On Broad Processes For Producing More Potent Dendritic Cells
Next Generation Technology Already In DCVax®-Direct; Will Be Applicable To All DCVax® Products
Building upon the pure immature dendritic cells, NW Bio's patented methods develop mature and activated dendritic cells that are far more potent than dendritic cells produced in the standard way. For example, NW Bio's dendritic cells produce as much as 10X or more the amount of signaling compounds which are key to mobilizing other active agents of the immune system, such as T cells (which infiltrate and attack tumors) and B cells (which produce antibodies).
NW Bio is already using these next generation methods for producing more potent dendritic cells in its production of DCVax-Direct. The same patented methods for activating dendritic cells were also used in the pre-clinical animal studies with DCVax-Direct. In those studies, injection of these potent dendritic cells into some of the tumors in each of the animals resulted in complete clearance of all tumors (both the tumors injected with DCVax-Direct and the tumors not injected) in 80-100% of the animals in the various studies, indicating a system-wide immune response.
?
Going forward, NW Bio's now patented methods of producing more potent dendritic cells will also enable development of the next generation of NW Bio's other two product lines: DCVax-L and DCVax-Prostate. The current DCVax-L and DCVax-Prostate products have already delivered striking results in clinical trials to date, extending the time to tumor recurrence and the patients' survival time by 1-1/2 to 2 years or more, with a substantial portion of patients going far beyond that. Incorporating NW Bio's patented methods for more potent dendritic cells will enable production of enhanced versions of these DCVax-L and DCVax-Prostate products.
https://prnewswire.com/news-releases/nw-bio-receives-us-patent-on-broad-processes-for-producing-more-potent-dendritic-cells-198760831.html
ATLnsider
Re: hyperopia post# 692199
Saturday, May 18, 2024 4:20:28 PM
Post#
692493
of 692641
Thanks hyperopia, you may also want to read 1 of my other previous posts regarding DCVax-L along with poly-ICLC:
hyperopia
Re: ATLnsider post# 692493
Sunday, May 19, 2024 2:00:26 PM
Post#
692615
of 692641
Right ATLnsider, as you indicate, in the UCLA combination studies with DCVax-L, the poly-ICLC is administered separately as an adjuvant. However, in the early studies with DCVax-Direct, it was used in the manufacturing process, which increased the potency. (poly I:C, or its derivative, poly-ICLC is included in the Direct patents, and the more recent hyperactive patents)
NW Bio Receives U.S. Patent On Broad Processes For Producing More Potent Dendritic Cells
Next Generation Technology Already In DCVax®-Direct; Will Be Applicable To All DCVax® Products
Building upon the pure immature dendritic cells, NW Bio's patented methods develop mature and activated dendritic cells that are far more potent than dendritic cells produced in the standard way. For example, NW Bio's dendritic cells produce as much as 10X or more the amount of signaling compounds which are key to mobilizing other active agents of the immune system, such as T cells (which infiltrate and attack tumors) and B cells (which produce antibodies).
NW Bio is already using these next generation methods for producing more potent dendritic cells in its production of DCVax-Direct. The same patented methods for activating dendritic cells were also used in the pre-clinical animal studies with DCVax-Direct. In those studies, injection of these potent dendritic cells into some of the tumors in each of the animals resulted in complete clearance of all tumors (both the tumors injected with DCVax-Direct and the tumors not injected) in 80-100% of the animals in the various studies, indicating a system-wide immune response.
?
Going forward, NW Bio's now patented methods of producing more potent dendritic cells will also enable development of the next generation of NW Bio's other two product lines: DCVax-L and DCVax-Prostate. The current DCVax-L and DCVax-Prostate products have already delivered striking results in clinical trials to date, extending the time to tumor recurrence and the patients' survival time by 1-1/2 to 2 years or more, with a substantial portion of patients going far beyond that. Incorporating NW Bio's patented methods for more potent dendritic cells will enable production of enhanced versions of these DCVax-L and DCVax-Prostate products.
https://www.prnewswire.com/news-releases/nw-bio-receives-us-patent-on-broad-processes-for-producing-more-potent-dendritic-cells-198760831.html
For DCVax-L, Northwest Bio uses the most common method of culturing PBMC’s to generate dendritic cells, which relies on the monocytes adhering to the bottom surface of the polystyrene culture vessel in a medium of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), which induces differentiation into dendritic cells, and activates them.
I believe this increased-potency knowledge was gained in the DCVax-Direct studies because they were attempting to overcome the tumor’s defenses by delaying the activation and maturation of the dendritic cells, which wasn’t possible using culture flasks that are used to produce DCVax-L, so they attempted culturing the monocytes in suspension, in a bagged system without IL-4, using only GM-CSF, and then various other mediums to optimize and activate the dendritic cells, including poly I:C.
Northwest Bio stated that they intended to use this higher potency method across all product lines for the next generation of DCVax, but the Flaskworks’ eden system uses a polystyrene culture cartridge (similar to the well plates used in manual culturing protocols) which the monocytes adhere to, so I’ve not seen evidence that they’ve actually changed the manufacturing method for the next generation of DCVax-L. I believe the method of combining poly-ICLC with DCVax-L requires separate (nearly simultaneous) injections to produce the more potent effect in vivo.
NWBO
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In this post, we can see that the original objective of the 2010 clinical trial, was to determine the best TLR agonist to add as a component in the DCVax-L formulation. Not as an adjuvant treatment, but as a DCVax formulation component. The poly-ICLC would be co-administered along with DCVax-L, at the same time. The poly-ICLC would send “the danger signal”, and would act as an in vivo maturation agent that would mature and hyperactivate the dendritic cell in situ:
https://classic.clinicaltrials.gov/ct2/show/NCT01204684
The main purpose of this study is to evaluate the most effective immunotherapy vaccine components in patients with malignant glioma.
The (sp) investigators previous phase I study (IRB #03-04-053) already confirmed that this vaccine procedure is safe in patients with malignant brain tumors, and with an indication of extended survival in several patients. However, the previous trial design did not allow us to test which formulation of the vaccine was the most effective. This phase II study will attempt to dissect out which components are most effective together
The first trial was a competition to decide which TLR agonist to add to the DCVax-L formulation, either TLR-3 (poly-ICLC) or TLR-7 (imiquimod / resiquimod), as a component in the DCVax-L formulation, to act as an in vivo maturation agent:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3568250/
In contrast, we administered the Toll-like receptor (TLR) agonists, imiquimod or poly ICLC, following intradermal injections of ATL-DC to induce DC maturation in vivo. We previously demonstrated in pre-clinical models that the utilization of TLR agonists could enhance the survival and trafficking of DC in situ and enhance the priming of tumor antigen-specific T lymphocytes 31. The findings from this current study suggest that the induction of patient-specific anti-tumor immunity using ATL-DC vaccination and in situ maturation with TLR agonists may represent a preferred formulation for DC-based therapies
The DCVax-L vaccine is administered intradermally, and the poly-ICLC is administered intramuscularly, as illustrated by Dr. Robert Prins in 2014:
Bullish
BULLISH
This is a PSA to all manipulative and collusive Market Makers, Hedge Funds and Short Sellers:
TIME IS RUNNING OUT. THE CLOCK IS TICKING!!!
beartrap12
Re: None
Saturday, May 18, 2024 10:05:57 AM
Post#
692432
of 692639
What are the possibilities that DCVax-L will be approved for new GBM and recurring GBM at one of the next four meeting of the UK’s CHM (Commission on Human Medicines).
They meet on these dates:
May 30-31
June 27-28
July 25-26
Aug. 29-30.
But which is most likely approval day?
Many have already discounted May 30-31, but I think it’s still possible. First, It fits with our submission deadline date of Dec. 25, 2023. May 25 is exactly 150 days from this deadline, but it’s a Saturday, so if UK’s CHM wants to move quickly on DCVax-L, it makes sense that they would approve it at 149 days, rather wait another month for when the commission meets next. Secondly, we received a “validation confirmation” date on May 7th, which just happens to be 2 days before we hit the 80-day completion of Phase 1 of the regulatory process, if you believe the clock started on Dec. 20 when we submitted our application.
But we have since learned that the clock starts when we receive “validation”. Our application passed validation on Jan. 24th, then our validation was “confirmed" on March 7th. The two different validations reported in the quarterly have created some confusion and speculation. I think there is a possibility that the second validation may represent when our application completed Phase I where we received NO request for info from CHM, which means NO need to stop the 150-day clock. Confirm means to check again. That fits with the idea that the first validation was quicker and less thorough and was followed by a more complete examination of the MAA, which ended in the March 7th confirmation and perhaps the end of Phase I. This hypothesis only works if we assume CHM ignored their very late (IMO) Jan. 24 validation and moved quickly to study our MAA.
The second possible CHM meeting on June 27-28 coincides with the first validation date of Jan. 24. 150 days out from Jan. 24 is actually June 22, another Saturday and 5 days before the start of this meeting. If we follow the 150-day law to the letter and believe there is no clock stop for answering questions, then this is the likeliest time to expect the UK to make a decision on our application.
The third possible CHM meeting date, July 25-26, somewhat aligns with the possibility that the clock started when our validation was “confirmed” on March 7th. 150 days out from that confirmation is actually July 29, a Monday. If CHM members wanted to move along our MAA, they could choose to make their decision the weekend before completion of the 150-day time table and hear it on July 25-26.
If they decide not to make a decision on our MAA a weekend early, they would have to wait almost a month until their next meeting on Aug. 29-30. I don’t see that happening unless they are trying to give our automated system another month to complete validation.
None of these dates address the possibility that NWBO may need to stop the clock to respond to a request for information letter from the UK. If this happens it could add 60 or even 120 days to the approval process. I think a clock stop at this point is unlikely, given the comments by Les and in the quarterly. Both were very bullish comments. NWBO has been very cagey about appearing bullish because so many negative shorts have attacked the company and even reported them to regulators when they’ve committed lesser infractions. If the clock started on Dec. 25 or on Jan. 24, in both cases, we would have completed the 80-day phase I long before the time we received the quarterly on May 10 and heard Les speak on May 9. If we accept the March 7th date for when the 150-day clock began, we would have been about 2 weeks before completion of Ph. 1 I believe the rules state that companies receive a request for info only after Ph 1 is complete. I think it’s unlikely Les would have been so bullish as to state “no news is good news” and the quarterly would have told us they had not changed a thing in our MAA if they knew the potential clock-stopping news was still in the future.
Of course, all of this assumes CHM doesn't set a special meeting for NWBO's application.
Bullish
BULLISH
ATLnsider
Re: hyperopia post# 692199
Saturday, May 18, 2024 4:20:28 PM
Post#
692493
of 692498
Thanks hyperopia, you may also want to read 1 of my other previous posts regarding DCVax-L along with poly-ICLC:
https://investorshub.advfn.com/boards/read_msg.aspx?message_id=174435365
In this post, we can see that the original objective of the 2010 clinical trial, was to determine the best TLR agonist to add as a component in the DCVax-L formulation. Not as an adjuvant treatment, but as a DCVax formulation component. The poly-ICLC would be co-administered along with DCVax-L, at the same time. The poly-ICLC would send “the danger signal”, and would act as an in vivo maturation agent that would mature and hyperactivate the dendritic cell in situ:
https://classic.clinicaltrials.gov/ct2/show/NCT01204684
The main purpose of this study is to evaluate the most effective immunotherapy vaccine components in patients with malignant glioma.
The (sp) investigators previous phase I study (IRB #03-04-053) already confirmed that this vaccine procedure is safe in patients with malignant brain tumors, and with an indication of extended survival in several patients. However, the previous trial design did not allow us to test which formulation of the vaccine was the most effective. This phase II study will attempt to dissect out which components are most effective together
The first trial was a competition to decide which TLR agonist to add to the DCVax-L formulation, either TLR-3 (poly-ICLC) or TLR-7 (imiquimod / resiquimod), as a component in the DCVax-L formulation, to act as an in vivo maturation agent:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3568250/
In contrast, we administered the Toll-like receptor (TLR) agonists, imiquimod or poly ICLC, following intradermal injections of ATL-DC to induce DC maturation in vivo. We previously demonstrated in pre-clinical models that the utilization of TLR agonists could enhance the survival and trafficking of DC in situ and enhance the priming of tumor antigen-specific T lymphocytes 31. The findings from this current study suggest that the induction of patient-specific anti-tumor immunity using ATL-DC vaccination and in situ maturation with TLR agonists may represent a preferred formulation for DC-based therapies
The DCVax-L vaccine is administered intradermally, and the poly-ICLC is administered intramuscularly, as illustrated by Dr. Robert Prins in 2014:
In the trial on subcutaneous keytruda mortality rate in the SC group is 5% higher than the IV group as pointed out by Doc logic on ihub. Merck is going to hold Oncology Investor Event at ASCO on June 3. Just wonder what Merck will comment on their progress on SC keytruda.… pic.twitter.com/TnnofMtLSR
— d_stock (@d_stock07734) May 18, 2024
$nwbo @alphavestcap @TiltMyBrain @glen_bwrhr42 @metacollectiveG @ATLnsider @SmithOnStocks1 @BrianEgolf2 @hoffmann6383 https://t.co/Zf8D8Xvwij
— alphavestcapital.com (@alphavestcap) May 18, 2024
@d_stock07734In the trial on subcutaneous keytruda mortality rate in the SC group is 5% higher than the IV group as pointed out by Doc… https://t.co/bdGHzzmFlV
Chiugray
Re: Red_Right_Hand post# 692286
Friday, May 17, 2024 3:33:44 PM
Post#
692372
of 692398
DCVax-L becomes the new SOC for GBM
- Successful DCVax-L Phase 3 clinical trial when comparing a) SOC vs b) SOC + DCVax-L.
- To be approved by MHRA soon
- Proves adding DCVax-L increases overall survival, with statistically significance and non-toxic.
Poly-ICLC adjuvant to DCVax-L becomes validation to doctors for Off-Label use in combination therapy for malignant brain tumors.
- The successful UCLA Phase 2 clinical trial compared a) DCVax-L vs b) DCVax-L + Poly-ICLC
- Nature publication
- Proves that adding Poly-ICLC increases overall survival, with statistical significance and non-toxic.
- Over time RWE makes this the new SOC for malignant brain tumors.
And when we look back in the future, this modular format for combination therapies with DCVax as the base layer, will be identified as the moment in history that the foundation for curing cancer was born. All IMHO.
Bullish
BULLISH
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When delivered, please post a transcript of Liau's speech. Thanks.
#dcvax $nwbo #gbm
— Peter Davis (@peter_brit) May 17, 2024
The amazing Linda Liau receives her 'Winn Prize' tomorrow, so very much deserved.
"To recognize outstanding, continuous commitment to research in the neurosciences by a neurological surgeon"https://t.co/5glLwExdO6 pic.twitter.com/Un7GDgKVng
The Medicare Bubble Has Burst
Government health-insurance program had been a gold mine for private insurers until recently
By
David Wainer
For years, the privately run Medicare Advantage business generated outsize profit growth for health-insurance giants.
With hundreds of billions of taxpayer dollars flowing to insurers in a fast-growing market buoyed by aging baby boomers, there was little not to like as far as Wall Street was concerned. Companies like UnitedHealth Group UNH 0.72%increase; green up pointing triangle and Humana HUM 2.32%increase; green up pointing triangle bet big on the program, and investors generally rewarded them for it. Medicare Advantage, in which the government pays insurers a set amount to manage the care of seniors, recently surpassed traditional Medicare’s share of beneficiaries. It was 30% a decade ago.
But the gold rush is over for investors, at least for now. After years of reports, lawsuits and whistleblower accounts accusing big insurers of gaming the system and overcharging the government, the Biden administration has made a series of policy changes that have negatively affected what the plans get paid. Meanwhile, a post-Covid surge in seniors’ medical costs caught insurers by surprise.
The stark drop in profitability is rattling corporate boards and investors. One of the biggest losers is CVS Health CVS 2.24%increase; green up pointing triangle, whose Aetna unit bet big on Medicare right before costs soared. To gain market share, CVS-Aetna offered generous plans this year, surprising some executives at rival insurers. While the move paid off in terms of membership count—CVS added more than 700,000 Medicare Advantage members this year—the company underestimated what it would cost to insure them. In its first-quarter earnings report earlier this month, CVS said the segment helped drive medical costs $900 million higher than the company had expected. Its shares had their largest one-day drop in almost 15 years in response and are down 26% for the year, giving it a market capitalization of just over $70 billion. That is roughly what it paid for Aetna back in 2018.
CVS isn’t the only one in trouble: Medicare-focused insurers, some of which had vastly outperformed the stock market in the past several years, are underperforming this year. Humana shares are down more than 20% this year and even industry leader UnitedHealth, which was more conservative in how it priced its plans, was down as much as 16% for the year in April before recouping much of the losses.
That by no means signals insurers are about to flee Medicare Advantage (though Cigna CI -1.17%decrease; red down pointing triangle did agree to sell its business earlier this year). Annual spending on the program, which is now hardwired into America’s healthcare system, is projected to approach $1 trillion by the start of the next decade. And while declining profit expectations have negatively affected share performances, there are still plenty of profits to be made.
Take hard-hit Humana, which is mostly focused on Medicare Advantage. The firm is expected to earn significantly less in 2024, but analysts polled by FactSet still see it making just over $16 per share this year. By 2026, analysts expect earnings to rise back to $26 per share—some $3 billion in net income.
The high cost of covering seniors is likely a temporary problem for insurers, who get to submit their bids to the Centers for Medicare and Medicaid Services every year. While they are limited in the changes they can make, CVS and others said they are planning to exit some counties and cut back on things such as vision benefits to boost margins.
“The goal for next year is margin over membership,” CVS Chief Financial Officer Thomas Cowhey said at a recent conference. “Could we lose up to 10% of our existing Medicare members next year? That’s entirely possible.” By all indications, other large players such as Humana will also be shifting from growth to profits. That could create an opportunity for leader UnitedHealth, which has relatively better profit margins, to grab more market share, argues Scott Fidel, an analyst at Stephens.
The tougher challenge is on the regulatory side. The Biden administration’s changes, from releasing stingier payment rates to changes in how programs can code patient risk, signal an era of tighter purse strings. With such a big part of their business at stake, the industry’s effort to sway public and policymakers’ opinions is expected to go into overdrive.
“I can assure you that the companies will be investing pretty heavily through campaign donations and lobbying to try to figure out what they can do from a public policy point of view,” said Wendell Potter, a former top communications employee at Cigna Group who is now a critic of the industry.
For decades, policymakers have sought to bring private insurers along as a way to manage soaring Medicare costs. In 2003, Congress passed the Medicare Modernization Act, which created Medicare Advantage as we know it. The idea, in a nutshell, is to bring down costs and improve care by allowing insurers to manage care, much like they do for the nation’s employers.
The high cost of covering seniors is likely a temporary problem for insurers. PHOTO: ANDREW HARRER/BLOOMBERG NEWS
But critics point to studies showing that Medicare Advantage plans cost the government and taxpayers billions of dollars more than traditional Medicare.
“For well over a decade, Medicare Advantage plans have been making extremely high profits. What’s going on now are long overdue policy changes to bring their pricing and coding practices back into line,” said Dr. Don Berwick, former head of the Centers for Medicare and Medicaid Services.
SHARE YOUR THOUGHTS
Do you have confidence in the future of Medicare Advantage? Why or why not? Join the conversation below.
In the near term, the best hope for a quick shift to insurers’ fortunes could be a Donald Trump win in the coming presidential elections, Fidel said. Republican administrations, which tend to favor privatization of government services, have been more favorable toward Medicare Advantage.
In either case, it isn’t going away—the business remains highly profitable. But the bonanza investors and health conglomerates got accustomed to in recent years has diminished for the foreseeable future.
Write to David Wainer at david.wainer@wsj.com
Copyright ©2024 Dow Jones & Co
CORRECTED COPY:
beartrap12: Thanks for the reply. As you might recall, in 5/21,Ashkan said in a Spotify medical podcast that his Specials patients treated with DC VAX L have been living 7-8 years. How could that be without poly ic lc being dosed in conjunction with dc vax L ? But, I certainly could not be sure at that 5/21 date . Then on 5/10/22 at the NYAS, I asked someone who would know the most about Ashkan's Specials data , how could Ashkan come to that conclusion . The reply: " Well, he has had 200-250 Specials patients.". AVC assumes he is up to 500 by now. Recently, a UK field agent speculated to me that Ashkan started to use poly iclc in '16. Is that true according to your records-contacts ? P.S., I believe ATLnsider has stated that when Liau-Ashkan state that dc vax L is used in a combo, it's implicit that poly iclc is bundled,i.e., by their policy,dc vax L is never given without poly iclc. I hope you can clarify , so I and others are not incorrect. Thanks so much .A.E.K.
Re: ae kusterer post# 692106
Thursday, May 16, 2024 6:06:28 PM
Post#
692147
of 692148
beartrap12 : how many of the c.500 Specials patients in the U.K. received poly iclc?
ae kusterer, that's another question we need to ask at the ASM!
Who wants startc a list ?
Bullish
beartrap12: Thanks for the reply. As you might recall, in 5/21,Ashkan said in a Spotify medical podcast ( @MB years. How could that be without poly iclc being dosed in conjunction with dc vax L ? But, I certainly could not be sure at that 5/21 date . Then on 5/10/22 at the NYAS, I asked someone who would know the most about Ashkan's Specials data , how could Ashkan come to that conclusion . The reply: " Well, he has had 200-250 Specials patients.". AVC assumes he is up to 500 by now. A UK field agent speculated to me that Ashkan started to use poly iclc in '16. Is that true according to your records-contacts ? P.S., I believe ATLnsider has stated that when Liau-Ashkan state that dc vax L is used in a combo, it's implicit that poly iclc is bundled,i.e., dc vax L is never given without poly iclc. I hope you can clarify , so I-others are not incorrect. Thanks so much .A.E.K.
Re: ae kusterer post# 692106
Thursday, May 16, 2024 6:06:28 PM
Post#
692147
of 692148
beartrap12 : how many of the c.500 Specials patients in the U.K. received poly iclc?
ae kusterer, that's another question we need to ask at the ASM!
Who wants startc a list ?
Bullish
Gary: thanks so much for this reply.A.E.K.
Re: ae kusterer post# 692104
Thursday, May 16, 2024 3:57:15 PM
Post#
692125
of 692132
There is no 60 day off clock period if no RFI is requested, the quarterly reinforced that no information was requested. If the clock didn't begin until the initial validation, we'd still be at the 150 day period before the end of June. There is nothing in the British system that says they must wait to day 150 to reveal their judgement. It's their ball game, if they must, they'll take more time and don't have to ask anyone's permission to do so.
I'd still not be surprised to hear from the company next week, but if not I'm really not certain that they'll actually set a date for the Annual Meeting until they do. If that meant moving the meeting into July, it wouldn't be the first time the company modified their guidance. I would hope that when the company gets the UK decision they not only PR it, but also schedule a webcast with investors and others to discuss it. The Annual Meeting, if not scheduled formally until they have the UK decision will probably be at least 20, and probably 30 days or more later.
I don't know if there have been any discussions between the UK authorities and the company during this period beyond what was said in the quarterly, where preliminary and full validation was confirmed. I don't believe investors realize how much communication there may be, but until something's official it may not mean much.
What actually may be of greater importance could be any discussions the company may be having with the other regulators as far as how they may act after a UK approval. I don't know that it's impossible that others, most likely Europe and Canada, may piggyback the UK approval with no or minimal effort required for additional approval.
It looks like the MM's are taking us down substantially at the close, after we went slightly green just minutes ago. If anyone wants to add shares, doing so now would get you a good price.
Gary
Gary
dstock07734
Re: hyperopia post# 692015
Thursday, May 16, 2024 3:43:55 PM
Post#
692116
of 692129
As I mentioned to beartrap, it is possible that Merck could use the combo trial on colorectal cancer to file for application too.biosectinvestor
The MHRA recognizes the potential use of external control arms in clinical trials, particularly when traditional randomized controlled trials (RCTs) are not feasible or ethical. This approach is notably applicable in the context of rare diseases or situations where patient recruitment for a control group is challenging. Exactly this case. Granted, this is all new, and same for their coordination with the FDA and EMA on adopting these policies in collaborative effort.
The MHRA also provides guidance on using real-world data (RWD) to support regulatory decisions, which includes the consideration of external control arms derived from sources such as historical clinical trials, electronic health records, or registries. Their focus is on ensuring that these external data sources are of high quality and appropriately matched to the trial population to minimize bias and ensure reliable results. NWBO went to great lengths and provided statistical and other data in spades to ensure this will be the case. Plus they have lots and lots of other data, side arms, clinical data from compassionate use and lots of supporting data.
Additionally, Health Technology Assessment (HTA) bodies across Europe, including the MHRA, increasingly accept evidence from single-arm trials complemented by external control arms. These studies also must employ rigorous analytical methods to address potential confounding factors and ensure the robustness of the evidence. Again, not really an issue in this case and they all consider historical data and this is contemporaneous data from the most important trials in this space. The individual patient data from THOSE trials is understandably, at this time, hard to come by and so the additional statistical comparisons and other work address that failing. And then remember, they hired a completely blind, external mathematical and epidemiological consulting firm filled with experts to match and provide all of this data. It is, in my opinion, very, very unlikely that besides possibly some questions, which is the job of regulators, they should find the various sources of evidence including NWBO’s scientific study on method of action, very satisfactory, compared to the other drugs previously approved in this space, most of which were in a far worse position and had no survival benefits to demonstrate.
Cool your horses.
Obviously there is never certainty with any of these trials, but there should be at least as much as with any of the other troubled trials that seemed to never bother any of the shorts before. Just their focus on this one that literally blows that old stuff out of the water in terms of the clinical evidence from numerous sources. There is even the UCLA Nature paper and UPenn confirmatory paper on scanning and measurement of results also confirming what NWBO said in its Phase 3 JAMA Oncology publication and what now everyone knows with a fair degree of comfort about immunotherapies, that they can cause pseudoprogression and also that patients do not want to join placebo trials when they have as certain a disease as GBM in terms of mortality. No one likes to be a Guinea Pig and there are more than enough Phase I and II trials these days that they do not need to be.
And let me close with this basic bit of information. It is no mystery to any doctor or patient or regulator in the field how fatal GBM is, how rapidly it kills and that the results seen in these trials is outstanding. While no doubt there could be formalists using formalism to hide their partisan and economic interests in any body, there should be no rational reason this is not ultimately approved, IMHO.
dennisdave
Re: hyperopia post# 692007
Thursday, May 16, 2024 2:27:06 PM
Post#
692067
of 692122
https://www.gov.uk/government/publications/mhra-performance-data-for-assessment-of-clinical-trials-and-established-medicines/mhra-performance-data-for-assessment-of-clinical-trials-and-established-medicines
Updated 15 May 2024
All eligible national Marketing Authorisation applications submitted before 1 January 2024 that were awaiting first assessment have been converted from the national route to recognition pathways, to speed up our regulatory assessments, where applicants requested this.
https://www.gov.uk/government/publications/international-recognition-procedure/international-recognition-procedure-supplementary-information
No delay to see here...
Re: beartrap12 post# 688309
Thursday, May 16, 2024 11:25:06 AM
Post#
692015
of 692124
beartrap, to market a new drug or biologic, regulations require adequate and well-controlled studies that distinguish the effect of a drug from other influences. And for regulatory approval of combination regimens, it is necessary to demonstrate the contribution effect of each monotherapy to the overall combination. This is why I believe that Northwest Bio’s first marketing application did not include other adjuvants.
ATLnsider has speculated that Northwest Bio is using poly-ICLC as a maturation and activation agent in the manufacturing process of DCVax-L and therefore, thinks it will be included in the marketing application. While I believe this manufacturing method may have been studied, I believe it was with DCVax-Direct, and not DCVax-L. Northwest Bio did not use this manufacturing method for the clinical trials with DCVax-L, so I do not believe the first marketing application includes poly-ICLC as a maturation/activation agent either.
As I’ve said, other than a few potential anecdotal cases, I don’t think Northwest Bio has enough (real-world) data to seek regulatory approval with Poly-ICLC at this time. I’m not aware of ANY actual evidence that studies with a significant number of patients were performed using this combination (or manufacturing method) for compassionate use, so this seems to be complete speculation. As far as Dr. Ashkan using it for compassionate use and collecting real-world data; I’ve seen a number his presentations about DCVax, and not once was poly-ICLC mentioned. The only data that I’m aware of with poly-ICLC, is the UCLA data, which I believe Northwest Bio intends to confirm through their own clinical studies,(both DCVax-L and Direct) as soon as possible. (after the first regulatory approval and in-licensing agreements are finalized) They might even run studies to determine the optimal method of combining poly-ICLC with DCVax; as a separate adjuvant, or as a maturation/activation agent in the manufacturing process.
BTW - I’ve read some of dstock’s posts, and in my opinion, his speculation seems rather far-fetched and not based in reality. For example, I don’t believe Merck or any Big Pharma company (except NW Bio’s CDMO, Advent) is constructing a manufacturing facility to manufacture DCVax, nor did Merck and Northwest Bio secretly run the combination trial for colorectal cancer. I highly doubt that any Big Pharma companies are currently hiring positions related to Northwest Bio, nor do I think that Northwest Bio is secretly applying for approval with poly-ICLC. Everyone is free to speculate and believe whatever they wish, so I mean no disrespect, but I’m just stating my personal opinion on this because you asked.
I think the extremely slow pace of DCVax development, and lack of communication, has led many of us to speculate that certain developments have occurred or could be happening behind the scenes. This very rarely proves true, and in most cases, Northwest Bio at least mentions, or alludes to their development plans in SEC filings or press releases, and announces significant milestones when they are achieved.
Re: beartrap12 post# 688309
Thursday, May 16, 2024 11:25:06 AM
Post#
692015
of 692109
beartrap, to market a new drug or biologic, regulations require adequate and well-controlled studies that distinguish the effect of a drug from other influences. And for regulatory approval of combination regimens, it is necessary to demonstrate the contribution effect of each monotherapy to the overall combination. This is why I believe that Northwest Bio’s first marketing application did not include other adjuvants.
ATLnsider has speculated that Northwest Bio is using poly-ICLC as a maturation and activation agent in the manufacturing process of DCVax-L and therefore, thinks it will be included in the marketing application. While I believe this manufacturing method may have been studied, I believe it was with DCVax-Direct, and not DCVax-L. Northwest Bio did not use this manufacturing method for the clinical trials with DCVax-L, so I do not believe the first marketing application includes poly-ICLC as a maturation/activation agent either.
As I’ve said, other than a few potential anecdotal cases, I don’t think Northwest Bio has enough (real-world) data to seek regulatory approval with Poly-ICLC at this time. I’m not aware of ANY actual evidence that studies with a significant number of patients were performed using this combination (or manufacturing method) for compassionate use, so this seems to be complete speculation. As far as Dr. Ashkan using it for compassionate use and collecting real-world data; I’ve seen a number his presentations about DCVax, and not once was poly-ICLC mentioned. The only data that I’m aware of with poly-ICLC, is the UCLA data, which I believe Northwest Bio intends to confirm through their own clinical studies,(both DCVax-L and Direct) as soon as possible. (after the first regulatory approval and in-licensing agreements are finalized) They might even run studies to determine the optimal method of combining poly-ICLC with DCVax; as a separate adjuvant, or as a maturation/activation agent in the manufacturing process.
BTW - I’ve read some of dstock’s posts, and in my opinion, his speculation seems rather far-fetched and not based in reality. For example, I don’t believe Merck or any Big Pharma company (except NW Bio’s CDMO, Advent) is constructing a manufacturing facility to manufacture DCVax, nor did Merck and Northwest Bio secretly run the combination trial for colorectal cancer. I highly doubt that any Big Pharma companies are currently hiring positions related to Northwest Bio, nor do I think that Northwest Bio is secretly applying for approval with poly-ICLC. Everyone is free to speculate and believe whatever they wish, so I mean no disrespect, but I’m just stating my personal opinion on this because you asked.
I think the extremely slow pace of DCVax development, and lack of communication, has led many of us to speculate that certain developments have occurred or could be happening behind the scenes. This very rarely proves true, and in most cases, Northwest Bio at least mentions, or alludes to their development plans in SEC filings or press releases, and announces significant milestones when they are achieved.
Re: hyperopia post# 692015
Thursday, May 16, 2024 1:26:32 PM
Post#
692054
of 692106
hyperopia, thanks again for a very thoughtful analysis of our situation. I also agree we are only going for nGBM and rGBM with NO poly ICLC in the current MAA in front of MHRA. I think it's imperative that DCVax-L be approved on its own to start. It looks like we already have a Ph I with poly-ICLC and also a clinical trial with Merck's Keytruda added, that involves poly-iclc. Details from the first trial already tells us poly-iclc is adding time to patients' lives. We're all hoping to hear more about the additio of Keytruda soon.
I also agree that poly-iclc could not have been added to our Ph III after the trial was started.
I look forward to hearing some figures from the company on how many patient have received DCVax-L + poly-iclc and how well they are doing in the real world.
It will be interesting to see if NWBO adds poly-iclc to patient treatments if and when we receive approval!
Thanks again!
hyperopia
Re: beartrap12 post# 688309
Thursday, May 16, 2024 11:25:06 AM
Post#
692015
of 692115
beartrap, to market a new drug or biologic, regulations require adequate and well-controlled studies that distinguish the effect of a drug from other influences. And for regulatory approval of combination regimens, it is necessary to demonstrate the contribution effect of each monotherapy to the overall combination. This is why I believe that Northwest Bio’s first marketing application did not include other adjuvants.
ATLnsider has speculated that Northwest Bio is using poly-ICLC as a maturation and activation agent in the manufacturing process of DCVax-L and therefore, thinks it will be included in the marketing application. While I believe this manufacturing method may have been studied, I believe it was with DCVax-Direct, and not DCVax-L. Northwest Bio did not use this manufacturing method for the clinical trials with DCVax-L, so I do not believe the first marketing application includes poly-ICLC as a maturation/activation agent either.
As I’ve said, other than a few potential anecdotal cases, I don’t think Northwest Bio has enough (real-world) data to seek regulatory approval with Poly-ICLC at this time. I’m not aware of ANY actual evidence that studies with a significant number of patients were performed using this combination (or manufacturing method) for compassionate use, so this seems to be complete speculation. As far as Dr. Ashkan using it for compassionate use and collecting real-world data; I’ve seen a number his presentations about DCVax, and not once was poly-ICLC mentioned. The only data that I’m aware of with poly-ICLC, is the UCLA data, which I believe Northwest Bio intends to confirm through their own clinical studies,(both DCVax-L and Direct) as soon as possible. (after the first regulatory approval and in-licensing agreements are finalized) They might even run studies to determine the optimal method of combining poly-ICLC with DCVax; as a separate adjuvant, or as a maturation/activation agent in the manufacturing process.
BTW - I’ve read some of dstock’s posts, and in my opinion, his speculation seems rather far-fetched and not based in reality. For example, I don’t believe Merck or any Big Pharma company (except NW Bio’s CDMO, Advent) is constructing a manufacturing facility to manufacture DCVax, nor did Merck and Northwest Bio secretly run the combination trial for colorectal cancer. I highly doubt that any Big Pharma companies are currently hiring positions related to Northwest Bio, nor do I think that Northwest Bio is secretly applying for approval with poly-ICLC. Everyone is free to speculate and believe whatever they wish, so I mean no disrespect, but I’m just stating my personal opinion on this because you asked.
I think the extremely slow pace of DCVax development, and lack of communication, has led many of us to speculate that certain developments have occurred or could be happening behind the scenes. This very rarely proves true, and in most cases, Northwest Bio at least mentions, or alludes to their development plans in SEC filings or press releases, and announces significant milestones when they are achieved.
attilathehunt
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attilathehunt
Re: norisknorewards post# 692005
Thursday, May 16, 2024 11:18:33 AM
Post#
692010
of 692116
It does say expired because it missed the deadline of 4/5/24...However, it looks like they resubmitted on 3/25/24 under a different number....Not sure if it matters if it is for adult or pediatric.
newman2021
Re: manibiotech post# 692017
Thursday, May 16, 2024 12:28:57 PM
Post#
692024
of 692117
hyperopia is not giving any credit to anything the 10Qs are not saying. Period. But we heard from Les the very first sentence was 'All the approvals are coming'; he also talked about the Nature publication which involves only PCLYC. So, we don't know what all goes behind the scenes. The effect of PCLYC is humongous. We don't need Keytrudas any more and so LP has changed her Cementing language to Broad Franchising. While everything is happening in front of our eyes, the shorts are covering as we speak. The share price is in a holding pattern even at these low volumes... is indicative of the shorts covering. But they don't have too much time even if we see the approval in the Fall looking at the number of shares they have to cover both legal and naked. .
beartrap12 : how many of the c.500 Specials patients in the U.K. received poly iclc?
Re: hyperopia post# 692015
Thursday, May 16, 2024 1:26:32 PM
Post#
692054
of 692105
hyperopia, thanks again for a very thoughtful analysis of our situation. I also agree we are only going for nGBM and rGBM with NO poly ICLC in the current MAA in front of MHRA. I think it's imperative that DCVax-L be approved on its own to start. It looks like we already have a Ph I with poly-ICLC and also a clinical trial with Merck's Keytruda added, that involves poly-iclc. Details from the first trial already tells us poly-iclc is adding time to patients' lives. We're all hoping to hear more about the additio of Keytruda soon.
I also agree that poly-iclc could not have been added to our Ph III after the trial was started.
I look forward to hearing some figures from the company on how many patient have received DCVax-L + poly-iclc and how well they are doing in the real world.
It will be interesting to see if NWBO adds poly-iclc to patient treatments if and when we receive approval!
Thanks again!
underpar71 : when did the inspections start?
Re: newman2021 post# 692092
Thursday, May 16, 2024 3:23:16 PM
Post#
692101
of 692102
Impossible. Validation was January 24th. 150 days would be end of June at earliest, if the clock on started that day. Pretty confident there is a 60 day clock off period underway as well. So, earliest would be end of August per guidelines. Earlier would be great but I have seen this movie too many times with this company, so Les was likely correct.
@peter_brit
"In summary, the protein biomarkers identified in these studies could synergize with technologies like DCVax by enabling earlier diagnosis, personalized risk assessment, rational selection of vaccine targets, and potentially predicting which patients will respond best to vaccination. Integrating cancer proteomics with immune-based prevention and treatment strategies holds exciting potential for the future of oncology. However, further research is still needed to validate the predictive value of these protein markers and establish how to optimally integrate them with vaccine approaches like DCVax"
Key findings
Of the 1,463 proteins analysed, 618 proteins were associated with 19 different types of cancer;
107 of these proteins were present in blood samples of people who developed cancer more than seven years before they received a diagnosis;
182 proteins were present in blood samples of people who developed cancer more than three years before they were diagnosed.
https://ndph.ox.ac.uk/news/blood-proteins-may-be-able-to-predict-risk-of-cancer-more-than-seven-years-before-it-is-diagnosed
https://nature.com/articles/s41467-024-48017-6
https://nature.com/articles/s41467-024-46834-3
$nwbo @alphavestcap @hoffmann6383 @BrianEgolf2 @ATLnsider @metacollectiveG @SmithOnStocks1
— alphavestcapital.com (@alphavestcap) May 16, 2024
Despite : MAA approval imminent (by 8/1/24), a combo partnership in the works(30 days), per 10-Q, $11 million straight debt raised just prior to the 5/10/24 10-Q in 24 hours ( 8%, 1-2… https://t.co/LiWd7yn3nQ
@peter_brit
#dcvax $nwbo #gbm
"In summary, the protein biomarkers identified in these studies could synergize with technologies like DCVax by enabling earlier diagnosis, personalized risk assessment, rational selection of vaccine targets, and potentially predicting which patients will respond best to vaccination. Integrating cancer proteomics with immune-based prevention and treatment strategies holds exciting potential for the future of oncology. However, further research is still needed to validate the predictive value of these protein markers and establish how to optimally integrate them with vaccine approaches like DCVax"
Key findings
Of the 1,463 proteins analysed, 618 proteins were associated with 19 different types of cancer;
107 of these proteins were present in blood samples of people who developed cancer more than seven years before they received a diagnosis;
182 proteins were present in blood samples of people who developed cancer more than three years before they were diagnosed.
https://ndph.ox.ac.uk/news/blood-proteins-may-be-able-to-predict-risk-of-cancer-more-than-seven-years-before-it-is-diagnosed
https://nature.com/articles/s41467-024-48017-6
https://nature.com/articles/s41467-024-46834-3
One such study has been submitted through the MHRA’s New Notification Scheme for low-risk phase 3 and 4 trials – those trials that compare new treatments with the best currently available treatment (phase 3) and after a drug has been licensed (phase 4).
A new, risk-proportionate process
The company applied for its phase 4 trial under the MHRA’s new Notification Scheme for lower-risk clinical trials.
Introduced in October 2023, the new scheme aims to reduce the time taken by the MHRA to approve the lowest-risk clinical trials by more than 50%.
The introduction of the new Notification Scheme followed the agency’s clinical trials consultation, which was endorsed by 74% of respondents. It forms a significant part of the MHRA’s overhaul of clinical trials regulation.
About 20% of UK initial clinical trial applications are expected to be eligible for the scheme. It is underpinned by a risk proportionate approach that relies on the sponsor demonstrating that the trial meets certain key criteria.
To access the scheme, sponsors register their interest directly with the MHRA. Guidance is then sent to the sponsor, detailing the information that is needed in a cover letter and notification scheme criteria form. This information is then submitted in the usual way to the Integrated Research Application System (IRAS) – the single system for applying for permissions and approvals in the UK.
Once received by the MHRA, the information is reviewed, and the trial is either accepted or objected to; the sponsor is updated within 14 days of the application’s effective date. If the trial is objected, the application progresses through the full combined review CTA assessment route, which would include the co-assessment of the application by a Research Ethics Committee, as set out in the process below.
Taking advantage of shorter timelines
As the trial submitted by Octapharma met the criteria for low-risk phase 4 trials, it was eligible for the new Notification Scheme. After being reviewed, the MHRA was able to notify Octapharma that the trial was acceptable under the notification scheme, by day 14.
This case study demonstrates the effective operation of the MHRA’s new, streamlined clinical trials system, in reducing the time taken to get the lowest-risk clinical trials up-and-running without undermining patient safety.Clinical trials regulation should be flexible and risk-proportionate so that the regulatory requirements are geared to the risk that a trial presents.This will help give UK patients quicker access to the potentially life-saving medicines being studied.
Andrea Manfrin, the MHRA’s Deputy Director, Clinical Investigations and Trials
https://gov.uk/government/case-studies/innovation-in-regulation-is-shortening-timelines-for-lowest-risk-clinical-trials
With all due respect to IHUB. commentators posting very astute observations, of which I do not want to loose track. I aplogize.A.E.K
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Alias Born 09/07/2014
beartrap12
Re: skitahoe post# 691935
Thursday, May 16, 2024 6:46:21 AM
Post#
691949
of 691964
First, Gary, Flaskworks is NWBO. We own the the Eden system. I think you mean that you believe NWBO will remain "quite small' in terms of personnel, not income. I think their income from DCVax will be huge and I think they'll bring on whatever personnel they need to do the job. I agree that we'll be paying CRL a decent amount for manufacturing and cryopresrvation, and that they stand to benefit finanially very well and couldl eventually probably do more of the work of manufacturing in the world than NWBO's one Sawston's plant. However, as the owner, we will do even better. It's a lot like selling photography. Most photographers send their pictures out to be printed by a lab, and they make less per picture. I do my own printing, so I save that cost, but it costs me more in time and labor. There were years when the manufacturing of my pictures cut into time I could have been out photographing. It's a trade-off.
I think NWBO is about to make themselves and a lot of other companies a lot of money. The word franchise means a lot of licensing out to other companies. Think of McDonalds. Do you really think the main company suffers from having thousands of individuals who own a franchise and run it themselves?
Bullish
BULLISH
Re: Horseb4CarT post# 691920
Wednesday, May 15, 2024 10:08:03 PM
Post#
691931
of 691964
I think the shorts know that approval is likely going to happen. I think their short position is far too large for them to just "go long and go for the ride". They have to buy to cover the huge short position, rather than to buy to go long, though maybe they are doing a little of both. The problem for them is that on the one hand they need to keep the price down and for news to be delayed for as long as possible for them to unwind their position. On the other hand the lack of news, uncertainty, and low share price keeps trading volume low, which means it takes longer to unwind. But that is all they have to work with. News along with huge volume and their game is over. I still have my fingers crossed for a rather large short squeeze when the ball gets rolling down hill.Re: beartrap12 post# 691926
Wednesday, May 15, 2024 10:57:21 PM
Post#
691935
of 691964
I agree Beartrap, I've always believed that CRL has the potential of providing the vaccine in much of the world, and the remainder handled in Sawston. The question in my mind is, does CRL pay NWBO for the rights to produce the vaccine and use the EDEN units, or is it the other way around with NWBO paying them to do it. The difference between on scenario and the other would be how much each receives for every batch of the vaccine that's made and distributed over time. There are virtually unlimited ways the agreement could be structured.
It's my belief that if this is done with CRL, it will be them, not NWBO, who'll be taking almost total responsibility for manufacturing, storage, distribution and sales in much of the world. CRL is already a large company, but some growth may be needed. NWBO on the other hand may minimize growth mainly to develop new trials and products. If NWBO chooses to continue to contract out much of their work, the growth of the company could be quite small, but FlaskWorks itself might grow dramatically depending on the demand for the EDEN unit, and perhaps other similar devices for producing other personalized products.
Gary
Wednesday, May 15, 2024 9:28:06 PM
Post#
691917
of 691964
Dstock, thanks for your DD. Maybe somebody who can read German can check the clinical trial site in Germany. I remember this trial and Merck pushing BMS out of the picture. That trial is 8 years old now if it started in 2016. It should have some decent long term results.
Let's ask this question at the ASM: What happened to the German trial between DCVax-L and Merck in Germany for colorectal cancer which started in 2016?
Bullish
BULLISHbeartrap12
Re: skitahoe post# 691918
Wednesday, May 15, 2024 9:54:56 PM
Post#
691926
of 691963
Skitahoe, all DCVax-L/polyiclc treatments will be processed at NWBO approved sites whether its for a clinical trial or not, according to everything I've read. After CRL is on line - contract is coming, IMO - there may eventually be Eden units in many or all of CRL's plants around the world, IMO. And once the Eden units are validated, there will be no need to go back to manual processing. I'm no expert, Gary, but this is what makes sense for now and the near future. Frankly, I don't see NWBO allowing any other company to take charge of the Eden units. Remember, with DCVax, the manufacturing is a large portion of the treatment, so we don't want to lose control of it.
Re: sentiment_stocks post# 691924
Wednesday, May 15, 2024 11:35:04 PM
Post#
691941
of 691964
I made the same mistake. That guidance is for clinical trial applications, not for new drug applications. Drug applications start after the validation date.
Validation
The assessment timetable for new active substances and biosimilar products or existing active substances will begin after the validation of the application.
https://www.gov.uk/guidance/guidance-on-150-day-assessment-for-national-applications-for-medicines
beartrap12, dstock :did the trail announced by nwbo 11/17/16 ever accrue patients? thanks.
Re: dstock07734 post# 691912
Wednesday, May 15, 2024 9:28:06 PM
Post#
691917
of 691923
Dstock, thanks for your DD. Maybe somebody who can read German can check the clinical trial site in Germany. I remember this trial and Merck pushing BMS out of the picture. That trial is 8 years old now if it started in 2016. It should have some decent long term results.
Let's ask this question at the ASM: What happened to the German trial between DCVax-L and Merck in Germany for colorectal cancer which started in 2016?
Bullish
17
NOV
2016
NW Bio Announces Phase II Clinical Trial Program Combining DCVax®-L and Pembrolizumab (Keytruda®) for Colorectal Cancer
Trial to Be Led By University Of Mainz Center of Excellence
BETHESDA, MD, November 17, 2016 – Northwest Biotherapeutics (NASDAQ: NWBO) (“NW Bio”), a U.S. biotechnology company developing DCVax® personalized immune therapies for solid tumor cancers, today joined the University Medical Center (UMC) of the Johannes Gutenberg University Mainz in Germany in announcing a Phase II clinical trial program combining DCVax®-L and Pembrolizumab (Keytruda®) for colorectal cancer with liver metastases.
The trial will be conducted as an investigator-initiated trial led by the University of Mainz, thereby substantially reducing the costs involved. NW Bio will provide the DCVax-L products and MSD Sharp & Dohme GmbH will provide the Pembrolizumab. (MSD Sharp & Dohme is the operating name used by U.S.-based Merck & Co., Inc. outside the U.S. and Canada.) All of the parties contributed to and approved the novel trial design.
This trial will combine a broad spectrum personalized vaccine (DCVax®-L) with a highly targeted checkpoint inhibitor drug (Pembrolizumab). In contrast, most combination trials to date have combined two specifically targeted agents, with no broad spectrum agent included.
Colorectal cancer represents a major unmet medical need. The number of new cases per year is second only to lung cancer, and colorectal cancer results in approximately 700,000 deaths per year in the Western world. Colorectal cancer is very heterogeneous and various therapies to date, including various immune therapies when used as a single agent, have been disappointing.
The UMC of Johannes Gutenberg University Mainz is a leading Center of Excellence for colorectal cancer, and especially liver metastases of colorectal cancer. The 1st Department of Medicine provides a wide range of treatments and conducts a variety of clinical trials, especially in gastrointestinal oncology.
After the parties complete certain preparatory activities, including obtaining regulatory approvals, the Phase II trial will treat patients for up to 6 months with DCVax®-L and Pembrolizumab, followed by long-term follow-up regarding patient survival.
Linda Powers, CEO of NW Bio, noted that, “More than 2 years of planning and work have gone into developing this Phase II trial program. We are excited to work with a leading team to test the potential synergies of combining broad spectrum DCVax-L and targeted Pembrolizumab to treat a heterogeneous and challenging disease such as metastatic colorectal cancer.”
The University of Mainz’s own announcement can be found on their website at https://www.unimedizin-mainz.de/home.html?L=1
About Northwest Biotherapeutics
Northwest Biotherapeutics is a biotechnology company focused on developing personalized immunotherapy products designed to treat cancers more effectively than current treatments, without toxicities of the kind associated with chemotherapies, and on a cost-effective basis, in both the United States and Europe. The Company has a broad platform technology for DCVax dendritic cell-based vaccines. The Company’s lead program is a 348-patient Phase III trial in newly diagnosed Glioblastoma multiforme (GBM), which is on a partial clinical hold in regard to new screening of patients. GBM is the most aggressive and lethal form of brain cancer, and is an “orphan disease.” The Company is under way with a Phase I/II trial with DCVax-Direct for inoperable solid tumor cancers. It has completed the 40-patient Phase I portion of the trial. The Company previously conducted a Phase I/II trial with DCVax-L for metastatic ovarian cancer together with the University of Pennsylvania. In Germany, the Company has received approval of a 5-year Hospital Exemption for the treatment of all gliomas (primary brain cancers) outside the clinical trial.
About MSD Sharp and Dohme GmbH
MSD Sharp and Dohme GmbH is the German subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA, a leading healthcare company that discovers, develops, manufactures and markets a wide range of innovative pharmaceutical products to improve human health and animal health. It focuses on several key therapeutic areas, including Cardiovascular, Diabetes, Immunology, Infectious Disease, Oncology and Women’s Health. It also has a strong pipeline in oncology with pembrolizumab (Keytruda®) being the most advanced pharmaceutical product.
Disclaimer
Statements made in this news release that are not historical facts, including statements concerning future treatment of patients using DCVax and future clinical trials, are forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Words such as “expect,” “believe,” “intend,” “design,” “plan,” “continue,” “may,” “will,” “anticipate,” and similar expressions are intended to identify forward-looking statements. Actual results may differ materially from those projected in any forward-looking statement. Specifically, there are a number of important factors that could cause actual results to differ materially from those anticipated, such as risks and uncertainties related to the actions and decisions of Nasdaq, the Company’s ongoing ability to raise additional capital, risks related to the Company’s ability to enroll patients in its clinical trials and complete the trials on a timely basis, uncertainties about the clinical trials process including the actions and decisions of the FDA and other regulators, uncertainties about the timely performance of third parties, risks related to whether the Company’s products will demonstrate safety and efficacy, risks related to the Company’s and Cognate’s abilities to carry out the intended manufacturing and expansions contemplated in the Cognate Agreements, risks related to the Company’s ability to carry out the Hospital Exemption program and risks related to possible reimbursement and pricing. Additional information on these and other factors, including Risk Factors, which could affect the Company’s results, is included in its Securities and Exchange Commission (“SEC”) filings. Finally, there may be other factors not mentioned above or included in the Company’s SEC filings that may cause actual results to differ materially from those projected in any forward-looking statement. You should not place undue reliance on any forward-looking statements. The Company assumes no obligation to update any forward-looking statements as a result of new information, future events or developments, except as required by securities laws.
CONTACT
Les Goldman
beartrap12
Re: dstock07734 post# 691896
Wednesday, May 15, 2024 7:48:11 PM
Post#
691907
of 691925
dstock, thanks for bringing up this trial in Germany back in 2016.
BETHESDA, MD, USA I November 17, 2016 I Northwest Biotherapeutics (NASDAQ: NWBO) (“NW Bio”), a U.S. biotechnology company developing DCVax® personalized immune therapies for solid tumor cancers, today joined the University Medical Center (UMC) of the Johannes Gutenberg University Mainz in Germany in announcing a Phase II clinical trial program combining DCVax®-L and Pembrolizumab (Keytruda®) for colorectal cancer with liver metastases.
I have no idea what's going on with this trial and whether it ever actually happened. Maybe somebody on the board knows something. I know that several trials proposed during that time frame did not go through.
If you're looking for a way that our FDA could approve DCVax-L without us knowing they were doing it, there's an easier way: We have reciprical agreements with numerous other countries' health agencies to accept their drug approvals if the FDA want to. I've forgotten the name of the program but it was discussed quite a bit on this forum in the last year when it was put into place. I think there's about 17 countries and the European Union that signed the agreement. So, technically, UK's approval could quickly trigger approvals by other countries. For example, I would not be surprised if Canada follows UK's lead with little or no examination of NWBO's MAA within a few months of UK approval. Would the US do this? I don't think they would, but we do have a president who's son died of brain cancer and who is pushing the cancer moonshot program. I would not be surprised to hear he knows about NWBO and DCVax and I would not be surprised if he urges FDA to approve DCVax-L as quickly as possible. Obviously, he has no control over FDA decisions. Personally, I would love to see Joe Biden use an approval of DCVax-L in the US as an example of his Cancer Moonshot's success.
meirluc: The SEC guy who posts on Twitter said companies in NWBO's position could forestall the ASM. until the end of the year.
Re: hankmanhub post# 691817
Wednesday, May 15, 2024 2:55:49 PM
Post#
691840
of 691919
You may be right Hankmanhub. If Flipper's idea of MHRA approval
in July were correct, the ASM would not have been scheduled
for late June but more likely thereafter, for late July, August or even early
September.
After LG's remarks, the only two logical timelines for an MHRA
approval seem to be:
1. Approval within the next few weeks followed by the scheduled ASM in
late June.
2. Approval in the fall which would have made a scheduled ASM thereafter
too late because the previous ASM was held almost 2 years before
that time (12/30/23). NWBO therefore felt forced to hold an ASM next month,
which unfortunately would amount to several months before the expected
MHRA fall approval.
I am guessing that if if we don't receive an approval PR in the next three weeks,
alternative 2 will become the most likely outcome.
dstock07734
Followers 55
Posts 3599
Boards Moderated 0
Alias Born 05/25/2021
dstock07734
Re: meirluc post# 691641
Tuesday, May 14, 2024 6:27:03 PM
Post#
691644
of 691746
Bingo! meirluc, you are asking the right questions.
If I may, I add two questions that we should ask. Why did NCI analyze our results without Dr. Bosch? Why did NIH sponsor the new patent development of Oncovir?
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE249282
https://patents.google.com/patent/WO2022031314A2/en?assignee=oncovir&oq=oncovirmeirluc
Re: flipper44 post# 691519
Tuesday, May 14, 2024 6:17:18 PM
Post#
691641
of 691744
If we were to expect a MHRA decision during sometime in the middle of
July or even August, why did NWBO schedule an ASM to no later than
the end of June? Why not hold off till July or August when approval is
in the bag and then schedule The ASM shortly thereafter?
Re: None
Tuesday, May 14, 2024 12:58:46 PM
Post#
691519
of 691745
Here’s what I think, and I’ll be off the board for a couple days.
IMO, Les thought there was a chance they might need to file an extension request to answer an RFI. He said they’d talk about “fall” a day later in the 10q. The 10q mentioned NOTHING about fall. Therefore, imo, their contractors did not need to file an extension request. Which means, imo, we are looking at somewhere around mid-July for a decision. (60 days earlier then Les thought) imo.
I think this because Les should have known what was in the 10q unless it was subject to change in the 24 hours or so after he spoke. He didn’t know, therefore I think they decided not to ask for an extension at some point after Les spoke.
Further, it seems they were already anticipating inspections by the 10q, but such inspections were not scheduled yet because, imo, an RFI response was turned in shortly before the 10q was filed, so phase II had not started until that response was filed. Therefore, it makes sense they’d not yet know when any inspections would occur. IMO.
Finally, if I’m right, it’s very positive, because NWBO and their contractors not asking for an extension would be very unusual, for them, and yet extremely confident.
https://twitter.com/alphavestcap/status/1790098243918917692 (please post transcript of LG presentation, i believe it begins at about the 25 minute mark.thanks.
$nwbo @alphavestcap
— alphavestcapital.com (@alphavestcap) May 11, 2024
The 2 most important sentences in the Q.
"On March 7, 2024, the Company received notification from the MHRA that the validation was confirmed. The Company did not make any amendment or addition to its MAA after the original December 20, 2023 submission."…
Nemesis : When the MAA is approved , a dagger will enter the heart of the vampire that lives within you. To try to destroy (https://twitter.com/AllenTurner206) is pure evil. DC VAX L will be a new paradigm in cancer therapy that will bring the gift of continued life to millions. Wishing that to not happen,as you and your benefactors have done for years, and to continue to do every day, is pure evil.I surmise that you and your associates will long be remebered for your nefartious ways.
https://investorshub.advfn.com/boards/read_msg.aspx?message_id=174400930
"This is total and utter bullshit. 1. The MHRA is not governed by NHS. 2. NHS is a CLINICAL governance entity meaning it governs Physician and other caregiver licensing, Hospital oversight, etc. It has nothing whatsoever to do with drug/treatment evaluation and approvals. 3. MHRA is governed by the Department of Health and Social Care and they do not interfere with MHRA’s regulatory approval process. They only help create and oversee strategic planning and operational directives. Why would anyone reply to that nut Nemesis in the first place?"
ae kusterer
Re: None
Sunday, May 12, 2024 7:27:59 AM
Post#
690802
of 690813
https://www.england.nhs.uk/about/nhs-england-board/
Nemesis18
Re: theorysuit post# 690596
Sunday, May 12, 2024 6:16:18 AM
Post#
690800
of 690802
Irrespective of the subjective speculation on this board by NWBO investors , there will be a binary event happening soon, that will singularly decide the immediate future of Northwest BioTherapeutics.
The granting, or not, of the MAA is that determinate event. This is indisputable.
BUT that decision is not ultimately for the Regulators to decide.
It’s guiding ‘authority’ can be set aside for many reasons at any stage by the NHS Governance Departments (https://www.england.nhs.uk/about/nhs-england-board/) imho.
Make of that as you will!
https://www.england.nhs.uk/about/nhs-england-board/
Nemesis18
Re: theorysuit post# 690596
Sunday, May 12, 2024 6:16:18 AM
Post#
690800
of 690802
Irrespective of the subjective speculation on this board by NWBO investors , there will be a binary event happening soon, that will singularly decide the immediate future of Northwest BioTherapeutics.
The granting, or not, of the MAA is that determinate event. This is indisputable.
BUT that decision is not ultimately for the Regulators to decide.
It’s guiding ‘authority’ can be set aside for many reasons at any stage by the NHS Governance Departments (https://www.england.nhs.uk/about/nhs-england-board/) imho.
Make of that as you will!
The 2 most important sentences in the Q.
"On March 7, 2024, the Company received notification from the MHRA that the validation was confirmed. The Company did not make any amendment or addition to its MAA after the original December 20, 2023 submission."
10-Q (5/10/24)SALIENTS: 1) "On March 7, 2024, the Company received notification from the MHRA that the validation was confirmed. The Company did not make any amendment or addition to its MAA after the original December 20, 2023 submission." 2) "The Company does not know when MAA - related inspections may take place." 10-Q-5/10/24: Item 2. Management’s Discussion and Analysis of Financial Condition and Results of Operations The following discussion and analysis of our financial condition and results of operations should be read in conjunction with our unaudited condensed consolidated financial statements and the notes to those statements included with this report. In addition to historical information, this report contains forward-looking statements within the meaning of Section 27A of the Securities Act of 1933, as amended (the “Securities Act”), and Section 21E of the Securities Exchange Act of 1934, as amended (the “Exchange Act”). Such forward-looking statements are subject to certain risks and uncertainties that could cause actual results to differ materially from those projected. The words “believe,” “expect,” “intend,” “anticipate,” and similar expressions are used to identify forward-looking statements, but some forward-looking statements are expressed differently. Many factors could affect our actual results, including those factors described under “Risk Factors” in our Form 10-K for the year ended December 31, 2023 and in Part II Item 1A of this report. These factors, among others, could cause results to differ materially from those presently anticipated by us. You should not place undue reliance on these forward-looking statements. Overview We are a biotechnology company focused on developing personalized immune therapies for cancer. We have developed a platform technology, DCVax®, which uses activated dendritic cells to mobilize a patient’s own immune system to attack their cancer. Our lead product, DCVax®-L, is designed to treat solid tumor cancers in which the tumor can be surgically removed. We have completed a 331-patient international Phase III trial of DCVax-L for Glioblastoma multiforme brain cancer (GBM), published the results in the JAMA Oncology peer reviewed journal, and on December 20, 2023 we submitted a Marketing Authorization Application (MAA) for commercial approval in the U.K. We plan to conduct clinical trials of DCVax-L for other solid tumor cancers in the future, when resources permit. Our second product, DCVax®-Direct, is designed to treat inoperable solid tumors. A 40-patient Phase I trial has been completed, and included treatment of a diverse range of more than a dozen types of cancers. We plan to work on preparations for Phase II trials of DCVax-Direct as resources permit. The Company’s programs and operations continue to be impacted by supply chain issues and backlogs which, surprisingly, have still not substantially resolved. These involve service firms and also vendors and suppliers of a wide variety of items, ranging from major equipment to particular reagents required for the manufacturing process. Shortages of certain key materials and supplies have also occurred. The Company is hopeful that the various backlog circumstances will improve over the course of 2024. During the first quarter of 2024, the Company continued its progress on multiple fronts, including the following. MAA Application. As previously reported, the Company filed a Marketing Authorization Application (MAA) to the U.K. Medicines and Healthcare Products Regulatory Agency (MHRA) on December 20, 2023, seeking regulatory approval for commercialization of DCVax - L for newly diagnosed and recurrent Glioblastoma (GBM). On January 24, 2024 the Company received notification from the MHRA that the MAA had passed validation. On March 7, 2024, the Company received notification from the MHRA that the validation was confirmed. The Company did not make any amendment or addition to its MAA after the original December 20, 2023 submission. As is typical, the Company does not plan to make any interim announcements while its MAA is going through the regulatory process. The Company will announce the results when the regulatory review and decision - making about the MAA is complete. Preparations for Regulatory Inspections. As anticipated in the Company’s prior reports, preparations for regulatory inspections associated with the MAA have continued to be a major focus of the Company’s activities this year to date. The Company has continued working intensively with teams of expert consultants in both the U.S. and U.K. on these preparations, and has also arranged for further mock inspections by specialists who were formerly inspectors for regulatory agencies. It is anticipated that teams of multiple inspectors for extended periods will conduct comprehensive inspections of all the key parties involved conducting in the Phase 3 trial and of all documentation and records. The Company does not know when MAA - related inspections may take place. Pediatric Glioma Clinical Trials. The Company continued its discussions with physicians about the two planned trials of DCVax - L for pediatric gliomas. The arrangements for the trials have not yet been finalized. 23 Table of Contents Flaskworks. The Flaskworks team and Advent BioServices continued to work with the specialized contractor on the development of the GMP-grade units of the Flaskworks system. Two key areas of focus in the work have been the ability to clean the machines to GMP requirements and additional safety features for protection of the operators in the GMP setting. Enabling all parts of the machines to be cleanable to the level of sterility required for GMP operations has required changing certain materials to achieve acceptable surface properties, constructing housings around certain portions of the machines or otherwise eliminating nooks and crannies while not interfering with the movements of the machines, and other such development aspects. The Company believes the work is on track for the GMP adaptation work to be finished in the near future, and the first GMP units to be delivered to the Company soon after that. Sawston Facility. The Company has commenced the design and build works for the construction of the first Grade C cleanroom at the Sawston facility and the contractor is onsite. The Company expects the Grade C cleanrooms to be important for scale - up, as they will accommodate the “closed” Flaskworks system, and be able to manufacture a number of patients’ products in the lab at the same time. In contrast, with the existing “open” process in the Grade B cleanrooms, products must be manufactured for one patient at a time and the whole lab must be cleaned and sterilized between each patient’s process. Since construction work in the facility can directly and/or indirectly impact the existing cleanrooms in the facility (e.g., by generating a lot of dust and particles) and prevent the existing Grade B cleanrooms from being able to operate in accordance with GMP sterility standards, the Company has been working with Advent BioServices and contractors to arrange for the construction work to take place as much as possible during periodic scheduled shutdowns of the existing clean rooms for deep cleaning and special maintenance which are required for GMP compliance. Intellectual Property and Collaborations. As previously reported, the Company filed new patent applications relating to the Flaskworks system, which the Company believes will be an important factor for facilitating scale - up and commercialization of DCVax - L products. In addition, multiple new patents were granted and 1 new patent was allowed from patent applications previously filed. The Company continued working with certain intellectual property that it has in - licensed as previously reported, on preparations for expansion of the Company’s pipeline with certain new clinical programs when bandwidth and resources permit. The Company also continued discussions and negotiations during the first quarter of 2024 relating to collaborations which the Company believes will help it build a broad franchise in dendritic cell-based immunotherapies. Litigation. Significant amounts of Company bandwidth and resources were spent on litigations during the first quarter of 2024 (and to date in the second quarter of 2024). The cases are described in Part II - Item 1, Legal Proceedings, below. Annual Shareholder Meeting. The Company plans to conduct its Annual Shareholder Meeting before the end of June 2024. Critical Accounting Policies and Estimates "I agree. The “validation confirmation” and no need to change anything in the MAA means to me that part is over and only confirmatory inspections and questions to the people involved ." P.S., With the 5/10/24 Q having de-risked the MAA approval, and the Nature paper having opened the door to RA approval for the commencement of combo trials,who will be the first cancer immunotherapy stategic players to consummate franchise agreements with LP? https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000741/nwbo-20240331x10q.htm…… https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000133/nwbo-20231231x10k.htm……$nwbo@alphavestcap"I agree. The “validation confirmation” and no need to change anything in the MAA means to me that part is over and only confirmatory inspections and questions to the people involved ."-Zivic AVC-P.S., With the 5/10/24 Q having de-risked the MAA approval, and the Nature paper having opened the door to RA approval for the commencement of combo trials,who will be the first cancer immunotherapy stategic players to consummate franchise agreements with LP? https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000741/nwbo-20240331x10q.htm…… https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000133/nwbo-20231231x10k.htm……@alphavestcapLast edited1:03 PM · May 11, 2024·
https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000741/nwbo-20240331x10q.htm
Also: ready for inspections:( Preparations for Regulatory Inspections. As anticipated in the Company’s prior reports, preparations for regulatory inspections associated with the MAA have continued to be a major focus of the Company’s activities this year to date. The Company has continued working intensively with teams of expert consultants in both the U.S. and U.K. on these preparations, and has also arranged for further mock inspections by specialists who were formerly inspectors for regulatory agencies. It is anticipated that teams of multiple inspectors for extended periods will conduct comprehensive inspections of all the key parties involved conducting in the Phase 3 trial and of all documentation and records. The Company does not know when MAA - related inspections may take place.)
And, Sawston getting ready for C Suite high speed production : (Flaskworks. The Flaskworks team and Advent BioServices continued to work with the specialized contractor on the development of the GMP-grade units of the Flaskworks system. Two key areas of focus in the work have been the ability to clean the machines to GMP requirements and additional safety features for protection of the operators in the GMP setting. Enabling all parts of the machines to be cleanable to the level of sterility required for GMP operations has required changing certain materials to achieve acceptable surface properties, constructing housings around certain portions of the machines or otherwise eliminating nooks and crannies while not interfering with the movements of the machines, and other such development aspects. The Company believes the work is on track for the GMP adaptation work to be finished in the near future, and the first GMP units to be delivered to the Company soon after that.
Sawston Facility. The Company has commenced the design and build works for the construction of the first Grade C cleanroom at the Sawston facility and the contractor is onsite. The Company expects the Grade C cleanrooms to be important for scale - up, as they will accommodate the “closed” Flaskworks system, and be able to manufacture a number of patients’ products in the lab at the same time. In contrast, with the existing “open” process in the Grade B cleanrooms, products must be manufactured for one patient at a time and the whole lab must be cleaned and sterilized between each patient’s process. Since construction work in the facility can directly and/or indirectly impact the existing cleanrooms in the facility (e.g., by generating a lot of dust and particles) and prevent the existing Grade B cleanrooms from being able to operate in accordance with GMP sterility standards, the Company has been working with Advent BioServices and contractors to arrange for the construction work to take place as much as possible during periodic scheduled shutdowns of the existing clean rooms for deep cleaning and special maintenance which are required for GMP compliance.)
https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000133/nwbo-20231231x10k.htm
poly iclc supply secured with in-licensing:
"we in-licensed patents and patent applications from other parties."
"Intellectual Property. We further strengthened our intellectual property portfolio in three ways during 2023: we achieved issuance of certain patents previously filed, we filed new patent applications based on our own work, and we in-licensed patents and patent applications from other parties. We are continuing to build an IP portfolio that we believe will provide a strong foundation to help us build a leading franchise in dendritic cells and active immunotherapies. This includes enhanced versions of dendritic cells, combinations of other agents with dendritic cells, manufacturing methods and processes, and related IP."
in-license of poly iclc patents would secure its supply.
n-License:
https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000133/nwbo-20231231x10k.htm
10-K-2023:" Intellectual Property. We further strengthened our intellectual property portfolio in three ways during 2023: we achieved issuance of certain patents previously filed, we filed new patent applications based on our own work, and we in-licensed patents and patent applications from other parties. We are continuing to build an IP portfolio that we believe will provide a strong foundation to help us build a leading franchise in dendritic cells and active immunotherapies. This includes enhanced versions of dendritic cells, combinations of other agents with dendritic cells, manufacturing methods and processes, and related IP."
https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000741/nwbo-20240331x10q.htm
5/10/24 10-Q:"Intellectual Property and Collaborations. As previously reported, the Company filed new patent applications relating to the Flaskworks system, which the Company believes will be an important factor for facilitating scale - up and commercialization of DCVax - L products. In addition, multiple new patents were granted and 1 new patent was allowed from patent applications previously filed. The Company continued working with certain intellectual property that it has in - licensed as previously reported (in 12/31/23 10-K), on preparations for expansion of the Company’s pipeline with certain new clinical programs when bandwidth and resources permit. The Company also continued discussions and negotiations during the first quarter of 2024 relating to collaborations which the Company believes will help it build a broad franchise in dendritic cell-based immunotherapies."
With the MAA application now acceptable to the MHRA, successful inspections need to be completed in order for the MAA to be granted.
SALIENTS IN THE 5/10/24 Q MD and A section:
Preparations for Regulatory Inspections. As anticipated in the Company’s prior reports, preparations for regulatory inspections associated with the MAA have continued to be a major focus of the Company’s activities this year to date. The Company has continued working intensively with teams of expert consultants in both the U.S. and U.K. on these preparations, and has also arranged for further mock inspections by specialists who were formerly inspectors for regulatory agencies. It is anticipated that teams of multiple inspectors for extended periods will conduct comprehensive inspections of all the key parties involved conducting in the Phase 3 trial and of all documentation and records. The Company does not know when MAA - related inspections may take place.
On March 7, 2024, the Company received notification from the MHRA that the validation was confirmed. The Company did not make any amendment or addition to its MAA after the original December 20, 2023 submission.
Sawston Facility. The Company has commenced the design and build works for the construction of the first Grade C cleanroom at the Sawston facility and the contractor is onsite.
tem 2. Management’s Discussion and Analysis of Financial Condition and Results of Operations
https://www.sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000741/nwbo-20240331x10q.htm
The following discussion and analysis of our financial condition and results of operations should be read in conjunction with our unaudited condensed consolidated financial statements and the notes to those statements included with this report. In addition to historical information, this report contains forward-looking statements within the meaning of Section 27A of the Securities Act of 1933, as amended (the “Securities Act”), and Section 21E of the Securities Exchange Act of 1934, as amended (the “Exchange Act”). Such forward-looking statements are subject to certain risks and uncertainties that could cause actual results to differ materially from those projected. The words “believe,” “expect,” “intend,” “anticipate,” and similar expressions are used to identify forward-looking statements, but some forward-looking statements are expressed differently. Many factors could affect our actual results, including those factors described under “Risk Factors” in our Form 10-K for the year ended December 31, 2023 and in Part II Item 1A of this report. These factors, among others, could cause results to differ materially from those presently anticipated by us. You should not place undue reliance on these forward-looking statements.
Overview
We are a biotechnology company focused on developing personalized immune therapies for cancer. We have developed a platform technology, DCVax®, which uses activated dendritic cells to mobilize a patient’s own immune system to attack their cancer.
Our lead product, DCVax®-L, is designed to treat solid tumor cancers in which the tumor can be surgically removed. We have completed a 331-patient international Phase III trial of DCVax-L for Glioblastoma multiforme brain cancer (GBM), published the results in the JAMA Oncology peer reviewed journal, and on December 20, 2023 we submitted a Marketing Authorization Application (MAA) for commercial approval in the U.K. We plan to conduct clinical trials of DCVax-L for other solid tumor cancers in the future, when resources permit. Our second product, DCVax®-Direct, is designed to treat inoperable solid tumors. A 40-patient Phase I trial has been completed, and included treatment of a diverse range of more than a dozen types of cancers. We plan to work on preparations for Phase II trials of DCVax-Direct as resources permit.
The Company’s programs and operations continue to be impacted by supply chain issues and backlogs which, surprisingly, have still not substantially resolved. These involve service firms and also vendors and suppliers of a wide variety of items, ranging from major equipment to particular reagents required for the manufacturing process. Shortages of certain key materials and supplies have also occurred. The Company is hopeful that the various backlog circumstances will improve over the course of 2024.
During the first quarter of 2024, the Company continued its progress on multiple fronts, including the following.
MAA Application. As previously reported, the Company filed a Marketing Authorization Application (MAA) to the U.K. Medicines and Healthcare Products Regulatory Agency (MHRA) on December 20, 2023, seeking regulatory approval for commercialization of DCVax - L for newly diagnosed and recurrent Glioblastoma (GBM). On January 24, 2024 the Company received notification from the MHRA that the MAA had passed validation. On March 7, 2024, the Company received notification from the MHRA that the validation was confirmed. The Company did not make any amendment or addition to its MAA after the original December 20, 2023 submission.
As is typical, the Company does not plan to make any interim announcements while its MAA is going through the regulatory process. The Company will announce the results when the regulatory review and decision - making about the MAA is complete.
Preparations for Regulatory Inspections. As anticipated in the Company’s prior reports, preparations for regulatory inspections associated with the MAA have continued to be a major focus of the Company’s activities this year to date. The Company has continued working intensively with teams of expert consultants in both the U.S. and U.K. on these preparations, and has also arranged for further mock inspections by specialists who were formerly inspectors for regulatory agencies. It is anticipated that teams of multiple inspectors for extended periods will conduct comprehensive inspections of all the key parties involved conducting in the Phase 3 trial and of all documentation and records. The Company does not know when MAA - related inspections may take place.
Pediatric Glioma Clinical Trials. The Company continued its discussions with physicians about the two planned trials of DCVax - L for pediatric gliomas. The arrangements for the trials have not yet been finalized.
23
Table of Contents
Flaskworks. The Flaskworks team and Advent BioServices continued to work with the specialized contractor on the development of the GMP-grade units of the Flaskworks system. Two key areas of focus in the work have been the ability to clean the machines to GMP requirements and additional safety features for protection of the operators in the GMP setting. Enabling all parts of the machines to be cleanable to the level of sterility required for GMP operations has required changing certain materials to achieve acceptable surface properties, constructing housings around certain portions of the machines or otherwise eliminating nooks and crannies while not interfering with the movements of the machines, and other such development aspects. The Company believes the work is on track for the GMP adaptation work to be finished in the near future, and the first GMP units to be delivered to the Company soon after that.
Sawston Facility. The Company has commenced the design and build works for the construction of the first Grade C cleanroom at the Sawston facility and the contractor is onsite. The Company expects the Grade C cleanrooms to be important for scale - up, as they will accommodate the “closed” Flaskworks system, and be able to manufacture a number of patients’ products in the lab at the same time. In contrast, with the existing “open” process in the Grade B cleanrooms, products must be manufactured for one patient at a time and the whole lab must be cleaned and sterilized between each patient’s process. Since construction work in the facility can directly and/or indirectly impact the existing cleanrooms in the facility (e.g., by generating a lot of dust and particles) and prevent the existing Grade B cleanrooms from being able to operate in accordance with GMP sterility standards, the Company has been working with Advent BioServices and contractors to arrange for the construction work to take place as much as possible during periodic scheduled shutdowns of the existing clean rooms for deep cleaning and special maintenance which are required for GMP compliance.
Intellectual Property and Collaborations. As previously reported, the Company filed new patent applications relating to the Flaskworks system, which the Company believes will be an important factor for facilitating scale - up and commercialization of DCVax - L products. In addition, multiple new patents were granted and 1 new patent was allowed from patent applications previously filed. The Company continued working with certain intellectual property that it has in - licensed as previously reported, on preparations for expansion of the Company’s pipeline with certain new clinical programs when bandwidth and resources permit. The Company also continued discussions and negotiations during the first quarter of 2024 relating to collaborations which the Company believes will help it build a broad franchise in dendritic cell-based immunotherapies.
Litigation. Significant amounts of Company bandwidth and resources were spent on litigations during the first quarter of 2024 (and to date in the second quarter of 2024). The cases are described in Part II - Item 1, Legal Proceedings, below.
Annual Shareholder Meeting. The Company plans to conduct its Annual Shareholder Meeting before the end of June 2024.
Critical Accounting Policies and Estimates
Our discussion and analysis of our financial condition and results of operations are based on our financial statements, which have been prepared in accordance with U.S. GAAP. The preparation of these financial statements requires us to make estimates and judgments that affect our reported amounts of assets, liabilities, revenues and expenses.
On an ongoing basis, we evaluate our estimates and judgments, including those related to derivative liabilities, accrued expenses and stock-based compensation. We based our estimates on historical experience and on various other assumptions that we believe to be reasonable under the circumstances, the results of which form the basis for making judgments about the carrying values of assets and liabilities and the reported amounts of revenues and expenses that are not readily apparent from other sources. Actual results could differ from those estimates.
Our critical accounting policies and significant estimates are detailed in our Annual Report on Form 10-K for the year ended December 31, 2023. Our critical accounting policies and significant estimates have not changed substantially from those previously disclosed in our Annual Report on Form 10-K for the year ended December 31, 2023.
Results of Operations
The 2 most important sentences in the Q.
"On March 7, 2024, the Company received notification from the MHRA that the validation was confirmed. The Company did not make any amendment or addition to its MAA after the original December 20, 2023 submission."
https://twitter.com/alphavestcap/status/1789355402829652006
"I agree. The “validation confirmation” and no need to change anything in the MAA means to me that part is over and only confirmatory inspections and questions to the people involved ."-Zivic
AVC-P.S., With the 5/10/24 Q having de-risked the MAA approval, and the Nature paper having opened the door to RA approval for the commencement of combo trials,who will be the first cancer immunotherapy stategic players to consummate franchise agreements with LP?
https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000741/nwbo-20240331x10q.htm…
https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000133/nwbo-20231231x10k.htm…
@alphavestcap
10-Q (5/10/24)SALIENTS:
1) "On March 7, 2024, the Company received notification from the MHRA that the validation was confirmed. The Company did not make any amendment or addition to its MAA after the original December 20, 2023 submission."
2) "The Company does not know when MAA - related inspections may take place."
10-Q-5/10/24: Item 2. Management’s Discussion and Analysis of Financial Condition and Results of Operations The following discussion and analysis of our financial condition and results of operations should be read in conjunction with our unaudited condensed consolidated financial statements and the notes to those statements included with this report. In addition to historical information, this report contains forward-looking statements within the meaning of Section 27A of the Securities Act of 1933, as amended (the “Securities Act”), and Section 21E of the Securities Exchange Act of 1934, as amended (the “Exchange Act”). Such forward-looking statements are subject to certain risks and uncertainties that could cause actual results to differ materially from those projected. The words “believe,” “expect,” “intend,” “anticipate,” and similar expressions are used to identify forward-looking statements, but some forward-looking statements are expressed differently. Many factors could affect our actual results, including those factors described under “Risk Factors” in our Form 10-K for the year ended December 31, 2023 and in Part II Item 1A of this report. These factors, among others, could cause results to differ materially from those presently anticipated by us. You should not place undue reliance on these forward-looking statements. Overview We are a biotechnology company focused on developing personalized immune therapies for cancer. We have developed a platform technology, DCVax®, which uses activated dendritic cells to mobilize a patient’s own immune system to attack their cancer. Our lead product, DCVax®-L, is designed to treat solid tumor cancers in which the tumor can be surgically removed. We have completed a 331-patient international Phase III trial of DCVax-L for Glioblastoma multiforme brain cancer (GBM), published the results in the JAMA Oncology peer reviewed journal, and on December 20, 2023 we submitted a Marketing Authorization Application (MAA) for commercial approval in the U.K. We plan to conduct clinical trials of DCVax-L for other solid tumor cancers in the future, when resources permit. Our second product, DCVax®-Direct, is designed to treat inoperable solid tumors. A 40-patient Phase I trial has been completed, and included treatment of a diverse range of more than a dozen types of cancers. We plan to work on preparations for Phase II trials of DCVax-Direct as resources permit. The Company’s programs and operations continue to be impacted by supply chain issues and backlogs which, surprisingly, have still not substantially resolved. These involve service firms and also vendors and suppliers of a wide variety of items, ranging from major equipment to particular reagents required for the manufacturing process. Shortages of certain key materials and supplies have also occurred. The Company is hopeful that the various backlog circumstances will improve over the course of 2024. During the first quarter of 2024, the Company continued its progress on multiple fronts, including the following. MAA Application. As previously reported, the Company filed a Marketing Authorization Application (MAA) to the U.K. Medicines and Healthcare Products Regulatory Agency (MHRA) on December 20, 2023, seeking regulatory approval for commercialization of DCVax - L for newly diagnosed and recurrent Glioblastoma (GBM). On January 24, 2024 the Company received notification from the MHRA that the MAA had passed validation. On March 7, 2024, the Company received notification from the MHRA that the validation was confirmed. The Company did not make any amendment or addition to its MAA after the original December 20, 2023 submission. As is typical, the Company does not plan to make any interim announcements while its MAA is going through the regulatory process. The Company will announce the results when the regulatory review and decision - making about the MAA is complete. Preparations for Regulatory Inspections. As anticipated in the Company’s prior reports, preparations for regulatory inspections associated with the MAA have continued to be a major focus of the Company’s activities this year to date. The Company has continued working intensively with teams of expert consultants in both the U.S. and U.K. on these preparations, and has also arranged for further mock inspections by specialists who were formerly inspectors for regulatory agencies. It is anticipated that teams of multiple inspectors for extended periods will conduct comprehensive inspections of all the key parties involved conducting in the Phase 3 trial and of all documentation and records. The Company does not know when MAA - related inspections may take place. Pediatric Glioma Clinical Trials. The Company continued its discussions with physicians about the two planned trials of DCVax - L for pediatric gliomas. The arrangements for the trials have not yet been finalized. 23 Table of Contents Flaskworks. The Flaskworks team and Advent BioServices continued to work with the specialized contractor on the development of the GMP-grade units of the Flaskworks system. Two key areas of focus in the work have been the ability to clean the machines to GMP requirements and additional safety features for protection of the operators in the GMP setting. Enabling all parts of the machines to be cleanable to the level of sterility required for GMP operations has required changing certain materials to achieve acceptable surface properties, constructing housings around certain portions of the machines or otherwise eliminating nooks and crannies while not interfering with the movements of the machines, and other such development aspects. The Company believes the work is on track for the GMP adaptation work to be finished in the near future, and the first GMP units to be delivered to the Company soon after that. Sawston Facility. The Company has commenced the design and build works for the construction of the first Grade C cleanroom at the Sawston facility and the contractor is onsite. The Company expects the Grade C cleanrooms to be important for scale - up, as they will accommodate the “closed” Flaskworks system, and be able to manufacture a number of patients’ products in the lab at the same time. In contrast, with the existing “open” process in the Grade B cleanrooms, products must be manufactured for one patient at a time and the whole lab must be cleaned and sterilized between each patient’s process. Since construction work in the facility can directly and/or indirectly impact the existing cleanrooms in the facility (e.g., by generating a lot of dust and particles) and prevent the existing Grade B cleanrooms from being able to operate in accordance with GMP sterility standards, the Company has been working with Advent BioServices and contractors to arrange for the construction work to take place as much as possible during periodic scheduled shutdowns of the existing clean rooms for deep cleaning and special maintenance which are required for GMP compliance. Intellectual Property and Collaborations. As previously reported, the Company filed new patent applications relating to the Flaskworks system, which the Company believes will be an important factor for facilitating scale - up and commercialization of DCVax - L products. In addition, multiple new patents were granted and 1 new patent was allowed from patent applications previously filed. The Company continued working with certain intellectual property that it has in - licensed as previously reported, on preparations for expansion of the Company’s pipeline with certain new clinical programs when bandwidth and resources permit. The Company also continued discussions and negotiations during the first quarter of 2024 relating to collaborations which the Company believes will help it build a broad franchise in dendritic cell-based immunotherapies. Litigation. Significant amounts of Company bandwidth and resources were spent on litigations during the first quarter of 2024 (and to date in the second quarter of 2024). The cases are described in Part II - Item 1, Legal Proceedings, below. Annual Shareholder Meeting. The Company plans to conduct its Annual Shareholder Meeting before the end of June 2024. Critical Accounting Policies and Estimates
"I agree. The “validation confirmation” and no need to change anything in the MAA means to me that part is over and only confirmatory inspections and questions to the people involved ."
P.S., With the 5/10/24 Q having de-risked the MAA approval, and the Nature paper having opened the door to RA approval for the commencement of combo trials,who will be the first cancer immunotherapy stategic players to consummate franchise agreements with LP?
https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000741/nwbo-20240331x10q.htm…
https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000133/nwbo-20231231x10k.htm…
https://twitter.com/alphavestcap/status/1789340496059347392
"I agree. The “validation confirmation” and no need to change anything in the MAA means to me that part is over and only confirmatory inspections and questions to the people involved ."-Zivic
AVC-P.S., With the 5/10/24 Q having de-risked the MAA approval, and the Nature paper having opened the door to RA approval for the commencement of combo trials,who will be the first cancer immunotherapy stategic players to consummate franchise agreements with LP?
https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000741/nwbo-20240331x10q.htm…
https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000133/nwbo-20231231x10k.htm…
@alphavestcap
10-Q (5/10/24)SALIENTS:
1) "On March 7, 2024, the Company received notification from the MHRA that the validation was confirmed. The Company did not make any amendment or addition to its MAA after the original December 20, 2023 submission."
2) "The Company does not know when MAA - related inspections may take place."
10-Q-5/10/24: Item 2. Management’s Discussion and Analysis of Financial Condition and Results of Operations The following discussion and analysis of our financial condition and results of operations should be read in conjunction with our unaudited condensed consolidated financial statements and the notes to those statements included with this report. In addition to historical information, this report contains forward-looking statements within the meaning of Section 27A of the Securities Act of 1933, as amended (the “Securities Act”), and Section 21E of the Securities Exchange Act of 1934, as amended (the “Exchange Act”). Such forward-looking statements are subject to certain risks and uncertainties that could cause actual results to differ materially from those projected. The words “believe,” “expect,” “intend,” “anticipate,” and similar expressions are used to identify forward-looking statements, but some forward-looking statements are expressed differently. Many factors could affect our actual results, including those factors described under “Risk Factors” in our Form 10-K for the year ended December 31, 2023 and in Part II Item 1A of this report. These factors, among others, could cause results to differ materially from those presently anticipated by us. You should not place undue reliance on these forward-looking statements. Overview We are a biotechnology company focused on developing personalized immune therapies for cancer. We have developed a platform technology, DCVax®, which uses activated dendritic cells to mobilize a patient’s own immune system to attack their cancer. Our lead product, DCVax®-L, is designed to treat solid tumor cancers in which the tumor can be surgically removed. We have completed a 331-patient international Phase III trial of DCVax-L for Glioblastoma multiforme brain cancer (GBM), published the results in the JAMA Oncology peer reviewed journal, and on December 20, 2023 we submitted a Marketing Authorization Application (MAA) for commercial approval in the U.K. We plan to conduct clinical trials of DCVax-L for other solid tumor cancers in the future, when resources permit. Our second product, DCVax®-Direct, is designed to treat inoperable solid tumors. A 40-patient Phase I trial has been completed, and included treatment of a diverse range of more than a dozen types of cancers. We plan to work on preparations for Phase II trials of DCVax-Direct as resources permit. The Company’s programs and operations continue to be impacted by supply chain issues and backlogs which, surprisingly, have still not substantially resolved. These involve service firms and also vendors and suppliers of a wide variety of items, ranging from major equipment to particular reagents required for the manufacturing process. Shortages of certain key materials and supplies have also occurred. The Company is hopeful that the various backlog circumstances will improve over the course of 2024. During the first quarter of 2024, the Company continued its progress on multiple fronts, including the following. MAA Application. As previously reported, the Company filed a Marketing Authorization Application (MAA) to the U.K. Medicines and Healthcare Products Regulatory Agency (MHRA) on December 20, 2023, seeking regulatory approval for commercialization of DCVax - L for newly diagnosed and recurrent Glioblastoma (GBM). On January 24, 2024 the Company received notification from the MHRA that the MAA had passed validation. On March 7, 2024, the Company received notification from the MHRA that the validation was confirmed. The Company did not make any amendment or addition to its MAA after the original December 20, 2023 submission. As is typical, the Company does not plan to make any interim announcements while its MAA is going through the regulatory process. The Company will announce the results when the regulatory review and decision - making about the MAA is complete. Preparations for Regulatory Inspections. As anticipated in the Company’s prior reports, preparations for regulatory inspections associated with the MAA have continued to be a major focus of the Company’s activities this year to date. The Company has continued working intensively with teams of expert consultants in both the U.S. and U.K. on these preparations, and has also arranged for further mock inspections by specialists who were formerly inspectors for regulatory agencies. It is anticipated that teams of multiple inspectors for extended periods will conduct comprehensive inspections of all the key parties involved conducting in the Phase 3 trial and of all documentation and records. The Company does not know when MAA - related inspections may take place. Pediatric Glioma Clinical Trials. The Company continued its discussions with physicians about the two planned trials of DCVax - L for pediatric gliomas. The arrangements for the trials have not yet been finalized. 23 Table of Contents Flaskworks. The Flaskworks team and Advent BioServices continued to work with the specialized contractor on the development of the GMP-grade units of the Flaskworks system. Two key areas of focus in the work have been the ability to clean the machines to GMP requirements and additional safety features for protection of the operators in the GMP setting. Enabling all parts of the machines to be cleanable to the level of sterility required for GMP operations has required changing certain materials to achieve acceptable surface properties, constructing housings around certain portions of the machines or otherwise eliminating nooks and crannies while not interfering with the movements of the machines, and other such development aspects. The Company believes the work is on track for the GMP adaptation work to be finished in the near future, and the first GMP units to be delivered to the Company soon after that. Sawston Facility. The Company has commenced the design and build works for the construction of the first Grade C cleanroom at the Sawston facility and the contractor is onsite. The Company expects the Grade C cleanrooms to be important for scale - up, as they will accommodate the “closed” Flaskworks system, and be able to manufacture a number of patients’ products in the lab at the same time. In contrast, with the existing “open” process in the Grade B cleanrooms, products must be manufactured for one patient at a time and the whole lab must be cleaned and sterilized between each patient’s process. Since construction work in the facility can directly and/or indirectly impact the existing cleanrooms in the facility (e.g., by generating a lot of dust and particles) and prevent the existing Grade B cleanrooms from being able to operate in accordance with GMP sterility standards, the Company has been working with Advent BioServices and contractors to arrange for the construction work to take place as much as possible during periodic scheduled shutdowns of the existing clean rooms for deep cleaning and special maintenance which are required for GMP compliance. Intellectual Property and Collaborations. As previously reported, the Company filed new patent applications relating to the Flaskworks system, which the Company believes will be an important factor for facilitating scale - up and commercialization of DCVax - L products. In addition, multiple new patents were granted and 1 new patent was allowed from patent applications previously filed. The Company continued working with certain intellectual property that it has in - licensed as previously reported, on preparations for expansion of the Company’s pipeline with certain new clinical programs when bandwidth and resources permit. The Company also continued discussions and negotiations during the first quarter of 2024 relating to collaborations which the Company believes will help it build a broad franchise in dendritic cell-based immunotherapies. Litigation. Significant amounts of Company bandwidth and resources were spent on litigations during the first quarter of 2024 (and to date in the second quarter of 2024). The cases are described in Part II - Item 1, Legal Proceedings, below. Annual Shareholder Meeting. The Company plans to conduct its Annual Shareholder Meeting before the end of June 2024. Critical Accounting Policies and Estimates
"I agree. The “validation confirmation” and no need to change anything in the MAA means to me that part is over and only confirmatory inspections and questions to the people involved ."
P.S., With the 5/10/24 Q having de-risked the MAA approval, and the Nature paper having opened the door to RA approval for the commencement of combo trials,who will be the first cancer immunotherapy stategic players to consummate franchise agreements with LP?
https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000741/nwbo-20240331x10q.htm…
https://sec.gov/ix?doc=/Archives/edgar/data/1072379/000141057824000133/nwbo-20231231x10k.htm…
WHAT UCLA HAD TO SAY ABOUT THE NATURE PUBLICATION:
https://www.researchsquare.com/article/rs-3287211/v1 (pre-print)
https://www.nature.com/articles/s41467-024-48073-y (5/8/24)
https://www.uclahealth.org/news/release/adding-immune-boosting-agent-personalized-cancer-vaccine
'Adding immune-boosting agent to personalized cancer vaccine supercharges the body's immune defense against malignant brain tumors
The combination of poly-ICLC with an immunotherapy vaccine utilizing dendritic cells is a promising approach for treating patients with deadly brain cancer
brain scans
Credit: iStock
May 9, 2024
By Denise Heady, MA
5 min read
Investigators at the UCLA Health Jonsson Comprehensive Cancer Center have pinpointed a combination immunotherapy treatment that enhances the immune response for people with malignant gliomas, an aggressive type of brain tumor that is fast growing and difficult to treat.
The study, published in Nature Communications, found that pairing a personalized dendritic cell vaccine with the immune-boosting substance poly-ICLC enhances the immune response and activity of T cells in patients with malignant glioma, and improves the dendritic cells’ ability to fight the brain tumor more effectively than the vaccine alone.
“Treating malignant gliomas is very complex and due to the infiltrative nature of these tumors and their location in the brain, these patients often have a poor prognosis,” said Robert Prins, a professor of molecular and medical pharmacology and of neurosurgery at the David Geffen School of Medicine at UCLA, and co-senior author of the study. “By improving the potency of the vaccine, we’re hoping it can induce more effective anti-tumor immune responses in patients diagnosed with malignant gliomas.”
The dendritic cell vaccine, pioneered at UCLA, uses a person’s own white blood cells to help activate the immune system to fight cancer. Dendritic cells typically alert the immune system when it detects a foreign invader.
The vaccine works by combining brain tumor protein antigens derived from surgically removed tumors with dendritic immune cells generated from the patient’s own blood. The dendritic cells train the immune system to recognize the tumor antigens so that when they are injected back into the patient the immune system will be educated to recognize and attack tumor cells.
While the vaccine has shown promise in treating patients with malignant gliomas, the treatment does not work for everyone.
To further amplify the anti-tumor immune response, researchers looked at adding toll-like receptor (TLR) agonists to the vaccine. TLR agonists bind and activate a family of evolutionarily conserved receptors expressed by dendritic cells and macrophages to help alert the immune system to foreign pathogens. By activating these TLRs on dendritic cells, the UCLA team theorized that the combination might then increase the frequency and infiltration of antitumor specific T cells, while reducing the suppressive capacity of the tumor microenvironment.
The team specifically looked at two different TLR agonists — poly-ICLC and resiquimod — to see which one would be safer and more effective in combination with the vaccine.
The team enrolled 23 patients, ranging from 26- to 72-years-old, with WHO Grade III-IV glioma who were randomized to receive either poly-ICLC, resiquimod or a placebo in addition to the personalized DC vaccine.
In order to determine the optimal therapeutic combination, the team performed a high-dimensional single-cell analysis to understand the systemic proteomic and transcriptomic changes induced by the TLR agonists. This type of analysis lets the researchers see how the TLR agonists affect the immune cell proteins throughout the body.
They discovered that poly-ICLC demonstrated superior effectiveness, triggering a stronger immune response compared to resiquimod or the vaccine alone. The researchers observed a marked increase in the activity of interferon genes and substantial alterations in immune cell behavior, indicative of enhanced antitumor activity.
Most notably, the expression of PD-1 surged in CD4+ T-cells, while CD38 and CD39 levels diminished in CD8+ T-cells. There was a notable rise in the number of monocytes, which are key players in the immune response.
The investigators also found that the response was specifically linked to interferon, a protein that plays a key role in the body’s defense against pathogens and is measurable in the patient’s peripheral blood. The stronger the interferon response after the treatment, the longer the patients survived. While this association was statistically significant and suggests a potential link between this treatment and improved survival rates, the study was not originally designed to measure survival rates of this treatment. As such, the authors emphasized the need for caution regarding true clinical benefits of this combination treatment.
“If further study confirms the link between systemic interferon activation and survival rates in malignant glioma patients, we could potentially use interferon activation as a biomarker,” said Willy Hugo, assistant professor of medicine in the division of dermatology at the David Geffen School of Medicine at UCLA and co-first author of the study. “This means we could test patients for this specific immune response, and if it’s strong, we know they are likely to respond well to the combined TLR agonist and dendritic cell vaccine therapy.”
Patients who show no or low interferon response after the therapy could be directed towards other treatments or clinical trials more quickly, saving valuable time in their fight against this aggressive form of brain cancer.
The team also noted that combining these treatments with immune checkpoint inhibitors, which are another type of immunotherapy, could be another promising approach. They have already started a new clinical trial to test this combination in patients with recurrent glioblastoma, which is supported by the UCLA Specialized Program of Research Excellence (SPORE) in Brain Cancer.
“This research is a step forward in the quest for more effective immunotherapy for gliomas, along with developing a potential blood-based test to determine if the patient’s immune system is responding to the vaccine in a way that will help in the fight against this devastating form of brain cancer,” said Dr. Richard Everson, assistant professor of neurosurgery and co-first author of the study.
The study’s other co-senior author is Dr. Linda Liau, professor and chair of neurosurgery. Other authors, all of UCLA, are Lu Sun, Joseph Antonios, Alexander Lee, Lizhong Ding, Melissa Bu, Sarah Khattab, Carolina Chavez, Emma Billingslea-Yoon, Benjamin Ellingson and Dr. Timothy Cloughesy. Prins, Hugo, Cloughesy, Ellingson, Everson and Liau are all members of the UCLA Health Jonsson Comprehensive Cancer Center.
This study was funded in part by a grant from the National Cancer Institute."
Published: 08 May 2024
TLR agonists polarize interferon responses in conjunction with dendritic cell vaccination in malignant glioma: a randomized phase II Trial
Richard G. Everson, Willy Hugo, Lu Sun, Joseph Antonios, Alexander Lee, Lizhong Ding, Melissa Bu, Sarah Khattab, Carolina Chavez, Emma Billingslea-Yoon, Andres Salazar, Benjamin M. Ellingson, Timothy F. Cloughesy, Linda M. Liau & Robert M. Prins
Nature Communications volume 15, Article number: 3882 (2024) Cite this article
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Abstract
In this randomized phase II clinical trial, we evaluated the effectiveness of adding the TLR agonists, poly-ICLC or resiquimod, to autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination in patients with newly-diagnosed or recurrent WHO Grade III-IV malignant gliomas. The primary endpoints were to assess the most effective combination of vaccine and adjuvant in order to enhance the immune potency, along with safety. The combination of ATL-DC vaccination and TLR agonist was safe and found to enhance systemic immune responses, as indicated by increased interferon gene expression and changes in immune cell activation. Specifically, PD-1 expression increases on CD4+?T-cells, while CD38 and CD39 expression are reduced on CD8+?T cells, alongside an increase in monocytes. Poly-ICLC treatment amplifies the induction of interferon-induced genes in monocytes and T lymphocytes. Patients that exhibit higher interferon response gene expression demonstrate prolonged survival and delayed disease progression. These findings suggest that combining ATL-DC with poly-ICLC can induce a polarized interferon response in circulating monocytes and CD8+?T cells, which may represent an important blood biomarker for immunotherapy in this patient population.Trial Registration: ClinicalTrials.gov Identifier: NCT01204684.
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Introduction
There have been significant advances in our genetic and immunologic understanding of primary brain tumors, such as malignant gliomas. Yet, it has still proven difficult to improve long-term outcomes in patients using standard-of-care therapies1. We and others have demonstrated that autologous tumor lysate (ATL) dendritic cell (DC) vaccination can induce local and systemic anti-tumor immune responses in malignant glioma patients, and clinical trials have suggested that this may improve survival in this deadly condition2,3,4,5,6. However, variable response rates in cancer immunotherapy trials have prompted the search for strategies to enhance cancer vaccine potency. In particular, agonists of a family of pattern-recognition receptors (PRR) called Toll-like receptors (TLR)7,8,9,10, which appear capable of activating of antigen-presenting (i.e., dendritic) cells, enhancing T-cell priming, and decreasing myeloid-derived suppressor cells (MDSC), are rational candidates for use in combination with ATL-DC vaccination to potentially enhance the antitumor immune response10,11.
TLR3 is an intracellular PRR that recognizes double-stranded RNA (dsRNA), usually associated with viral infection, and induces high levels of IFN-a/ß and pro-inflammatory cytokines when activated. TLR-3 is predominantly expressed by macrophages, plasmacytoid DC and myeloid DC12,13, as well as microglial cells14,15. It has also been shown that astrocytes16,17,18 and malignant gliomas19 also respond similarly to TLR3-induced signaling. Polyinosinic acid-polycytidylic acid stabilized with polylysine (poly-ICLC) is a multi-dimensional synthetic dsRNA analogue and viral mimic that signals via TLR3, MDA5 and other dsRNA-dependent PRR signaling, induces type I-II IFNs20,21, promotes the infiltration of effector T cells in pre-clinical glioma models22, and upregulates genes associated with chemokine activity, T-cell activation, and antigen presentation23. Poly-ICLC has been tested as a single-agent therapeutic for multiple malignancies24, including malignant glioma patients25, in whom it has demonstrated adequate safety, but limited survival benefit in combination with standard-of-care therapies26.
Similarly, TLR7 and TLR8 are other intracellular PRRs that recognize single-stranded RNA (ssRNA), which subsequently induces proinflammatory cytokines, chemokines, and type I interferons (IFNs)27. In pre-clinical work, we previously demonstrated that DC injected into imiquimod (TLR7 agonist)-pre-treated sites acquired lymph node migratory capacity and enhanced T-cell priming28. Our early phase clinical trials demonstrated that DC vaccination with adjuvant topical imiquimod, a TLR-7 agonist, was safe and feasible in glioblastoma patients3. Resiquimod is a newer imidazoquinoline agonist that shows enhanced transdermal delivery, activates TLR7/8 to enhance T-cell responses and TH1-type cytokine secretion by DC29,30,31,32, and may have greater potency as an immune modulator.
In this study, we report the long-term results of 23 malignant glioma patients enrolled in a phase II randomized clinical trial where patients were randomized to receive Poly-ICLC, Resiquimod or a placebo in addition to ATL-DC. The trial was designed to evaluate the immunologic effects of the addition of the TLR agonists and compare the safety, immune responses, and potential efficacy. Post-hoc analysis using cytometry by time-of-flight (CyTOF) and bulk and single-cell RNA sequencing (scRNAseq) technologies were used to detect the cellular and molecular immune signatures from peripheral blood mononuclear cells (PBMCs) pre- and post-treatment.
Results
Patient characteristics and safety
A total of 23 patients with resection-eligible WHO grade III or IV glioma were enrolled and randomized between September 2010 and August 2014. All patients received ATL-DC vaccination as an initial series of 3 biweekly bilateral upper extremity injections of 2.5x10e6 ATL-DCs followed by up to 7 booster injections at 4-month intervals. Randomization allocated nine into the adjuvant TLR-7/8 agonist (resiquimod, 0.2% gel, 3M, applied to ATL-DC injection site days 0, 2, 4 post-DC injection) group, nine into the adjuvant TLR-3 agonist (poly-ICLC, 20?mcg/kg IM, Oncovir, upper extremity, at time of DC injection) group, and five to the adjuvant placebo arm where patients received either carrier gel without resiquimod or IM saline injection. (Fig. 1A, Supplementary Fig. 1). All patients were followed for clinical evaluations, toxicity, survival, imaging changes, as well as in-depth systemic immune monitoring. Baseline patient characteristics are presented and segregated by treatment group in Table 1 (see also Supplementary Data 1). The median age was 45.3 (range 26.2–72.8) years, and 57% of the enrolled patients were male. Patients were enrolled prior to the 2016 update to the WHO classification of central nervous system tumors; 65% (n?=?15) had histopathological diagnoses of WHO Grade IV glioblastoma (now consistent with IDH wild-type glioblastoma), while 35% (n?=?8) of the patients were WHO Grade III (all of which would now classify as IDH-mutant astrocytoma or oligodendroglioma). Fifty-two percent (n?=?12) of patients were treated following recurrence, while 48% (n?=?11) were treated in the newly diagnosed setting. All patients were treated following surgical resection and standard-of-care treatment. The molecular characteristics of the patient tumors are outlined in Table 1. Overall, MGMT methylation was seen in 35% (n?=?8), IDH mutations were observed in 35% (n?=?8, all grade III), and EGFR amplification was seen in 44% (n?=?10, all glioblastoma) of patients, consistent with the heterogenous population of malignant glioma patients. There were no statistically significant differences in age, sex, Karnofsky performance status, MGMT methylation status, pre- or postsurgery enhancing tumor volume, nor steroid administration at enrollment. No statistically significant differences were observed between the molecular characteristics, although the number of patients in each treatment group was small.
Fig. 1: Combination of ATL-DC vaccine and TLR agonists results in a robust interferon pathway activation in the patient PBMCs.
figure 1
A Timeline of PBMC acquisition and analysis using CyTOF and/or RNAseq. V = vaccine, D = Day. (Figure created with the help of BioRender). B Schematic of differential gene expression analysis performed on pre-treatment and post-treatment PBMCs of indicated treatment groups. Differentially expressed genes (DEGs) in TLR agonist-treated groups are compared against their changes in the placebo group to identify DEGs specific to the TLR-agonist groups. C, D Enriched gene set terms in Gene Ontology Biological Process (C) or ARCHS4 TF Coexp (D) datasets that significantly overlap with the union of DEGs from ATL-DC + poly-ICLC and ATL-DC + resiquimod groups (P values, FDR-adjusted, two-sided fisher exact test). E Differential gene expression (pre vs. post-treatment fold change, in log2) of representative antigen presentation and IFN-related genes across treatment groups (P values, two-sided Welch t test). F Gene set enrichment score differences (pre vs. post-treatment, delta GSVA score) of representative IFN-related genesets across treatment groups (P values, two-sided Welch t test). G Heatmap of single-sample, gene set enrichment scores (GSVA) of type I and type II interferon genesets in pre-treatment, ATL-DC + placebo, ATL-DC+poly-ICLC and ATL-DC+resiquimod samples. The number of sample pairs analyzed in panels E and F are: ATL-DC+placebo, 5 pairs; ATL-DC+poly-ICLC, 8 pairs; ATL-DC+resiquimod, 8 pairs. The rectangular box in each boxplot represents the interquartile range (IQR), spanning from the first quartile (25th percentile, bottom of box) to the third quartile (75th percentile top of box). Inside the box, the median (50th percentile) is marked. The whiskers (shown as lines extending from the box) extend to the largest and smallest non-outlier values within 1.5 times the IQR, while outliers lie beyond the whiskers.
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Table 1 Baseline patient characteristics
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Overall, the addition of a TLR agonist-induced only Grade 1-2 treatment-related adverse events (TRAEs), and all adverse events reported resolved without further treatment or hospitalization (Table 2). The most common TRAEs were rash (39%), fever (35%), and fatigue (26%; see Table 2), and were more common in patients treated with resiquimod and poly-ICLC. 88.9% of patients who received resiquimod reported a temporary localized, cutaneous rash that resolved without further treatment. Other observed adverse events were not uncommon in the setting of postoperative central nervous system (CNS) tumor treatment. However, no serious adverse events (Grade 3-4) attributable to the treatment were observed. As such, the addition of a TLR agonist to ATL-DC vaccination in malignant glioma patients was found to be safe and tolerable.
Table 2 Adverse events across treatment cohorts
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Adjuvant TLR agonist treatment induces systemic expression of type I and type II interferon downstream genes
The primary endpoint of this clinical trial was to evaluate systemic immune response changes induced by ATL-DC vaccination with and without TLR agonist administration. As such, we collected PBMCs at baseline (pre-treatment), one day after the vaccination (on treatment), and then following the completion of the treatment cycle (post-treatment) of each patient (Fig. 1A). We aimed to understand how the adjuvant administration of TLR agonists modified the immune response in comparison with ATL-DC vaccination alone (placebo control).
We first performed paired bulk RNA-seq on patient-matched, pre-treatment and post-treatment PBMC samples that passed QC (see sample list in Supplementary Data 1C). For each gene, we computed the difference between its expression in the pre- and post-samples of patients in each treatment group: ATL-DC+placebo (n?=?5 pairs); ATL-DC+poly-ICLC (n?=?8 pairs); ATL-DC+resiquimod (n?=?8 pairs); for brevity, we refer to them as placebo, poly-ICLC and resiquimod, respectively. To identify expression changes specific to the TLR agonist groups, we identified genes whose average upregulation in the TLR agonist pairs (poly-ICLC or resiquimod) were at least two-fold higher than the placebo pairs (Fig. 1B, Supplementary Data 2A, see Methods).
Genes upregulated in the TLR agonist groups were involved in antigen processing and were enriched with known interferon-stimulated genes (ISGs) (Fig. 1C–E, Supplementary Data 2B, C). This observation was also confirmed by per-sample gene set enrichment analysis, where the TLR agonist-treated groups displayed higher enrichment of both type I and II interferon downstream gene sets compared to ATL-DC/placebo (Fig. 1F, Supplementary Data 2D, E). PBMC samples with higher absolute enrichment scores of interferon gene sets were dominated by post-treatment samples from both grade III and IV glioma patients in the TLR-agonist-treated groups (Fig. 1G). The two TLR agonist-treated groups showed a largely similar trend in treatment-induced gene expression changes, which included a measurable increase in the expression of ISGs in the peripheral blood of malignant glioma patients. However, we noted that the resiquimod group had a more heterogenous response, which resulted in a lower degree of statistical significance compared to that of the poly-ICLC group.
TLR agonist treatment induces systemic T cell activation, monocyte proliferation and interferon responses in myeloid and lymphoid populations
We performed CyTOF on PBMC timepoints with a 27-marker heavy metal antibody-conjugated panel for 20 of the 23 patients where sufficient material was available (placebo, n?=?4 pairs; poly-ICLC, n?=?9 pairs; resiquimod, n?=?7 pairs; see Supplementary Data 1C, 3A, 3B). The panel was selected to be able to broadly characterize different immune cell types, activation/effector, memory, and exhaustion phenotypes, with a bias towards T-cell relevant markers. The different immune cell type populations were visualized by the uniform manifold approximation and projection (UMAP) method (Fig. 2A), which we broadly assigned to seven different major immune populations based off the normalized heatmap marker expression (Fig. 2B).
Fig. 2: Single cell analysis reveals activation of systemic T cells and monocytes as a part of interferon pathway activation in all myeloid and lymphoid populations.
figure 2
A A UMAP projection of the pre- and post-treatment PBMC sample pairs from twenty patients (placebo, n?=?4 pairs; poly-ICLC, n?=?9 pairs; resiquimod, n?=?7 pairs). Clustering was performed with a random sampling of 5,000 cells from each patient. B Heatmap of normalized expression of all 27 cell markers within cell populations identified in the patient PBMCs. C, D Normalized expression of indicated markers in monocyte (C), or T cell populations (D) within the PBMC samples of patients from indicated treatment groups. P values, two-sided Wilcoxon rank sum test. E, UMAP projection of the PBMC-derived single cells (n?=?99,590). The immune subset associated with each cluster is inferred based on the cluster’s differentially expressed transcripts. Canonical markers of known immune subsets are shown. F, G Heatmaps showing the union of recurrent DEGs computed between ATL-DC treated samples (combined with placebo, resiquimod or poly-ICLC) and pre-treatment samples in the myeloid populations (F) or lymphocyte populations (G). Shown in the heatmaps are the log fold change values of the DEGs in each cell population grouped by their treatment groups. The number of sample pairs analyzed in C and D are: ATL-DC+placebo, 4 pairs; ATL-DC+poly-ICLC, 9 pairs; ATL-DC+resiquimod, 7 pairs. The rectangular box in each boxplot represents the interquartile range (IQR), spanning from the first quartile (25th percentile, bottom of box) to the third quartile (75th percentile the top of box). Inside the box, the median (50th percentile) is marked. The whiskers (shown as lines extending from the box) extend to the largest and smallest non-outlier values within 1.5 times the IQR, while outliers lie beyond the whiskers.
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After 3 cycles of treatment, the post-treatment samples of patients in the TLR agonist groups showed a significant increase in the proportion of proliferating Ki67?+?CD14+ classical monocytes (Fig. 2C, Supplementary Data 3C). Such findings were corroborated by the increased monocyte fraction and CD14 transcript expression after ATL-DC?+?TLR agonist-treated samples (Supplementary Fig. 2A, B, Supplementary Data 3D). ATL-DC?+?TLR agonist treatment induced PD-1 expression in CD4 T cell population and increased the T-cell normalized expression of PDCD1 (the transcript that encodes PD-1 protein) and TCF7 (a marker of progenitor-like T cells) (Fig. 2D, Supplementary Fig. 2C). Moreover, expression of markers associated with irreversible T cell exhaustion, such as CD38 and CD3933,34, were also significantly reduced after ATL-DC?+?TLR agonist treatment (Fig. 2D, Supplementary Fig. 2D). Increased expression of PD-1 and decreased expression of CD38 and CD39 suggest the addition of the TLR agonists led to enhanced systemic T cell activity and cellular fitness in the patient.
To delineate the changes induced by ATL-DC and TLR agonist treatment in discrete peripheral blood immune cell subsets, we performed single-cell RNA-seq on selected patients at baseline and then following the completion of therapy. We analyzed two representative sample pairs from each cohort (placebo, poly-ICLC, and resiquimod) (Supplementary Data 1C, 3E). We identified a total of twelve clusters from the total PBMC immune cell population and annotated these clusters based on differentially expressed gene markers in each cluster. From the initial clustering, we were able to identify multiple populations of CD4+ and CD8+ T cells, two populations of NK cells, three monocytic cell populations, B cell, and dendritic cells (type 2 conventional dendritic cells (cDC2) and plasmacytoid dendritic cells (pDCs), in accordance with the previous characterization of these cell types in peripheral blood (Fig. 2E and Supplementary Fig. 2E, F).
Differential gene expression analysis across the different lymphoid and myeloid populations revealed concordant upregulation of known ISGs (e.g. IFI6/35/44?L, ISG15/20, IFIT3, IFITM1/3, GBP1/5, MX1, STAT1, and CXCL10) and antigen presentation-related proteasomes (PSMB9 and PSME2) in both TLR agonist sample pairs. The magnitude of induction was weaker in the paired PBMC samples obtained from the resiquimod group compared to the poly-ICLC group (Fig. 2F, G).
Thus, our combination of high dimensional proteomics, bulk and single-cell RNAseq demonstrates how adjuvant TLR administration in conjunction with ATL-DC reproducibly increases the proportion of canonical CD14+ monocytes within the systemic blood circulation. This TLR agonist administration was also associated with enhanced T cell activity, coupled with decreased expression of CD38 and CD39 and their downstream T cell-suppressive adenosine pathway33,34,35. ATL-DC?+?TLR agonist-driven induction of ISGs across lymphoid and myeloid populations identified in our scRNAseq analysis corroborated our bulk transcriptomic analysis. Given the consistent changes observed with TLR agonist administration, we examined whether these systemic measurements correlated the observed progression-free and overall-survival differences between these patient populations to speculate on their contribution.
Long-term clinical outcomes of malignant glioma patients treated with ATL-DC vaccination plus adjuvant TLR agonists
Median follow-up of patients treated on this clinical trial was 2.2 years after surgery, although the long-term survivors have now been followed for over 10 years. Median progression-free survival (PFS) was 8.1 months; and median overall survival (OS) was 26.6 months. Although this clinical trial was not designed or powered to detect effects of these treatments on survival between the treatment groups, there were noticeable differences in median survival between the treatments groups for both OS (placebo: 7.7 months, poly-ICLC: 52.5 months, and resiquimod: 16.7 months; log-rank P?=?0.017) and PFS (placebo: 5.5 months, poly-ICLC: 31.4 months and resiquimod: 8.1 months; log-rank P?=?0.0012) (Fig. 3A). Because the trial included patients with both grade III and IV tumors, we stratified our analysis based on tumor grade. When we analyzed only the grade IV (GBM) patients, we observed a trend towards improved PFS (log-rank P?=?0.068) and OS (P not significant) (Fig. 3B). Interestingly, for the IDH mutant/Grade III cohort, all four patients that received ATL-DC + poly-ICLC treatment are still alive at the data cutoff date (three of the patients have survival > 120 months and one > 112 months), and they have significantly longer OS and PFS compared to the other (n?=?4) grade III patients who received ATL-DC + resiquimod or ATL-DC alone where median OS was 15.73 months (Fig. 3C).
Fig. 3: Combined ATL-DC vaccine and TLR agonist treatment show trends of improved tumor control and patient survival.
figure 3
A–C Progression-free survival (PFS, top) and overall survival (OS, bottom) of all patients (A), patient subset with GBM (B), or grade III glioma (C) in indicated treatment groups. P values, log-rank test. D, E, Multivariate Cox proportional hazards analysis assessing the hazard ratios of tumor progression in TLR agonist treatment groups against placebo in all patients (D) or GBM subset (E) after adjusting for other clinical covariates (Tx_Group=treatment group, RecurNum=number of recurrences prior to ATL-DC treatment). In the forest plot, the squares are the hazard ratio (HR) estimates, the error bars are 95% confidence interval (CI) of the HR, the P value of each covariate is based on its Wald statistics, the P values are not adjusted. In D, the sample distribution in each covariate is Tx_Group: placebo=5, poly-ICLC?=?9, resiquimod=9; Grade: III?=?8, IV?=?15; MGMT_methylation: True=8, False=15. In E, Tx_Group: placebo=4, poly-ICLC?=?5, resiquimod=6. F, MR-computed volumes of post-treatment, recurrent tumors in indicated treatment groups. Treatment groups: Placebo (n?=?5), Resiquimod (n?=?8); Poly ICLC (n?=?9). P values, unpaired, two-sided Wilcoxon rank sum test.
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We performed multivariate Cox proportional hazard (PH) analysis, adjusting for clinical variables that are significantly correlated with OS or PFS as a single variable (tumor grade, MGMT methylation status, and number of recurrences). Our analysis confirmed that patients in the poly-ICLC and resiquimod treatment groups had a lower risk of progression that was independent of grade, MGMT methylation, and number of recurrences (Fig. 3D). Risk of death was significantly lower in the poly-ICLC group, while the resiquimod group showed a similar trend that was not statistically significant (Supplementary Fig. 3A). In the GBM patient subset, TLR agonist treatment also significantly lowered risk of recurrence, but not risk of death (Fig. 3E, Supplementary Fig. 3B).
To determine whether this treatment directly impacted tumor volume, MR imaging was performed, and contrast-enhancing tumor volume was quantified over time. We noted that the rate of tumor volume increase over time in the ATL-DC/placebo treatment cohort was higher than in the ATL-DC/resiquimod treatment (p?=?0.022) and the ATL-DC/poly-ICLC treatment groups (P?<?0.001; Fig. 3F). Anecdotally, we observed an increased T2/FLAIR MRI signal after completion of the vaccine series in two of the four long-term survivors who received ATL-DC/poly-ICLC (Supplementary Fig. 3C, D), although such findings are potentially confounded by prior radiation therapy, and thus we cannot ascribe such changes solely to the vaccine/TLR agonist intervention. However, this increased post-vaccination T2/FLAIR on MRI was not seen in patients who did not receive poly-ICLC (not shown).
Interferon activation score in the peripheral blood immune cells is a significant predictor of survival after ATL-DC therapy
Finally, we asked if the magnitude of interferon pathway induction by the adjuvant TLR agonist treatment directly correlated with OS or PFS. This could allow for the use of an interferon activity score as a biomarker for productive anti-tumor immune responses following ATL-DC immunotherapy. To this end, we stratified the patients by the median GSVA score of the “HALLMARK INTERFERON GAMMA RESPONSE” gene set in post-treatment PBMC samples. We confirmed that patients whose post-treatment samples displayed higher interferon gene set scores (=median) had longer OS and PFS than those with lower scores (Fig. 4A, Supplementary Fig. 4A). Separate analyses on the grade IV (GBM) and grade III glioma patients showed a concordant trend but with a lower degree of statistical significance; this was likely caused by the small sample sizes. Notably, multivariate Cox PH analysis strongly suggested that the interferon gene set score is a significant predictor of tumor recurrence (Fig. 4B, C) and death (Supplementary Fig. 4B), even after adjusting for other potentially confounding clinical variables. To ensure that the correlation is not specific to this single gene set, we confirmed that the gene set scores of other interferon gene sets after ATL-DC treatment are also positively correlated with the patients OS and PFS (Supplementary Data 4A, B). Such findings can be confirmed in larger subsequent studies.
Fig. 4: IFN pathway activation is a positive predictor of survival after ATL-DC vaccine and TLR agonist therapy.
figure 4
A Kaplan-Meier progression-free survival curves of all patients (left), GBM (center), and Grade III glioma subsets (right) stratified by their HALLMARK_INTERFERON_GAMMA_RESPONSE GSVA scores in their post-treatment PBMCs. P values, log-rank test. B, C Multivariate Cox proportional hazards analysis assessing hazard ratios of tumor progression in patients with high HALLMARK_INTERFERON_GAMMA_RESPONSE GSVA score in all patients (B) or GBM subset (C) after adjusting for other clinical covariates. In the forest plot, the squares are the hazard ratio (HR) estimates, the error bars are 95% confidence interval (CI) of the HR, the P value of each covariate is based on its Wald statistics, the P values are not adjusted. In B, the sample distribution in each covariate is GSVA score (post-Tx): <median=10, =median=11; Grade: III?=?7, IV?=?14; MGMT_methylation: True=7, False=14. In (C), GSVA score (post-Tx): <median=7, =median=7; MGMT_methylation: True=3, False=11.
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Taken together, these data suggest that the addition of TLR agonists to ATL-DC vaccination shifts towards an interferon-induced immune response in both lymphoid and myeloid cells. Poly-ICLC and resiquimod appear to upregulate similar ISGs but with different magnitude. Enhancing systemic ISG-signaling may reflect an environment more favorable towards the generation of an antitumor immune response and clinical effects.
Discussion
We report herein that ATL-DC vaccination with adjuvant poly-ICLC or resiquimod is overall safe and well-tolerated in patients with malignant glioma. To achieve the primary immunological endpoints of this study, we utilized high-dimensional single-cell analysis to understand the systemic proteomic and transcriptomic changes induced by TLR agonists in order to rationally determine the optimal therapeutic combination.
Our study is the first high-dimensional single-cell analysis done in a clinical trial for malignant glioma patients treated with dendritic cell vaccination and TLR agonists. Although our study was not designed to examine what happens in the tumor microenvironment, our results indicate that we are able to sensitively detect systemic changes in the blood after intradermal autologous dendritic cell vaccination with and without TLR agonists. Adjuvant TLR agonist treatment promotes the expression of IFNa/ß and IFN?-induced genes on the peripheral lymphoid and myeloid cells, and GSVA further confirmed increased expression of the IFNa and IFN? downstream genes, including IFN-induced proteins ISG15 and STAT1. Other genes that were significantly upregulated by TLR agonist treatment include PARP9-DTX3L, and this heterodimer is also known to amplify interferon signalling36. Our current observation of increased mRNA expression of interferon downstream genes may represent activation of either type I or type II interferons or both; type I and II interferon downstream gene signatures significantly overlap with one another, and additional follow-up study is required to dissect the individual contribution of type I and type II interferons in the efficacy of ATL-DC therapy. Regardless, our results support the conclusion that DC vaccination with poly-ICLC induces Type I and Type II IFN responses more effectively than with adjuvant resiquimod or a dendritic cell vaccine alone. Similar to our results, additional studies have identified poly-ICLC as the most effective TLR/PRR agonist when compared with others37,38. The downstream effect of this signaling in the lymphoid cell population appears to be increased T-cell activity, as well as decreased T-cell exhaustion phenotype. Together, these effects may enhance the activity of tumor antigen-specific T cells generated from an active vaccine.
It is also important to recognize that, in contrast to resiquimod and even plain poly-IC, poly-ICLC signals through various PRRs in addition to TLR3, consistent with its role as an authentic viral mimic. The poly-lysine stabilizer also functions as a transfection agent. Specifically, poly-lysine bursts the endosome through a proton sponge effect and releases the dsRNA into the cytoplasm, where it then preferentially activates MDA5, OAS, PKR, and other cytoplasmic dsRNA-dependent systems21. Among the actions generated putatively through MDA5 are a further increase in Type 1 IFN, depression of MDSC, expansion of CD8 T cell populations through IL-15, CD8 targeting and infiltration of tumor through CXCL10, and a direct Type 1 IFN-dependent effect on tumor endothelium through VCAM-139. These effects are best seen with systemic (intramuscular or intravenous) rather than local (subcutaneous) administration, as we have done in the current study. Such adjuvant responses induced by poly-ICLC may play a role in the longer-term maintenance of the immune responses generated by ATL-DC vaccination, but further studies are required to verify these findings.
While malignant gliomas are usually conceived of as a locoregional disease with essentially no capacity to spread outside the central nervous system, there has been a growing understanding of the role that systemic tissues play in priming, developing and/or suppressing an immune response in the brain. The catalog of known pervasive systemic immune deficits in glioblastoma patients is continually growing40. The failure of immune checkpoint inhibitor therapy in malignant gliomas has led many to conclude that immune cells in the tumor microenvironment of cancers unresponsive to these checkpoint inhibitors may exist in an irreversible, terminally exhausted state41,42. The generation of de novo tumor antigen-specific immune responses in the periphery that lead to new T-cell infiltration into the tumor microenvironment may be required to overcome this barrier43. Dendritic cell vaccines are a robust example of an agent capable of mediating the initiation of such a T-cell response.
The fraction of monocytes in the systemic circulation is known to be an important biomarker for the response to PD-1 checkpoint blockade immunotherapy44. Our data is consistent with other findings that TLR agonists can induce a higher fraction of CD14+ classical monocytes in the blood. These findings further suggest that the combination of ATL-DC?+?TLR agonist with immune checkpoint blockade may be a rational choice. In fact, we have now initiated a phase I trial combining ATL-DC+Poly-ICLC with pembrolizumab in recurrent glioblastoma patients (NCT04201873). Our data also reveals increased relative expression of PDCD1 and TCF7 after ATL-DC/poly-ICLC. The abundance of TCF7?+?PD1?+?CD8 T cells was associated with better response to PD-1 blockade in melanoma and non-small cell lung cancer45,46.
While encouraging, our clinical findings must be interpreted with caution. Even though this was a randomized clinical trial (randomization software assigned patients to TLR agonist/placebo groups), the small number of patients enrolled contributed to an imbalance in patient selection between the treatment groups. Such effects are inherent in trials with small numbers of patients. The patients randomized to the resiquimod group and poly ICLC group were approximately consistent, but the patients in the ATL-DC + placebo had more unfavorable clinical characteristics; the distribution of patient age and disease grade in the placebo group are more unfavorable. We found that ATL-DC vaccinated patients randomized to receive adjuvant TLR agonists demonstrated a statistically significant extended overall time to tumor progression and slower rates of tumor growth, compared with those who received an adjuvant placebo. The poly-ICLC group was further associated with a statistically significant increase in overall median survival. Some of the grade III gliomas treated with ATL-DC/poly-ICLC exhibited unique T2/FLAIR changes on brain MRI scans following DC vaccination, but such findings were confounded by previous radiation therapy, even though such changes were not seen in the other patients. The significance of these imaging findings is not clear and needs to be replicated.
In conclusion, we demonstrate that autologous dendritic cell vaccination plus TLR agonists in patients with malignant gliomas generates a systemic interferon activation signature in the peripheral blood that is correlated with overall survival. Although this was a randomized study, it was powered for immune biomarker analysis, not for survival. As such, the clinical efficacy outcomes should be interpreted with caution. Given the noted long-term survival with the adjuvant use of poly-ICLC with DC vaccination, particularly in the grade III cohort of patients, further clinical trials that incorporate these combinations of immunotherapeutic agents are warranted.
Methods
Study design
This was a single-center, randomized, open-label multi-arm phase II clinical trial. The study protocol was approved by an independent ethics committee, institutional review board and internal scientific peer review committee at the University of California, Los Angeles. Patients were recruited and completed treatment between 2010 and 2014, with survival follow-up until the present date. Subjects were not compensated, and all patients gave written informed consent before enrollment.
Twenty-three patients with high-grade WHO Grade III or IV gliomas were enrolled in this protocol. To be eligible for the primary cohort, patients had to be >18 years and have newly diagnosed or recurrent WHO Grade III or IV malignant glioma, as determined through central pathology review. For all patients, a Karnofsky Performance Score (KPS) of =60, adequate bone marrow, liver, and renal function, life expectancy of =8 weeks, no other prior malignancy within the last 5 years, no active viral infections, and sufficient resected tumor material to produce the autologous vaccine were required. All newly diagnosed patients underwent surgical resection followed by radiation and chemotherapy with temozolomide for 6 weeks, per standard of care. Patients in the recurrent setting proceeded to trial treatment after recovery from surgery. All patients were scheduled to receive ATL-DC. Patients were then randomized to receive either placebo, resiquimod (topical 0.2%, 3?M), or poly-ICLC (20 µg/kg i.m., Oncovir) as an adjuvant to the DC vaccine. Patients underwent leukapheresis to obtain adequate numbers of PBMC for DC generation. For the study treatment, we processed the resected tumor tissue into a lysate, then prepared and cryopreserved the autologous DCs as we previously described2,3. Patients were then treated with three intradermal injections of autologous tumor lysate-pulsed DC plus adjuvant TLRs/placebo on days 0, 14, and 28. The TLR agonists were delivered as separate injections. Poly ICLC (20 ug/kg) was given as an intramuscular injection at the same site as the intradermal ATL-DC vaccine. Resiquimod (0.2%) was applied as a topical gel directly over the intradermal ATL-DC vaccine site. The placebo was a gel without any resiquimod and administered similarly over the intradermal vaccine site. All vaccines were administered on the upper arm. Follow-up for patients was conducted at the study site for vital signs, KPS, hematology and serum chemistries, as well as neurological and physical examinations.
Clinical assessments
Safety was assessed on the basis of occurrence of adverse events, which were categorized according to the NCI Common Toxicity Criteria for Adverse Events v. 4.0. Safety assessments were performed on the day of vaccination and 1 week after each vaccination during the treatment phase, and every 2 months thereafter until tumor progression or death.
Anatomic MR images were acquired prior to DC + adjuvant treatment and at 2-month intervals for all patients using the standardized brain tumor imaging protocol (BTIP)47, including three-dimensional pre- and post-contrast T1-weighted images at 1-1.5?mm isotropic resolution, two-dimensional T2-weighted and T2-weighted fluid-attenuated inversion recovery (FLAIR) images with 3-4?mm slice thickness and no interslice gap, and diffusion-weighted images with b?=?0, 500, and 1000?s/mm2, 3–4?mm slice thickness and no interslice gap. Disease progression was determined using the modified RANO criteria48. Additionally, post-hoc quantitative tumor volumetric analysis was performed using contrast-enhanced T1-weighted digital subtraction maps and segmentation techniques described previously49,50,51. Briefly, linear registration was first performed between all images, including contrast-enhanced T1-weighted images and T2-weighted and/or FLAIR images to nonenhanced T1-weighted images using a 12-degree-of-freedom transformation and a correlation coefficient cost function. Next, intensity normalization and bias field correction were performed for both nonenhanced and contrast-enhanced T1-weighted images, and voxel-by-voxel subtraction between normalized nonenhanced and contrast-enhanced T1-weighted images was performed. Image voxels with a positive (greater than zero) before-to-after change in normalized contrast enhancement signal intensity (i.e., voxels increasing in MR signal after contrast agent administration) within T2-weighted FLAIR hyperintense regions were isolated to create the final T1 subtraction maps. Estimates of tumor volume included areas of contrast enhancement on T1 subtraction maps, including central necrosis (defined as being enclosed by contiguous, positive-enhancing disease).
Patient samples
Heparinized peripheral blood was collected at the baseline visit and at each treatment visit for immune monitoring. Peripheral blood mononuclear cells were collected in CPT tubes (BD Biosciences, cat: 362753), isolated according to the manufacturer’s protocol, placed in freezing media made of 90% human AB serum (Fisher Scientific, cat. MT35060CI) and 10% dimethyl sulfoxide (Sigma, cat. C6295-50ML) and stored in liquid nitrogen until the time of analysis. On the day of data acquisition, samples were thawed in a 37?°C water bath and washed in RPMI-1640 media (Genesse Scientific, cat: 25-506) supplemented with FBS and penicillin and streptomycin. Patient tumor samples were attained immediately following surgery.
Generation of autologous dendritic cell vaccines
Monocyte-derived DCs were established from adherent peripheral blood mononuclear cells (PBMC) obtained via leukapheresis performed at the UCLA Hemapheresis Unit, as we have published previously3,6,52. All ex vivo DC preparations were performed in the UCLA-Jonsson Cancer Center GMP facility under sterile and monitored conditions. In brief, dendritic cells were prepared by culturing adherent cells from peripheral blood in RPMI-1640 (Gibco) and supplemented with 10% autologous serum, 500?U/mL GM-CSF (Leukine®, Amgen, Thousand Oaks, CA) and 500?U/mL of IL-4 (CellGenix), using techniques described previously2. Following culture, DCs were collected by vigorous rinsing and washed with sterile 0.9% NaCl solution. The purity and phenotype of each DC lot was also determined by flow cytometry (FACScan flow cytometer; BD Biosciences, San Jose, CA). Cells were stained with FITC-conjugated CD83, PE-conjugated CD86 and PerCP-conjugated HLA-DR mAb’s (BD Biosciences). Release criteria were >70% viable by trypan blue exclusion, and >30% of the large cell gate being CD86+ and HLA-DR+. One day before each vaccination, DC were pulsed (co-cultured) with tumor lysate overnight, washed, and the final product was tested for sterility by Gram stain, mycoplasma, and endotoxin testing prior to injection.
Molecular and immune analyses
CyTOF
Cells for mass cytometry analysis were prepared according to the Maxpar cell surface staining protocol. Briefly, 0.5 to 3?×?106 cells were washed with PBS and treated with 0.1?mg/mL of DNAse I Solution (StemCell Technologies, cat: 07900) for 15?minutes at room temperature. Cells were then resuspended in 5?µM Cell-ID cisplatin (Fluidigm, cat: 201064) as a live/dead marker for 5?minutes at room temperature. After quenching with the Maxpar cell staining buffer (Fluidigm, cat: 201068), the cells were incubated with a 27-marker panel for 30?minutes at room temperature. After washing, cells were incubated overnight in 125?nM iridium intercalation solution (1000X dilution of 125?µM Cell-ID Intercalator-Ir; Fluidigm, cat: 201192?A) in Maxpar Fix and Perm Buffer (Fluidigm, cat: 201067) to label intracellular DNA. The next morning, cells were washed with cell staining buffer and distilled water. The samples were processed on a Helios mass cytometer (Fluidigm) in the University of California, Los Angeles Jonsson Comprehensive Cancer Center Flow Cytometry core.
The CyTOF data was normalized utilizing EQ four-element calibration beads (Fluidigm, cat: 201078) with the R package premessa (version 0.2.4, Parker Institute for Cancer Immunotherapy) following removal of dead cells. A total of 5,000 cells were subsampled from each sample (except for sample S16-07-2-Day 1 where we only had 4,861 cells). Subsequently, bead normalized data from 45 samples were integrated as described previously53. Briefly, flow cytometry standard (FCS) files were loaded into R with the flowCore package (version 2.8.0).
Raw marker intensities were transformed utilizing hyperbolic inverse sine (arcsinh) with cofactor of 5. Cell population identification was carried out using unsupervised clustering using FlowSOM package (version 2.4.0) and subsequent metaclustering using ConsensusClusterPlus package (version 1.60.0). The metaclusters were manually curated to identify canonical populations in Fig. 2B (including one unknown cluster with little/no marker expression). The high dimensional data was visualized with the Uniform Manifold Approximation and Projection (UMAP). Differential marker analysis across treatment groups were first performed using the linear mixed model analysis pipeline as described53. Markers with nominally significant p-values in one or more cell populations (P?=?0.05; e.g CD39, CD38, Ki-67, PD-1) were visualized in boxplots; statistical significance computed using the linear mixed model were further confirmed using non-parametric Wilcoxon rank sum test.
Bulk RNAseq
Total RNA was isolated from frozen PBMC of the patients isolated at baseline and after three biweekly vaccines with ATL-DC plus adjuvant using the ZYMO quick RNA extraction kit. We utilized the TruSeq RNA exome kit to construct the RNA sequencing libraries in samples that passed QC (placebo: 5 pairs, resiquimod: 8 pairs, and poly-ICLC: 8 pairs; see Supplementary Data 1C). Paired-end, 2 × 100 base pair (bp) transcriptome reads were mapped to the Genome Reference Consortium Human Build 38 (GRCh38) reference genome using HISAT2 (version 2.0.6)54. The gene-level counts were generated by the HTSeq-count program (version 0.5.4p5)55. We utilized the DESeq2 R package’s counts function (version 1.24.0)56 to compute the normalized gene expression values from the raw gene expression counts. DESeq2 normalized gene expression was log2 transformed after adding a pseudo count of 1. For subsequent differentially expressed genes (DEGs) and gene set enrichment analyses, we only included the known genes (i.e., genes with RefSeq transcripts ID starting with “NM_“, that satisfy: 1) normalized expression IQR?=?1; and 2) normalized log2 expression =1 in at least one of the samples.
Based on the filtered gene list, we first obtained the patient-specific, log2 fold change of each gene before and after the ATL-DC vaccine treatment. Next, the mean of the log2 fold changes in the poly-ICLC or resiquimod group is compared to those in the placebo group. Genes showing at least 2-fold upregulation in any of the TLR agonist-treated group (resiquimod or poly-ICLC, nominal t-test p value?=?0.05) with respect to the placebo were tested for significant overlap with gene ontology and known gene sets using the web-based tools, ENRICHR57.
To calculate single sample gene set enrichment of the interferon-related genes, we used the Gene Set Variation Analysis (GSVA) package (version 1.32.0)58. To compute the GSVA scores, the filtered, log2 normalized gene expression were supplied to the GSVA program using the ‘kcdf=Gaussian’ mode. We manually selected gene sets that are related to interferon pathway activation from the c2.cgp, c6, c7, and hallmark geneset collections of the Broad Institute’s Molecular Signatures Database (version 7.0)59.
Single-cell RNA-seq sample processing and data analysis
The cells for scRNAseq analysis were resuspended in PBS at a concentration of 1,000 cells/µl. We only selected representative patients from each treatment group whose PBMC quality were sufficient for single-cell RNAseq processing. Cell preparation, library preparation, and sequencing were carried out according to Chromium product-based manufacturer protocols (10X Genomics), targeting for a total of 10,000 cells sequenced. Single-cell RNA sequencing was carried out on a Novaseq 6000 S2 2 x 50?bp flow cell (Illumina) utilizing the Chromium single cell 3’ gene expression library preparation (10X Genomics).
The data was aligned with Cell Ranger (version 3.1.0) and aligned to the Genome Reference Consortium Human Build 38 (GRCh38). Data was imported into R (version 4.2.1) and analyzed with the Seurat package (version 4.2.0)60. For quality assurance, cells with greater than 20% mitochondrial features were excluded from further analysis. We analyzed a total of 99,590 cells after the QC step. The Seurat data object from each sample were then integrated and scaled, regressing out the percent mitochondrial features and cell cycle score difference, as described (https://satijalab.org/seurat/index.html). We manually identified each cluster using the genes that were differentially expressed as determined by FindAllMarker function; they are visualized using R’s ggplot2 and pheatmap packages. Differentially expressed genes (DEGs) corresponding to each treatment group (Placebo vs. Poly-ICLC vs. resiquimod) were computed by first computing cluster-specific DEGs between each group against the pre-treatment (Day 0) samples. To account for intrapatient correlation among cells from the same patient, we computed the DEGs using the FindAllMarker function setting the use.method parameter to MAST and the latent.vars parameter to the patient IDs. The union of cluster-specific DEGs that were seen in at least 25% of all comparisons (the total number of comparisons is the number of treatment groups (3 groups) times the number of lymphoid or myeloid clusters) were selected as recurrent DEGs shown in the heatmaps of Fig. 2F and G.
Statistical analysis
For the percentage comparisons in the CyTOF analysis, we used the Wilcoxon rank sum test for non-parametric data for 2 independent samples and compared the baseline (Day 0) to Day 1 or Day 29. We performed Fisher’s exact test for testing the null of independence of the phenotypic and genotypic characteristics and treatments using the stats package in R. Differences in transcript expression log2 fold changes and GSVA scores in the bulk RNA-seq data were calculated with unpaired T test with nonequal variances (two-sided Welch t test). The differences in overall survival or time to progression following treatment (either combination of ATL-DC and placebo, ATL-DC and adjuvant poly-ICLC or ATL-DC and adjuvant resiquimod treatment) were compared using the log-rank test and graphical evaluation of these curves were assessed using the methods of Kaplan and Meier (survminer R package). We further performed multivariable cox proportional hazard (cox PH) regression analysis with HRs (95% CIs) to determine if any of the treatment regimens were significantly predictive of overall survival or time to progression after adjusting for clinical covariates, such as WHO grade, number of recurrences, and MGMT status. The association between interferon pathway score and overall survival or time to progression was analyzed similarly using log-rank (univariate) and Cox PH (multivariate) analyses.
Data availability
Bulk and single-cell RNA sequencing data are available at Gene Expression Omnibus under accession ID GSE237581. The CyTOF data is uploaded to flowRepository with accession ID FR-FCM-Z6LY.
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Acknowledgements
This study was funded in part by the National Institutes of Health NIH/NCI grant (R01CA123396), the UCLA SPORE in Brain Cancer (P50CA211015), NIH National Center for Advancing Translational Science - UCLA CTSI (UL1TR001881), the Parker Institute for Cancer Immunotherapy, and the Brain Tumor Funder’s Collaborative. Mass cytometry was performed in the UCLA Jonsson Comprehensive Cancer Center (JCCC) Flow Cytometry Core Facility that is supported by NIH award P30 CA016042. W.H is supported by the NIH/NCI grant (1R01CA236910), Jonsson Comprehensive Cancer Center and the Parker Institute for Cancer Immunotherapy at UCLA. L.S. was supported by a Career Enhancement Program award from the UCLA SPORE in Brain Cancer. A.L. was supported by the UCLA Tumor Immunology Training Grant (USHHS Ruth L. Kirschstein Institutional National Research Service Award # T32 CA009120). L.D is supported by grant from Parker Institute for Cancer Immunotherapy at UCLA and a postdoctoral fellowship from National Cancer Center.
Author information
Author notes
These authors contributed equally: Richard G. Everson, Willy Hugo.
Authors and Affiliations
Department of Neurosurgery, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, CA, 90095, USA
Richard G. Everson, Lu Sun, Joseph Antonios, Alexander Lee, Sarah Khattab, Emma Billingslea-Yoon, Linda M. Liau & Robert M. Prins
Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, CA, 90095, USA
Richard G. Everson, Willy Hugo, Benjamin M. Ellingson, Linda M. Liau & Robert M. Prins
Department of Medicine, Division of Dermatology, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, CA, 90095, USA
Willy Hugo, Lizhong Ding & Melissa Bu
Parker Institute for Cancer Immunotherapy, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, CA, 90095, USA
Willy Hugo & Robert M. Prins
Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, CA, 90095, USA
Alexander Lee, Carolina Chavez, Timothy F. Cloughesy & Robert M. Prins
Oncovir, Inc., Winchester, VA, USA
Andres Salazar
Department of Radiological Sciences, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, CA, 90095, USA
Benjamin M. Ellingson
Department of Neurology/Neuro-Oncology, David Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, CA, 90095, USA
Timothy F. Cloughesy
Contributions
R.M.P., L.M.L, A.S., and T.F.C. designed the clinical study. L.M.L., R.G.E., R.M.P., E.B-Y, collected clinical data. W.H., L.S. designed the overall computational analyses. L.D. and W.H. analyzed the bulk RNA-seq. L.S., A.L., R.G.E. M.B., S.K. and C.C. performed single-cell RNA-seq and CyTOF analyses. B.M.E, J.A., and R.G.E. analyzed the MRI data. R.G.E., W.H., L.M.L., and R.M.P. wrote and edited the manuscript. All authors reviewed and approved the manuscript.
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Correspondence to Linda M. Liau or Robert M. Prins.
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Andres Salazar is the Founder, CEO and Scientific Director for Oncovir, which provided the TLR agonist (Poly-ICLC) used in the trial. Linda M. Liau is a member of the Scientific Advisory Board for Northwest Bioetherapeutics, Inc, which has licensed the DC vaccine technology. All others declare no competing interests.
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Everson, R.G., Hugo, W., Sun, L. et al. TLR agonists polarize interferon responses in conjunction with dendritic cell vaccination in malignant glioma: a randomized phase II Trial. Nat Commun 15, 3882 (2024). https://doi.org/10.1038/s41467-024-48073-y
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NATURE 5/8/24:
"While malignant gliomas are usually conceived of as a locoregional disease with essentially no capacity to spread outside the central nervous system, there has been a growing understanding of the role that systemic tissues play in priming, developing and/or suppressing an immune response in the brain. The catalog of known pervasive systemic immune deficits in glioblastoma patients is continually growing40. The failure of immune checkpoint inhibitor therapy in malignant gliomas has led many to conclude that immune cells in the tumor microenvironment of cancers unresponsive to these checkpoint inhibitors may exist in an irreversible, terminally exhausted state41,42. The generation of de novo tumor antigen-specific immune responses in the periphery that lead to new T-cell infiltration into the tumor microenvironment may be required to overcome this barrier43. Dendritic cell vaccines are a robust example of an agent capable of mediating the initiation of such a T-cell response.
https://www.researchsquare.com/article/rs-3287211/v1 (pre-print)
https://www.nature.com/articles/s41467-024-48073-y (5/8/24)
https://gritdaily.com/cancer-therapy-oncovirs-hiltonol/
The two links below are the preprint(9/23) and today's final publication.Does anyone have access to a highlight machine which can highlight in yellow text deletions and the highlight in red additions ?
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https://www.nature.com/articles/s41467-024-48073-y (5/8/24)
PREPRINT:
Abstract
Autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination is a promising immunotherapy for patients with high grade gliomas, but responses have not been demonstrated in all patients. To determine the most effective combination of autologous tumor lysate-pulsed DC vaccination, with or without the adjuvant toll-like receptor (TLR) agonists poly-ICLC or resiquimod, we conducted a Phase 2 clinical trial in 23 patients with newly diagnosed or recurrent WHO Grade III-IV malignant gliomas. We then performed deep, high-dimensional immune profiling of these patients to better understand how TLR agonists may influence the systemic immune responses induced by ATL-DC vaccination. Bulk RNAseq data demonstrated highly significant upregulation of type 1 and type 2 interferon gene expression selectively in patients who received adjuvant a TLR agonist together with ATL-DC. CyTOF analysis of patient peripheral blood mononuclear cells (PBMCs) showed increased expression of PD-1 on CD4+ T-cells, decreases in CD38 and CD39 on CD8+ T cells and elevated proportion of monocytes after ATL-DC + TLR agonist administration. In addition, scRNA-seq demonstrated a higher expression fold change of IFN-induced genes with poly-ICLC treatment in both peripheral blood monocytes and T lymphocytes. Patients who had higher expression of interferon response genes lived significantly longer and had longer time to progression compared to those with lower expression. The results suggest that ATL-DC in conjunction with adjuvant poly-ICLC induces a polarized interferon response in circulating monocytes and specific activation of a CD8+ T cell population, which may represent an important blood biomarker for immunotherapy in this patient population.
TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01204684
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Dendritic Cell Vaccination in Conjunction with a TLR Agonist Polarizes Interferon Immune Responses in Malignant Glioma Patients
Robert Prins, Richard Everson, Willy Hugo, Lu Sun, Joseph Antonios, and 10 more
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Abstract
Autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination is a promising immunotherapy for patients with high grade gliomas, but responses have not been demonstrated in all patients. To determine the most effective combination of autologous tumor lysate-pulsed DC vaccination, with or without the adjuvant toll-like receptor (TLR) agonists poly-ICLC or resiquimod, we conducted a Phase 2 clinical trial in 23 patients with newly diagnosed or recurrent WHO Grade III-IV malignant gliomas. We then performed deep, high-dimensional immune profiling of these patients to better understand how TLR agonists may influence the systemic immune responses induced by ATL-DC vaccination. Bulk RNAseq data demonstrated highly significant upregulation of type 1 and type 2 interferon gene expression selectively in patients who received adjuvant a TLR agonist together with ATL-DC. CyTOF analysis of patient peripheral blood mononuclear cells (PBMCs) showed increased expression of PD-1 on CD4+ T-cells, decreases in CD38 and CD39 on CD8+ T cells and elevated proportion of monocytes after ATL-DC + TLR agonist administration. In addition, scRNA-seq demonstrated a higher expression fold change of IFN-induced genes with poly-ICLC treatment in both peripheral blood monocytes and T lymphocytes. Patients who had higher expression of interferon response genes lived significantly longer and had longer time to progression compared to those with lower expression. The results suggest that ATL-DC in conjunction with adjuvant poly-ICLC induces a polarized interferon response in circulating monocytes and specific activation of a CD8+ T cell population, which may represent an important blood biomarker for immunotherapy in this patient population.
TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01204684
Biological sciences/Cancer/CNS cancer
Biological sciences/Immunology/Tumour immunology
brain cancer
immunotherapy
glioma
TLR agonist
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Dendritic Cell Vaccination in Conjunction with a TLRAgonist Polarizes Interferon Immune Responses inMalignant Glioma PatientsRobert Prins David Geffen School of Medicine at UCLA https://orcid.org/0000-0002-6282-6583Richard Everson University of California, Los Angeles https://orcid.org/0000-0003-2809-3061Willy Hugo UCLA https://orcid.org/0000-0002-1426-7190Lu Sun David Geffen School of Medicine at UCLAJoseph Antonios David Geffen School of Medicine at UCLAAlexander Lee University of California Los Angeles https://orcid.org/0000-0002-4583-0740Lizhong Ding University of California Los AngelesMelissa Bu University of California Los AngelesSara Khattab University of California Los AngelesCarolina Chavez University of California, Los AngelesEmma Billingslea-Yoon University of California, Los AngelesAndres Salazar Oncovir (United States)Benjamin Ellingson UCLATimothy Cloughesy https://orcid.org/0000-0002-8656-7483Linda Liau
UCLA https://orcid.org/0000-0002-4053-0052ArticleKeywords: brain cancer, immunotherapy, glioma, TLR agonistPosted Date: September 12th, 2023DOI:https://doi.org/10.21203/rs.3.rs-3287211/v1License:?? This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full LicenseAdditional Declarations:Yes there is potential Competing Interest. Dr. Salazar is an employee of Oncovir,Inc., which makes poly ICLC (Hiltonol).Version of Record: A version of this preprint was published at Nature Communications on May 8th, 2024.See the published version at https://doi.org/10.1038/s41467-024-48073-y.
Dendritic Cell Vaccination in Conjunction with a TLR Agonist Polarizes Interferon Immune Responses in Malignant Glioma PatientsRichard G. Everson 1,2, 9, Willy Hugo 2,3,4,9, Lu Sun1, Joseph Antonios 1, Alexander Lee1,5, Lizhong Ding3, Melissa Bu3, Sarah Khattab1, Carolina Chavez 5, Emma Billingslea-Yoon 1, Andres Salazar6, Benjamin M. Ellingson 2,7, Timothy F. Cloughesy 5,8, Linda M. Liau 1,2,* and Robert M. Prins 1,2,4,5,*1Department of Neurosurgery2Jonsson Comprehensive Cancer Center 3Department of Medicine, Division of Dermatology4Parker Institute for Cancer Immunotherapy5Department of Molecular and Medical Pharmacology6Oncovir, Inc., Winchester, VA7Department of Radiological Sciences8Department of Neurology/Neuro-Oncology9Richard Everson and Willy Hugo contributed equally to this work as first authorsDavid Geffen School of Medicine at UCLA, University of California Los Angeles, Los Angeles, California, 90095, U.S.A. *Correspondence: Robert M. Prins, Ph.D.: RPrins@mednet.ucla.eduor
Linda M. Liau, M.D., Ph.D.: lliau@mednet.ucla.eduRunning Title: Dendritic cell vaccination and TLR agonists for malignant gliomaAbbreviations: GBM, Glioblastoma; TLR, toll-like receptor; DC, dendritic cellKey words: brain cancer, immunotherapy, glioma, TLR agonist
ABSTRACT (Short)Autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccination is a promising immunotherapy for patients with high grade gliomas, but responses have not been demonstrated in all patients. To determine the most effective combination of autologous tumor lysate-pulsed DC vaccination, with or without the adjuvant toll-like receptor (TLR) agonists poly-ICLC or resiquimod, we conducted a Phase 2 clinical trial in 23 patients with newly diagnosed or recurrent WHO Grade III-IV malignant gliomas. We then performed deep, high-dimensional immune profiling of these patients to better understand how TLR agonists may influence the systemic immune responses induced by ATL-DC vaccination. Bulk RNAseq data demonstrated highly significant upregulation of type 1 and type 2 interferon gene expression selectively in patients who received adjuvant a TLR agonist together with ATL-DC. CyTOF analysis of patient peripheral blood mononuclear cells (PBMCs) showed increased expression of PD-1 on CD4+ T-cells, decreases in CD38 and CD39 on CD8+ T cells and elevated proportion of monocytes after ATL-DC + TLR agonist administration. In addition, scRNA-seq demonstrated a higher expression fold change of IFN-induced genes with poly-ICLC treatment in both peripheral blood monocytes and T lymphocytes.Patients who had higher expression of interferon response genes lived significantly longer and had longer time to progression compared to those with lower expression. The results suggest that ATL-DC in conjunction with adjuvant poly-ICLC induces a polarized interferon response in circulating monocytes and specific activation of a CD8+ T cell population, which may represent an important blood biomarker for immunotherapy in this patient population. TRIAL REGISTRATION:ClinicalTrials.gov Identifier: NCT01204684INTRODUCTIONThere have been significant advances in our genetic and immunologic understanding of primary brain tumors, such as malignant gliomas. Yet, it has still proven difficult to improve long-term
outcomes in patients using standard of care therapies1. We and others have demonstrated that autologous tumor lysate (ATL) dendritic cell (DC) vaccination can induce local and systemic anti-tumor immune responses in malignant glioma patients, and clinical trials have suggested that this may extend survival in this deadly condition2-6. However, variable response rates in cancer immunotherapy trials have prompted the search for potential strategies to enhance theimmune effects of dendritic cell vaccines. In particular, agonists of a family of pattern-recognition receptors (PRR) called Toll-like receptors (TLR)7- 10, which appear capable of activating of antigen-presenting cells (i.e., dendritic cells), enhancing T-cell priming, and decreasing myeloid-derived suppressor cells (MDSC), are strong candidates for use in combination with ATL-DC vaccination to potentially enhance the anti-tumor immune response.10,11TLR3 is an intracellular PRR that recognizes double-stranded RNA (dsRNA), usually associated with viral infection, and induces high levels of IFN-a/ß and pro-inflammatory cytokines when activated. TLR-3 is predominantly expressed by macrophages, plasmacytoid DC and myeloid DC 12,13, but also by microglial cells 14,15. It has also been shown that astrocytes16-18 and malignant gliomas19 also respond similarly to TLR3-induced signaling. Polyinosinic acid-polycytidylic acid stabilized with polylysine (poly-ICLC) is a multi-dimensional synthetic dsRNA analogue and viral mimic that signals via TLR3, MDA5 and other dsRNA-dependent PRR signaling, induces type I-II IFNs20,21, promotes the infiltration of effector T cells in pre-clinical glioma models22, and upregulates genes associated with chemokine activity, T-cell activation, and antigen presentation23. Poly-ICLC has been tested as a single-agent therapeutic for multiple malignancies24, including malignant glioma patients 25, in whom it has demonstrated adequatesafety, but limited survival benefit in combination with standard therapies26. Similarly, TLR7 and TLR8 are other intracellular PRRs that recognize single-stranded RNA (ssRNA), which subsequently induces proinflammatory cytokines, chemokines, and type I
interferons (IFNs) 27. In pre-clinical work, we previously demonstrated that DC injected into imiquimod (TLR7 agonist)-pre-treated sites acquired lymph node migratory capacity and enhanced T-cell priming 28. Our early phase clinical trials demonstrated that DC vaccination with adjuvant topical imiquimod, a TLR-7 agonist, was feasible and safe in glioblastoma patients 3. Resiquimod is a newer imidazoquinoline agonist that shows enhanced transdermal delivery, activates TLR7/8 to enhance T-cell responses and TH1-type cytokine secretion by DC 29- 32, and may havegreaterpotency as an immune modulator.In this study, we report the long-term results of 23 malignant glioma patients enrolled in a phase II randomized clinical trial designed to compare the safety, immune responses, and potentialefficacy of ATL-DC vaccination combined with placebo, poly-ICLC, or resiquimod. Post-hoc analysis using cytometry by time-of-flight (CyTOF) and bulk and single-cell RNA sequencing (scRNAseq) technologies were used to detect the cellular and molecular immune signatures from peripheral blood mononuclear cells (PBMCs) pre- and post-treatment.
RESULTSPatient Characteristics and SafetyA total of 23 patients with WHO grade III or IV glioma were enrolled and randomized between September 2010 and August 2014. All patients received ATL-DC vaccination. Nine patients were randomized into the adjuvant TLR-7 /8 agonist (r esiquimod, 3M) group, nine into the adjuvant TLR-3 agonist (poly-ICLC, Oncovir) group, and five received adjuvant placebo (Figure 1A , Supplementary Figure 1). All patients were followed for survival, imaging changes, as well as high dimensional, in-depth systemic immune monitoring. Baseline patient characteristics are presented and segregated by treatment group in Table 1 (see also Supplementary Table 1). The median age was 46.6 (S.D. 11.9) years and 57% of the enrolled patients were male. 65 % (n=15) had diagnoses of IDH wild type glioblastoma (WHO Grade IV), while 35 % (n=8) of the patients had a diagnosis of IDH mutant malignant glioma (WHO Grade III). 52% (n=12) of patients were treated following recurrence, while 48% (n=11) were treated in the newly diagnosed setting. All patients were treated following surgical resection and standard of care treatment. The molecular characteristics of the patient tumors are outlined in Table 1. Overall, MGMT methylation was seen in 35% (n=8), IDH mutations were observed in 35% (n=8, all grade III), and EGFR amplification was seen in 44% (n=10, all glioblastoma) of patients, consistent with the heterogenous population of malignant glioma patients. There were no statistic ally significant differences in age, sex, Karnofsky performance status, MGMT methylation status, pre- or post-surgery enhancing tumor volume, nor steroid administration at enrollment. No statistically significant differences were observed between the molecular characteristics, although the number of patients in each treatment group was small. Overall, the addition of a TLR agonist induced only Grade 1-2 treatment-related adverse events (TRAEs), and all adverse events reported resolved without further treatment or hospitalization (Table 2). The most common TRAEs were rash (39%), fever (35%), and fatigue (26%; see
Table 2), and were more common in patients treated with resiquimod and poly-ICLC. Other observed adverse events were not uncommon in the setting of post-operative central nervous system (CNS) tumor treatment. Additionally, 88.9% of patients who received resiquimod reported a temporary localized, cutaneous rash that resolved without further treatment. However, no serious adverse events (Grade 3-4) attributable to the treatment were observed. As such, the addition of a TLR agonist to ATL-DC vaccination in malignant glioma patients was found to be safe and tolerable. Adjuvant TLR agonist treat ment induces systemic exp ression of type I and type II interferon downstream g ene s. The primary scientific endpoint of this clinical trial was to evaluate the systemic immune response changes induced by ATL-DC vaccination with and without TLR agonist administration. As such, we collected PBMCs at baseline (pre-treatment), one day after the vaccination (on treatment), and then following the completion of the treatment cycle (post-treatment) of each patient (Figure 1A). We aimed to understand how the adjuvant administration of TLR agonists modified the immune response in comparison with ATL-DC vaccination alone (placebo control).We first performed paired bulk RNA-seq on patient-matched, pre-treatment and post-treatmentPBMC samples. For each gene, we computed the difference between its expression in the pre- and post- samples of patients in each treatment group: ATL-DC+placebo ( n=5 pairs); ATL-DC+poly-ICLC ( n=8 pairs); ATL-DC+resiquimod ( n=8 pairs) ; for brevity, we refer to them as placebo, poly-ICLC and resiquimod, respectively. To identify expression changes specific to the TLR agonist groups, we identified genes whose average upregulation in the TLR agonist pairs (poly-ICLC or resiquimod) were at least two-fold higher than the placebo pairs (Figure 1B , Supplementary Table 2A, see Methods).
Genes upregulated in the TLR agonist groups were involved in antigen processing and wereenriched with known interferon stimulated genes (ISGs) (Figure 1C-E , Supplementary Table 2B-C). This observation was also confirmed by per-sample gene set enrichment analysis, where the TLR agonist-treated groups displayed higher enrichment of type I and II interferon downstream gene sets compared to ATL-DC/placebo (Figure 1F , Supplementary Table 2D-E). PBMC samples with higher absolute enrichment scores of interferon gene sets were dominated by post-treatment samples from both grade III and IV glioma patients in the TLR-agonist treated groups (Figure 1G). We noted that the resiquimod group had a more heterogenous response, which resulted in a lower degree of statistical significance compared to that of poly-ICLC group. Nonetheless, the two TLR agonist-treated groups showed a largely similar trend in treatment-induced gene expression changes, which included a measurable increase in the expression of ISGs in the peripheral blood of malignant glioma patients.TLR agonist treatment induces systemic T cell activation, monocyte proliferation and interferon responses in myeloid and lymphoid populations.We performed CyTOF on PBMC timepoints with a 27- marker heavy metal antibody-conjugated panel for 20 of the 23 patients ( placebo, n=4 pairs; poly-ICLC, n=9 pairs; resiquimod, n=7 pairs; see Supplementary Table 3A, B). The panel was selected to be able to broadly characterize different immune cell types, activation/effector, memory and exhaustion phenotypes, with a bias towards T-cell relevant markers. The different immune cell type populations were visualized by the uniform manifold approximation and projection (UMAP) method (Figure 2A), which we broadly assigned to seven different major immune populations based off the normalized heatmap marker expression (Figure 2B). After 3 cycles of treatment, the post-treatment samples of patients in the TLR agonist groups showed a significant increase in the proportion of proliferating Ki67+ CD14+ classical
monocytes (Figure 2C, Supplementary Table 3C). Such findings were supported by the increased monocyte fraction and CD14 transcript expression after ATL-DC+TLR agonist-treated samples (Supplementary Figure 2A, B, Supplementary Table 3D). ATL-DC+TLR agonist treatment induced PD-1 expression in CD4 T cell population and increased the T-cellnormalized expression of PDCD1 ( the transcript that encodes PD-1 protein) (Figure 2D, Supplementary Figure 2C). Moreover, expression of markers associated with irreversible T cell exhaustion, such as CD38 and CD3933,34, were also reduced after ATL-DC+TLR agonist treatment (Figure 2D, Supplementary Figure 2D). Increased expression of PD-1 and decreased expression of CD38 and CD39 suggest the ATL-DC+TLR agonist combination therapy can improve systemic T cell activity and cellular fitness in the patient.To delineate the changes induced by ATL-DC and TLR agonist treatment in discrete peripheral blood immune cell subsets, we performed single cell RNA-seq on selected patients at baseline and then following the completion of therapy. We analyzed two representative sample pairs from each cohort (placebo, poly-ICLC, and resiquimod) (Supplementary Table 3E). We identified a total of twelve clusters from the total PBMC immune cell population and annotated these clusters based on differentially expressed gene markers in each cluster. From the initial clustering, we were able to identify multiple populations of CD4+ and CD8+ T cells, two populations of NK cells, three monocytic cell populations, B cell, and dendritic cells (type 2 conventional dendritic cells (cDC2) and plasmacytoid dendritic cells (pDCs), in accordance withthe previous characterization of these cell types in peripheral blood (Figure 2D and Supplementary Figure 2E, F). Differential gene expression analysis across the different lymphoid and myeloid populations revealed concordant upregulation of known ISGs (e.g. IFI6/35/44L, ISG15/20, IFIT3, IFITM1/3,
GBP1/5, MX1, STAT1 and CXCL10) and antigen presentation related proteasomes (PSMB9and PSME2) in both TLR agonist sample pairs. The induction was weaker in the paired PBMC samples obtained from the resiquimod group compared to the poly-ICLC group (Figure 2F, G). Thus, our combination of high dimensional proteomics, bulk and single cell RNAseq demonstrates how adjuvant TLR administration in conjunction with ATL-DC reproducibly increases the proportion of canonical CD14+ monocytes within the systemic blood circulation. This TLR agonist administration was also associated with enhanced T cell activity, coupled with decreased expression of CD38 and CD39 and their downstream T cell-suppressive adenosine pathway 33-35 . ATL-DC+TLR agonist-driven induction of ISGs across lymphoid and myeloid populations identified in our scRNAseq analysis corroborated our bulk transcriptomic analysis.Given the consistent systemic changes observed with TLR agonist administration, we wondered if there were survival differences between these patient populations.Long-term clinical outcomes of malignant glioma patients treated with ATL-DC vaccination plus adjuvant TLR agonists. Median follow-up of patients treated on this clinical trial was 2.2 years after surgery, although the long-term survivors have now been followed for over 10 years. Median progression-free survival (PFS) was 8.1 months; and median overall survival (OS) was 26.6 months. Although this clinical trial was not designed to be statistically powered to detect changes in survival between the treatment groups, we nonetheless noted statistically significant differences in median survival between the treatments groups for both OS (placebo: 7.7 months, poly-ICLC: 52.5 months, and resiquimod: 16.7 months; log-rank P=0.017) and PFS (placebo: 5.5 months, poly-ICLC: 31.4 months and resiquimod: 8.1 months; log-rank P=0.0012) (Figure 3A). Because the trial included patients with both grade III and IV tumors, we stratified our analysis based on tumor grade. When we analyzed only the grade IV (GBM) patients, we observed a trend towards improved PFS (log-
rank P=0.068) and OS (P not significant) (Figure 3B). Interestingly, for the IDH mutant/Grade III cohort, all four patients that received ATL-DC + poly-ICLC treatment are still alive at the data cutoff date (three of the patients have survival > 120 months and one > 112 months), and they have significantly longer OS and PFS compared to the other grade III patients who received ATL-DC + resiquimod or ATL-DC alone (Figure 3C). We performed multivariate Cox proportional hazard (PH) analysis, adjusting for clinical variables that are significantly correlated with OS or PFS as a single variable (tumor grade, MGMT methylation status, and number of recurrences). Our analysis confirmed that patients in either the poly-ICLC or resiquimod treatment group had a lower risk of progression that wasindependent of grade, MGMT methylation, and number of recurrences (Figure 3D). Risk of death was significantly lower in the poly-ICLC group, while the resiquimod group showed a similar trend that was not statistically significant (Supplementary Figure 3A). In the GBM patient subset, TLR agonist treatment also significantly lowered risk of recurrence, but not risk of death (Figure 3E, Supplementary Figure 3B). To determine whether this treatment directly impacted tumor volume, MR imaging was performed, and contrast-enhancing tumor volume was quantified over time. We noted that the rate of tumor volume increase over time in the ATL-DC/placebo treatment cohort was higher than in the ATL-DC/resiquimod treatment (p=0.022) and the ATL-DC/poly-ICLC treatment groups (P < 0.001; Figure 3F). Interestingly, we observed an increased T2/FLAIR MRI signal after completion of the vaccine series two of the four long-term survivors who received ATL-DC/poly-ICLC (Supplementary Figure 3C-D), but such findings are potentially confounded by prior radiation therapy, and thus we cannot ascribe such changes solely to the vaccine/TLR agonist intervention. However, this increased post-vaccination T2/FLAIR on MRI was not seen in patients who did not receive poly-ICLC (not shown).
Interferon activation score in the peripheral blood immune cells is a significant predictor of survival after ATL-DC therapy.Finally, we asked if the magnitude of interferon pathway induction by the adjuvant TLR agonist treatment directly correlated with OS or PFS. This could allow for the use of an interferon activity score as a biomarker for productive anti-tumor immune responses following ATL-DC immunotherapy. To this end, we stratified the patients by the median GSVA score of the “HALLMARK INTERFERON GAMMA RESPONSE” gene set in their p ost-treatment PBMC samples. We confirmed that patients whose post-treatment samples displayed higher interferon gene set scores (= median) have longer OS and PFS than those with lower scores (Figure 4A , Supplementary Figure 4A). Separate analyses on the grade IV (GBM) and grade III glioma patients showed a concordant trend but with a lower degree of statistical significance; this was likely caused by the small sample sizes. Notably, multivariate Cox PH analysis confirmed that the interferon gene set score is a significant predictor of tumor recurrence (Figure 4B, C) and death (Supplementary Figure 4B). To ensure that the correlation is not specific to this single gene set, we confirmed that the gene set scores of other interferon gene sets after ATL-DC treatment are also positively correlated with the patients OS and PFS (Supplementary Table 4A, B). Taken together, these data suggest that the addition of TLR agonists to ATL-DC vaccination shifts towards an interferon-induced immune response in both lymphoid and myeloid cells. The magnitude of this induction may be correlated with the systemic immune response and clinical outcome.DISCUSSION
We report herein that ATL-DC vaccination with adjuvant poly-ICLC or resiquimod is overall safe and well-tolerated in patients with malignant glioma. To achieve the primary immunological endpoints of this study, we utilized high-dimensional single-cell analysis to understand the systemic proteomic and transcriptomic changes induced by agonists of TLRs and other PRR in order to rationally determine the optimal therapeutic combination. Our study is the first high dimensional single-cell analysis done in a clinical trial for malignant glioma patients treated with dendritic cell vaccination and TLR agonists. Although our study was not designed to examine what happens in the tumor microenvironment, our results indicate that we are able to sensitively detect systemic changes in the blood after intradermal autologous dendritic cell vaccination with and without TLR agonists. Adjuvant TLR agonist treatment promotes the expression of IFNa/ß and IFNg-induced genes on the peripheral lymphoid and myeloid cells, and GSEA further confirmed increased expression of the IFNa and IFN? downstream genes, including IFNa/ß-induced proteins ISG15 and STAT1. ISG15 stimulates IFNg from lymphocytes 36 and negatively regulates IFNa/ß signaling 37, and type I IFN maintenance of STAT1 expression induces IFN? signaling 38. Other genes that were significantly upregulated by TLR agonist treatment include PARP9-DTX3L, and this heterodimeris also known to amplify interferon signalling.39 Our results support the conclusion that DC vaccination with poly-ICLC induces Type I and Type II IFN responses more effectively than withadjuvant resiquimod or a dendritic cell vaccine alone. Similar to our results, additional studies have identified poly-ICLC as the most effective TLR/PRR agonist when compared with others40,41. The downstream effect of this signaling in the lymphoid compartment appears to be increased T-cell activity, as well as decreased T cell exhaustion phenotype. Together, these effects may enhance of the activity of tumor antigen specific T cells generated from an activevaccine.
It is also important to recognize that, in contrast to resiquimod and even plain poly-IC, poly-ICLC signals through various PRRs in addition to TLR3, consistent with its role as an authentic viral mimic. The poly-lysine stabilizer also functions as a transfection agent. Specifically, poly-lysinebursts the endosome through a proton sponge effect and releases the dsRNA into the cytoplasm, where it then preferentially activates MDA5, OAS, PKR and other cytoplasmic dsRNA dependent systems 21. Among the actions generated putatively through MDA5 are a further increase in Type 1 IFN, depression of MDSC, expansion of CD8 T cell populations through IL-15, CD8 targeting and infiltration of tumor through CXCL10, and a direct Type 1 IFN-dependent effect on tumor endothelium through VCAM-1 42. These effects are best seen with systemic (intramuscular or intravenous) rather than local (subcutaneous) administration, as we have done in the current study. Such adjuvant responses induced by poly ICLC may play a role in the longer-term maintenance of the immune responses generated by ATL-DC vaccination, but further studies are required to verify these findings. While malignant gliomas are usually conceived of as a locoregional disease with essentially no capacity to spread outside the central nervous system, there has been a growing understanding of the role that systemic tissues play in priming, developing and/or suppressing an immune response in the brain. The catalog of known pervasive systemic immune deficits in glioblastoma patients is continually growing 43. The failure of immune checkpoint inhibitor therapy in malignant gliomas has led many to conclude that immune cells in the tumor microenvironment of cancers unresponsive to these checkpoint inhibitors may exist in an irreversible, terminally exhausted state44,45. The generation of de novo tumor antigen-specific immune responses in the periphery that lead to new T-cell infiltration into the tumor microenvironment may be required to overcome this barrier46. Dendritic cell vaccines are a robust example of an agent capable of mediating the initiation of such a T-cell response.
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Chiugray
Re: TiltMyBrain post# 689739
Wednesday, May 08, 2024 11:26:29 PM
Post#
689874
of 689913
TiltMyBrain, DCVax is about to give a knockout 1-2 punch against brain tumors and will mark a historic moment to the joy of cancer patients and oncologists.
1. Regulatory approvals (first UK, then globally) DCVax-L for stage 4 brain tumors/GBM
(replaces standard of care, generates robust immune memory, potential for cure, non-toxic)
2. Validation for off-label to add Poly-ICLC (adjuvant to DCVax-L therapy) for stage 3 brain tumors
(UCLA phase 2 clinical trial results, Nature scientific publication)
Excerpts from this Nature publication on the UCLA Phase 2 clinical trial:
- "results support the conclusion that DC vaccination with poly-ICLC induces Type I and Type II IFN responses more effectively than with adjuvant resiquimod or a dendritic cell vaccine alone"
- "combination of ATL-DC vaccination and TLR agonist was safe and found to enhance systemic immune responses"
- "placebo: 7.7 months, poly-ICLC: 52.5 months"
- "for the IDH mutant/Grade III cohort, all four patients that received ATL-DC + poly-ICLC treatment are still alive at the cutoff date (three of the patients have survival >120 months and one > 112 months), and they have significantly longer OS and PFS compared to... ATL-DC + resiquimod or ATL-DC alone"
Off-label drug use is common in cancer treatment because
- many cancer drugs are effective against more than one type of cancer
- cancer treatment often involves the use of combination chemotherapy
...
The FDA usually does not approve combinations of chemotherapy. There are so many of them that it would not be practical to approve each combination.
Health insurance coverage of off-label drugs in cancer treatment
Medicare and many insurance companies pay for off-label drugs for cancer treatment, as long as the off-label uses are listed in an approved compendium. A compendium is a collection of drug summaries put together by experts who have reviewed data about the drug’s use in patients.
https://www.cancer.gov/about-cancer/treatment/drugs/off-label
maverick_1
Re: maverick_1 post# 688732
Thursday, May 09, 2024 6:31:15 AM
Post#
689911
of 689914
The MAESTRO’s 1st TRIFECTA Set on SAME Day🔥🙏
Time’ s up to STOP LENDING YOUR Shares
MHRA approval before? ASCO 2024.
Merge RevImmune: NAKED no longer
COLLABORATIONS PARTNERSHIPS
UPLIST
VC $FUNDING MILESTONES HIT
dstock07734
Re: iclight post# 689884
Thursday, May 09, 2024 12:18:25 AM
Post#
689889
of 689915
BTW, I mentioned Professor Lisa Butterfield. I think you didn't pay any attention.
Professor Lisa Butterfield is a well-renowned scientist on dendritic cell vaccine too. She used to be the chair of Cellular, Tissues and Gene Therapies Advisory Committee of FDA for four years before she joined Merck last March. She ran a clinical trial using her own version of DC vaccine on melanoma. But she didn't make any breakthrough. Together with Antoni Ribas, she published plenty papers dendritic cells and immunotherapy. I really doubt that Merck would develop its own DC vaccine since the patent wall built by LP over the past many years would block any BP from such an attempt.
Immuno-metabolic dendritic cell vaccine signatures associate with overall survival in vaccinated melanoma patients
https://www.nature.com/articles/s41467-023-42881-4
Multiple Antigen-Engineered DC Vaccine for Melanoma
https://clinicaltrials.gov/study/NCT01622933?cond=NCT01622933&rank=1
https://scholar.google.com/scholar?hl=en&as_sdt=0%2C44&q=Lisa+butterfield+%2B+Antoni+Ribas&btnG=
The two links below are the preprint(9/23) and today's final publication.Does anyone have access to a highlight machine which can highlight in yellow text deletions and the highlight in red additions ?
https://www.researchsquare.com/article/rs-3287211/v1 (pre-print)
https://www.nature.com/articles/s41467-024-48073-y (5/8/24)
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