Early signs of clinical activity were observed in adult patients with relapsed/refractory CD22-positive B-cell acute lymphoblastic leukemia (ALL) who were treated with an investigational allogeneic off-the-shelf CD22-directed therapy. Findings from the phase 1 dose-escalation/expansion BALL1-01 study (NCT04150497) of UCART22 were presented during the 2020 ASH Annual Meeting demonstrating safety of the T-cell product across dose levels.1

Two patients achieved a complete remission (CR) with incomplete hematologic recovery on day 28 at the 1 x 105 cells/kg dose level. One of these patients attained a minimal residual disease (MRD)positive CR at day 42 followed by subsequent inotuzumab ozogamicin (Besponsa) and then transplant.

One patient at dose level 2, 1 x 106 cells/kg, experienced a significant bone marrow blast reduction at day 28, followed by disease progression.

No patients experienced dose-limiting toxicities (DLTs), immune effector cellassociated neurotoxicity syndrome (ICANS), graft-versus-host disease (GVHD), adverse effects (AE) of special interest (AESI), a UCART22-related AE that was grade 3 or higher, or a serious AE (SAE).

UCART22 showed no unexpected toxicities at the doses of 1 x 105 cells/kg and 1 x 106 cells/kg with fludarabine and cyclophosphamide lymphodepletion, lead study author Nitin Jain, MD, an assistant professor in the Department of Leukemia, The University of Texas MD Anderson Cancer Center, said in a virtual presentation during the meeting. Host immune recovery was observed early, and the addition of alemtuzumab [Lemtrada] to fludarabine and cyclophosphamide lymphodepletion is currently being explored with the goal to achieve deeper and more sustained T-cell depletion and to promote expansion and persistence of UCART22.

Standard treatment for adult patients with B-cell ALL includes multiagent chemotherapy with or without allogeneic stem cell transplant. However, 30% to 60% of patients with newly diagnosed B-cell ALL who achieve a CR will relapse, and the expected 5-year survival rate for those with relapsed/refractory disease is approximately 10%.

Previously, UCART19, when paired with lymphodepletion using fludarabine, cyclophosphamide, and alemtuzumab, was found to show efficacy in this patient population.2

CD22 is an FDA-approved therapeutic target in B-cell ALL. UCART22 is an immediately available, standardized, manufactured agent with the ability to re-dose, and its CAR expression redirects T cells to tumor antigens, Jain explained.

Moreover, through its mechanism of action, TRAC becomes disrupted using Transcription activator-like effector nucleases (Talen) technology to eliminate TCR from cell surface and reduce the risk of GVHD. CD52 is also disrupted with the use of Talen to eliminate sensitivity to lymphodepletion with alemtuzumab. Finally, there is a CD20 mimotope for rituximab (Rituxan) as a safety switch, Jain added.

UCART22 has also demonstrated in vivo antitumor activity in immune-compromised mice that were engrafted with CD22-positive Burkitt lymphoma cells in a dose-dependent manner.

In the dose-escalation/dose-expansion BALLI-01 study, investigators are enrolling up to 30 patients in a modified Toxicity Probability Interval design. There are 3 cohorts, which have 2 to 4 patients on each cohort: 1 x 105 cells/kg (dose level 1), 1 x 106 cells/kg (dose level 2), and 5 x 106 cells/kg. The focus of the dose-escalation phase of the trial was to determine the maximum-tolerated dose (MTD) and the recommended phase 2 dose (RP2D) before heading into the dose-expansion portion of the trial.

To be eligible for enrollment, patients must have been between 18 and 70 years old, have acceptable organ function, an ECOG performance status of 0 or 1, at least 90% of B-cell ALL blast CD22 expression, and had previously received at least 1 standard chemotherapy regimen and at least 1 salvage regimen.

End points of the trial included safety and tolerability, MTD/R2PD, investigator-assessed response, immune reconstitution, and UCART22 expansion and persistence.

The lymphodepletion regimens were comprised of fludarabine (at 30 mg/m2 x 4 days) plus cyclophosphamide (1 g/m2 x 3 days); the study has since been amended to include the regimen of fludarabine (at 30 mg/m2 x 3 days), cyclophosphamide (500 g/m2 x 3 days), and alemtuzumab (20 mg/day x 3 days) and is currently enrolling patients.

Following screening, lymphodepletion, and UCART22 infusion, patients underwent an observation period for DLTs with a primary disease evaluation at 28 days; additional efficacy evaluations occurred at 56 days and 84 days. Patients were followed for 2 years and continued to be assessed for long-term follow-up.

As of July 1, 2020, 7 patients were screened, of which 1 patient failed and 6 were therefore enrolled on the study. One patient discontinued therapy before receiving UCART22 due to hypoxia from pneumonitis that was linked with lymphodepletion. Five patients were treated with UCART22 at dose level 1 (n = 3) and dose level 2 (n = 2).

The median age of participants was 24 years (range, 22-52), 3 of the 5 patients were male, and 3 had an ECOG performance status of 0. The median number of prior therapies was 3 (range, 2-6), and there were a median 35% bone marrow blasts (range, 10%-78%) prior to lymphodepletion.

Three patients had complex karyotype and 2 had diploid cytogenetics. One patient each had the following molecular abnormalities: CRLF2, CRLF2 and JAK2, CDKN2A loss, KRAS and PTPN11, and IKZF1. Only 1 patient had undergone haploidentical transplant. Four patients previously received prior CD19- or CD22-directed therapy, including blinatumomab (Blincyto), inotuzumab ozogamicin (Besponsa), and CD19-directed CAR T-cell therapy. At study entry, 3 patients had refractory disease and 2 patients had relapsed disease.

Grade 3 or higher treatment-emergent AEs (TEAEs), which were unrelated to study treatment, included hypokalemia, anemia, increased bilirubin, and acute hypoxic respiratory failure. Also not related to UCART22, 3 patients experienced 4 treatment-emergent SAEs: porta-hepatis hematoma, sepsis, bleeding, and sepsis in the context of disease progression. No treatment discontinuations due to a treatment-related TEAE were reported.

The patient who achieved a CR followed by transplant was a 22-year-old male who had undergone 2 prior treatments for B-cell ALL and received UCART22 at a dose of 1 x 105 cells/kg. He did not experience CRS, ICANS, GVHD, nor a SAE, and all TEAEs were grade 1.

Jain also noted that host T-cell constitution was observed in all patients within the DLT observation period. UCART22 was also not detectable through flow cytometry or molecular analysis, the latter of which was at dose level 1 only.

References

1. Jain N, Roboz GJ, Konopleva M, et al. Preliminary results of BALLI-O1: a phase I study of UCART22 (allogeneic engineered T cells expressing anti-CD22 chimeric antigen receptor) in adult patients with relapsed/refractory anti-CD22+ B-cell acute lymphoblastic leukemia (NCT04150497). Presented at: 2020 ASH Annual Meeting and Exposition; December 4-8, 2020; Virtual. Abstract 163.

2. Benjamin R, Graham C, Yallop D, et al. Preliminary data on safety, cellular kinetics and anti-leukemic activity of UCART19, an allogeneic anti-CD19 CAR T-cell product, in a pool of adult and pediatric patients with high-risk CD19+ relapsed/refractory b-cell acute lymphoblastic leukemia. Blood. 2018;132(suppl 1):896. doi:10.1182/blood-2018-99-111356

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UCART22 Safe and Active in CD22-Expressing B-Cell ALLs - Targeted Oncology

Bone Marrow Stem Cells Comments Off on UCART22 Safe and Active in CD22-Expressing B-Cell ALLs – Targeted Oncology | December 6th, 2020

Newswise Researchers from The University of Kansas Cancer Center are involved in the presentation of nearly 50 research studies at the 62ndAmerican Society of Hematology (ASH) Annual Meeting, to be held virtually Dec. 5-8 because of the COVID-19 pandemic. With more than 18,000 members from nearly 100 countries, the ASH is the world's largest professional society serving both clinicians and scientists around the world who are working to conquer blood diseases.

The KU Cancer Center is one of only 71 cancer centers designated by the National Cancer Institute because they meet rigorous standards for transdisciplinary, state-of-the-art research focused on developing new and better approaches to preventing, diagnosing and treating cancer. Its catchment area includes the state of Kansas as well as western Missouri.

These 49 research studies represent the hard work of our many researchers focused on blood diseases, said Roy Jensen, M.D., director of the KU Cancer Center. This includes innovations in immunotherapy, advances in leukemia and significant work in stem cell transplants. While the conference is virtual this year, the KU Cancer Center will be well represented.

While a full list of abstracts involving KU Cancer Center researchers can be found online, three of the most significant are listed below.

# # #

About The University of Kansas Cancer Center:

The University of Kansas Cancer Center is transforming cancer research and clinical care by linking an innovative approach to drug discovery, delivery and development to a nationally-accredited patient care program. Our consortium center includes cancer research and health care professionals associated with the University of Kansas Medical Center and The University of Kansas Health System; the University of Kansas, Lawrence; The Stowers Institute for Medical Research; Childrens Mercy; and in partnership with members of the Masonic Cancer Alliance.

About the University of Kansas Medical Center:

The University of Kansas Medical Centers mission is to educate exceptional health care professionals through a full range of undergraduate, graduate, professional, postdoctoral and continuing education programs in the schools of Medicine, Nursing and Health Professions. KU Medical Center also advances the health sciences through world-class research programs; provides compassionate and state-of-the-art patient care in an academic medical center environment; and works with communities in every Kansas county to improve the health of Kansans.

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Cancer center is a contributor to 49 research studies at the 62nd American Society of Hematology Annual Meeting - Newswise

Bone Marrow Stem Cells Comments Off on Cancer center is a contributor to 49 research studies at the 62nd American Society of Hematology Annual Meeting – Newswise | December 5th, 2020

DUBLIN--(BUSINESS WIRE)--Dec 4, 2020--

The "Multiple Myeloma Drugs Market: Global Industry Trends, Share, Size, Growth, Opportunity and Forecast 2020-2025" report has been added to ResearchAndMarkets.com's offering.

The global multiple myeloma drugs market grew at a CAGR of around 9% during 2014-2019. Looking forward, the publisher expects the market to witness moderate growth during the next five years.

Multiple myeloma, or Kahler's disease, refers to a form of blood cancer that primarily affects the plasma cells. Some of the most common types of multiple myeloma drugs include chemotherapeutic agents, corticosteroids and immunomodulatory agents. These pharmaceutical drugs aid in promoting bone healing, prevent hypercalcemia, bone fracture, spinal cord compression and anemia, while minimizing the need for chemotherapy. The chemotherapeutic agents include various anthracycline antibiotics and alkylating agents, such as melphalan, doxorubicin, vincristine and liposomal doxorubicin. The targeted therapy drugs include proteasome inhibitor, such as bortezomib, and various other compounds, including dexamethasone, prednisone and thalidomide.

Significant developments in the healthcare sector, along with the increasing prevalence of hematological cancer, is one of the key factors driving the growth of the market. Multiple myeloma is usually caused by specific genetic abnormalities, and the treatment of this disease involves drugs that modulate the immune system and aid in enhancing the efficiency of chemotherapies, radiation therapies, stem cell transplants and platelet transfusion.

Furthermore, rising consumer awareness regarding the benefits of biologic therapy drugs, which utilize the body's immune system to identify and attack the myeloma cells, is also providing a boost to the market growth. Additionally, various technological advancements, such as the development of microRNA therapeutics and nanomedicines for the treatment of multiple myeloma, is acting as another growth-inducing factor. These medicines are used to facilitate the delivery of macromolecular agents into the bone marrow and catalyze antitumor responses. Other factors, including the rising healthcare expenditures and extensive research and development (R&D) activities in the field of medical sciences, are projected to drive the market further.

Companies Mentioned

Key Questions Answered in This Report:

Key Topics Covered:

1 Preface

2 Scope and Methodology

2.1 Objectives of the Study

2.2 Stakeholders

2.3 Data Sources

2.3.1 Primary Sources

2.3.2 Secondary Sources

2.4 Market Estimation

2.4.1 Bottom-Up Approach

2.4.2 Top-Down Approach

2.5 Forecasting Methodology

3 Executive Summary

4 Introduction

4.1 Overview

4.2 Key Industry Trends

5 Global Multiple Myeloma Drugs Market

5.1 Market Overview

5.2 Market Performance

5.3 Market Forecast

6 Market Breakup by Therapy

6.1 Targeted Therapy

6.1.1 Market Trends

6.1.2 Market Forecast

6.2 Biologic Therapy

6.2.1 Market Trends

6.2.2 Market Forecast

6.3 Chemotherapy

6.3.1 Market Trends

6.3.2 Market Forecast

6.4 Others

6.4.1 Market Trends

6.4.2 Market Forecast

7 Market Breakup by Drug Type

7.1 Immunomodulatory Drugs

7.1.1 Market Trends

7.1.2 Market Forecast

7.2 Proteasome Inhibitors

7.2.1 Market Trends

7.2.2 Market Forecast

7.3 Histone Deacetylase Inhibitors

7.3.1 Market Trends

7.3.2 Market Forecast

7.4 Monoclonal Antibody Drugs

7.4.1 Market Trends

7.4.2 Market Forecast

7.5 Steroids

7.5.1 Market Trends

7.5.2 Market Forecast

7.6 Others

7.6.1 Market Trends

7.6.2 Market Forecast

8 Market Breakup by End-User

8.1 Men

8.1.1 Market Trends

8.1.2 Market Forecast

8.2 Women

8.2.1 Market Trends

8.2.2 Market Forecast

9 Market Breakup by Distribution Channel

9.1 Hospital Pharmacies

9.1.1 Market Trends

9.1.2 Market Forecast

9.2 Retail Pharmacies

9.2.1 Market Trends

9.2.2 Market Forecast

9.3 Online Pharmacies

9.3.1 Market Trends

9.3.2 Market Forecast

9.4 Others

9.4.1 Market Trends

9.4.2 Market Forecast

10 Market Breakup by Region

10.1 North America

10.2 Asia Pacific

10.3 Europe

10.4 Latin America

10.5 Middle East and Africa

11 SWOT Analysis

12 Value Chain Analysis

13 Porters Five Forces Analysis

14 Price Indicators

15 Competitive Landscape

15.1 Market Structure

15.2 Key Players

15.3 Profiles of Key Players

For more information about this report visit https://www.researchandmarkets.com/r/8yriem

View source version on businesswire.com:https://www.businesswire.com/news/home/20201204005505/en/

CONTACT: ResearchAndMarkets.com

Laura Wood, Senior Press Manager

press@researchandmarkets.com

For E.S.T Office Hours Call 1-917-300-0470

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Outlook on the Multiple Myeloma Drugs Global Market to 2025 - by Therapy, Drug Type, End-user, Distribution Channel and Region -...

Bone Marrow Stem Cells Comments Off on Outlook on the Multiple Myeloma Drugs Global Market to 2025 – by Therapy, Drug Type, End-user, Distribution Channel and Region -… | December 5th, 2020

Groundbreaking research in stem cells has propelled scientists understanding of neurodegenerative diseases, including Parksinsons. Could stem cell therapies one day help cure Alzheimers?

Clinical trials of stem cell therapies are now underway to repair the damaged cells of people with Parkinsons disease and age-related macular degeneration. Being Patient spoke with Jack Price, professor of developmental neurobiology at Kings College London and author of the book The Future of Brain Repair, about the potential and challenges of repairing the brain with stem cell therapy.

Being Patient: What is stem cell therapy?

Prof. Jack Price: Its the transplantation of stem cells, either directly into the brain or in a way that gives them access to the brain and influence the brain, to bring about a therapeutic effect.

Being Patient: Are there stem cells in the brain?

Prof. Jack Price: For many years, neuroscientists didnt think there were stem cells in the brain. We now know there are. We know about a population [of stem cells] thats become very important in our understanding of Alzheimers disease and in mood disorders like anxiety and depression. These are stem cells that are found in a part of the brain called the hippocampus.

But by and large, the brain doesnt have stem cells, unlike skin and other tissues in the body. The blood is the classic [example]: Theres a population of stem cells in the bone marrow that regenerates blood all the time.

Being Patient: What makes stem cells so special and why are they a focus of research?

Prof. Jack Price: The definition of stem cells is a population of cells that gives rise to other types of cells. In neural stem cells, precursor cells can make adult brain cells, nerve cells, glial cells, all the different cell types that make up the brain. If you have a disease like Alzheimers or any other neurodegenerative disease, where we know the key pathology is the loss of nerve cells, your brain doesnt normally have the ability to replace those lost brain cells. The idea was [that] if you put stem cells where the loss of brain cells has taken place, maybe those stem cells would replace the lost cells.

Being Patient: What is the potential of stem cell therapy in treating neurodegenerative diseases?

Prof. Jack Price: Theres a piece of absolutely brilliant stem cell science that was done by Shinya Yamanaka in Kyoto in 2006. He showed you could effectively take any cell through a very straightforward genetic manipulation that he discovered, [and] turn them into what we call pluripotent stem cells, which are cells that can make any cell type in the body. They also have an ability that other stem cells generally dont: They can build tissue. If you grow them in a little culture dish, they can start to make little pieces of brain called organoids or cerebroids. This was a groundbreaking technology.

In Parkinsons disease, theres enormous progress and clinical trials are underway now. We know more about the pathology of Parkinsons disease [than in Alzheimers]. The pathology of Alzheimers turns out to be quite complex, and weve had, over the years, quite a few ideas about how it worked. But [turning] those into actual therapies hasnt quite worked as we expected, and we keep having to go back and rethink whats going on in Alzheimers.

The pathology of Parkinsons disease is also difficult. Its not trivial. But at the same time, one thing is clear: a lot of the pathology is associated with the loss of a particular population of nerve cells the midbrain dopaminergic cells. We can start with these pluripotent stem cells and make them make precisely the right type of dopaminergic cell that we know is lost in Parkinsons disease.

This is built on 30 [to] 40 years of research of people trying to find exactly the right cell type to work [with] in Parkinsons disease. They had some early success and fell backwards. But this technology looks much more precise than everything anybodys ever tried before.

In age-related macular degeneration, the disease of the eye where you lose your retinal photoreceptors, there are very clever strategies now where people are using these pluripotent stem cells to make a thing called a retinal pigment epithelium. It lies behind the retina, but its what supports the photoreceptors. It turns out, thats what goes wrong in age-related macular degeneration.

Being Patient: Are there any stem cell therapy approved to treat brain disorders?

Prof. Jack Price: There are no licensed stem cell therapy for any brain disorders anywhere in the world for the simple reason [that] nobody has shown one works. There are a lot of stem cell clinics in the U.S. and somewhat fewer elsewhere who are offering cell therapies that are untested. Theyll put stem cells into you for any disorder youve got. Those cell therapies do not work.

A lot of genuine companies are trying to get these cell therapies to work in clinical trials and falling flat on their face quite often, despite their best efforts. 90% of clinical trials fail, and thats clinical trials of conventional drugs by drug companies that know what theyre doing.

What do you suppose is the chance with a stem cell therapy [that] we dont really understand how it works, [that] we dont quite know how to manufacture it properly, [and that] we dont quite know what cells we really want, of working? The chance is almost zero. These companies know that, which is why theyre not going to clinical trials.

The interview has been edited for length and clarity.

Contact Nicholas Chan at nicholas@beingpatient.com

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Repairing the Brain With Stem Cells? A Conversation With Prof. Jack Price - Being Patient

Bone Marrow Stem Cells Comments Off on Repairing the Brain With Stem Cells? A Conversation With Prof. Jack Price – Being Patient | December 3rd, 2020

An effective new treatment to restore vision is on the horizon that works by injecting genetically modified stem cells into the eye to mend the damaged retina.

Researchers found that the cells of damaged retinas send out a rescue signal to attract the stem cells that repair eye damage.

The i newsletter latest news and analysis

They identified two of these cell signals known as Ccr5 and Cxcr6 and then genetically engineered the stem cells to make them more sensitive to those signals.

When these modified stem cells were transplanted back into mice and human tissue samples in the lab they flocked to the retina cells in much greater numbers, keeping the tissue of the damaged retina alive and functioning.

The technique holds promise for improving sight in people with poor vision and potentially even to cure blindness altogether but the researchers cautioned that any such development was some years away and required much bigger studies to confirm their findings.

One of the main hurdles in using stem cells to treat damaged eyesight is low cell migration and integration in the retina, says Pia Cosma, at the Centre for Genomic Regulation in Barcelona.

After the cells are transplanted they need to reach the retina and integrate through its layers. Here we have found a way to enhance this process using stem cells commonly found in the bone marrow, but in principle can be used with any transplanted cells, Dr Cosma said.

There is still considerable work to be done, but our findings could make stem cell transplants a feasible and realistic option for treating visual impairment and restoring eyesight, she said.

Retinal damage, which is currently incurable, inevitably leads to visual disabilities and in most cases blindness. With a growing and ageing population, the number of people affected by retinal damage is estimated to increase dramatically over the next few decades.

Stem cell therapies have been touted as one way of treating degenerative retinal conditions. Stem cells can be transplanted into the eye, releasing therapeutic molecules with neuroprotective and anti-inflammatory properties that promote the survival, proliferation and self-repair of retinal cells. The stem cells can also generate new retinal cells, replacing lost or damaged ones.

The researchers used mesenchymal stem cells, which are found in bone marrow and can differentiate into lots of types of cells, including retinal cells that respond to light.

Mesenchymal stem cells can also be easily grown outside an organism, providing abundant starting material for transplantation compared to other cell sources such as hematopoietic stem cells.

The study is published in the journal Molecular Therapy.

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Treatment to restore vision by injecting stem cells into the eye could help people with damaged eyesight - iNews

Bone Marrow Stem Cells Comments Off on Treatment to restore vision by injecting stem cells into the eye could help people with damaged eyesight – iNews | December 3rd, 2020

This article was originally published here

J Pharmacol Exp Ther. 2020 Nov 30:JPET-AR-2020-000277. doi: 10.1124/jpet.120.000277. Online ahead of print.

ABSTRACT

ONO-4641 is a second-generation sphingosine 1-phosphate (S1P) receptor modulator that exhibits selectivity for S1P receptors 1 and 5. Treatment with ONO-4641 leads to a reduction in magnetic resonance imaging disease measures in patients with relapsing-remitting multiple sclerosis. The objective of this study was to explore the potential impact of ONO-4641 treatment based on its immunomodulatory effects. Severe aplastic anemia is a bone marrow (BM) failure disease, typically caused by aberrant immune destruction of blood progenitors. Although the T helper type-1-mediated pathology is well described for aplastic anemia, the molecular mechanisms driving disease progression remain undefined. We evaluated the efficacy of ONO-4641 in a mouse model of aplastic anemia. ONO-4641 reduced the severity of BM failure in a dose-dependent manner, resulting in higher blood and BM cell counts. By evaluating the mode of action, we found that ONO-4641 inhibited the infiltration of donor-derived T lymphocytes to the BM. ONO-4641 also induced the accumulation of hematopoietic stem cells in the BM of mice. These observations indicate, for the first time, that S1P receptor modulators demonstrate efficacy in the mouse model of aplastic anemia and suggest that treatment with ONO-4641 might delay the progression of aplastic anemia. Significance Statement ONO-4641 is a second-generation sphingosine 1-phosphate (S1P) receptor modulator selective for S1P receptors 1 and 5. In this study, we demonstrated that ONO-4641 regulates the trafficking of T lymphocytes along with hematopoietic stem and progenitor cells leading to alleviation of pancytopenia and destruction of bone marrow in a bone marrow failure-induced mouse model mimicking human aplastic anemia.

PMID:33257316 | DOI:10.1124/jpet.120.000277

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Sphingosine 1-phosphate Receptor Modulator ONO-4641 Regulates Trafficking of T Lymphocytes and Hematopoietic Stem Cells and Alleviates Immune-Mediated...

Bone Marrow Stem Cells Comments Off on Sphingosine 1-phosphate Receptor Modulator ONO-4641 Regulates Trafficking of T Lymphocytes and Hematopoietic Stem Cells and Alleviates Immune-Mediated… | December 3rd, 2020

NEW YORK, Dec. 03, 2020 (GLOBE NEWSWIRE) -- IN8bio, Inc., a clinical-stage biotechnology company focused on developing innovative allogeneic, autologous and genetically modified gamma-delta T cell therapies for the treatment of cancers (IN8bio or the Company), today announced an upcoming presentation that provides an update of the ongoing Phase I clinical trial of their product candidate INB-100 at the 62nd American Society of Hematology Annual Meeting & Exposition (ASH), which will take place virtually from December 5 to 8, 2020. INB-100 is designed for the treatment of patients with leukemia undergoing hematopoietic stem cell transplantation with haploidentical donors.

The poster and accompanying narrated slide presentation is titled, First-in-Human Phase I Trial of Adoptive Immunotherapy with Ex Vivo Expanded and Activated gamma delta T-Cells Following Haploidentical Bone Marrow Transplantation and Post-BMT Cyclophosphamide and reviews the study design and provides a brief update on enrollment and patient status.

The company reported that, as of abstract submission, three female subjects with acute leukemia had been enrolled in the INB-100 Phase 1 trial, of whom two had been dosed, and that no treatment-related adverse events had been recorded. The trial is continuing to enroll and treat patients. The abstract for the presentation can be found at https://ash.confex.com/ash/2020/webprogram/Paper142876.html.

The poster and slide presentation are jointly authored by the scientific and physician investigators from IN8bio and The University of Kansas Cancer Center (KU Cancer Center), and will be presented by the studys Principal Investigator, Dr. Joseph McGuirk, Schutte-Speas Professor of Hematology-Oncology, Division Director of Hematological Malignancies and Cellular Therapeutics and Medical Director, Blood and Marrow Transplant at KU Cancer Center.

This preliminary data report from KU Cancer Center with our allogeneic product candidate, INB-100, demonstrates the absence of significant GvHD in these initial patients, said William Ho, Chief Executive Officer of IN8bio. This suggests that gamma delta T-cells delivered as an off-the-shelf allogeneic cell therapy may be well tolerated and have significant potential to treat patients with serious and life-threatening cancers.

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Dr. McGuirk, commented, Potentially curative stem cell transplants using partially matched donors -- called haploidentical transplants have greatly expanded access to stem cell transplantation. The infusion of donor-derived gamma delta T-cells from the stem cell donor, offers the hope of diminishing this risk of relapse and curing more patients.

About IN8bioIN8bio is a clinical-stage biotechnology company focused on developing novel therapies for the treatment of cancers, including solid tumors, by employing allogeneic, autologous and genetically modified gamma-delta T cells. IN8bios technology incorporates drug-resistant immunotherapy (DRI), which has been shown in preclinical studies to function in combination with therapeutic levels of chemotherapy. IN8bio is currently conducting two investigator-initiated Phase 1 clinical trials for its lead gamma-delta T cell product candidates: INB-200 for the treatment of newly diagnosed glioblastoma, which is a difficult to treat brain tumor that progresses rapidly, and INB-100 for the treatment of patients with acute leukemia undergoing hematopoietic stem cell transplantation. For more information about the Company and its programs, visit http://www.IN8bio.com.

Forward Looking StatementsCertain statements herein concerning the Companys future expectations, plans and prospects, including without limitation, the Companys current expectations regarding the curative potential of its product candidates, constitute forward-looking statements. The use of words such as may, might, will, should, expect, plan, anticipate, believe, estimate, project, intend, future, potential, or continue, the negative of these and other similar expressions are intended to identify such forward looking statements. Such statements, based as they are on the current expectations of management, inherently involve numerous risks and uncertainties, known and unknown, many of which are beyond the Companys control. Consequently, actual future results may differ materially from the anticipated results expressed in such statements. Specific risks which could cause actual results to differ materially from the Companys current expectations include: scientific, regulatory and technical developments; failure to demonstrate safety, tolerability and efficacy; final and quality controlled verification of data and the related analyses; expense and uncertainty of obtaining regulatory approval, including from the U.S. Food and Drug Administration; and the Companys reliance on third parties, including licensors and clinical research organizations. Do not place undue reliance on any forward-looking statements included herein, which speak only as of the date hereof and which the Company is under no obligation to update or revise as a result of any event, circumstances or otherwise, unless required by applicable law.

Contact:IN8bio, Inc.Kate Rochlin, Ph.D.+1 646.933.5605info@IN8bio.com

Investor Contact:Julia Balanova+ 1 646.378.2936jbalanova@soleburytrout.com

Media Contact:Ryo Imai / Robert Flamm, Ph.D.Burns McClellan, Inc.212-213-0006 ext. 315 / 364Rimai@burnsmc.com / rflamm@burnsmc.com

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IN8bio announces first-in-human Phase 1 trial Update from The University of Kansas Cancer Center using INB-100, IN8bios Gamma Delta T-cell product...

Bone Marrow Stem Cells Comments Off on IN8bio announces first-in-human Phase 1 trial Update from The University of Kansas Cancer Center using INB-100, IN8bios Gamma Delta T-cell product… | December 3rd, 2020

This article was originally published here

J Vasc Interv Radiol. 2020 Nov 25:S1051-0443(20)30769-7. doi: 10.1016/j.jvir.2020.09.003. Online ahead of print.

ABSTRACT

PURPOSE: To evaluate safety and efficacy of angiogenesis induced by intraarterial autologous bone marrow-derived stem cell (BMSC) injection in patients with severe peripheral arterial disease (PAD).

MATERIALS AND METHODS: Eighty-one patients with severe PAD (77 men), including 56 with critical limb ischemia (CLI) and 25 with severe claudication, were randomized to receive sham injection (group A) or intraarterial BMSC injection at the site of occlusion (group B). Primary endpoints included improvement in ankle-brachial index (ABI) of > 0.1 and transcutaneous pressure of oxygen (TcPO2) of > 15% at mid- and lower foot at 6 mo. Secondary endpoints included relief from rest pain, > 30% reduction in ulcer size, and reduction in major amputation in patients with CLI and > 50% improvement in pain-free walking distance in patients with severe claudication.

RESULTS: Technical success was achieved in all patients, without complications. At 6 mo, group B showed more improvements in ABI of > 0.1 (35 of 41 [85.37%] vs 13 of 40 [32.50%]; P < .0001) and TcPO2 of > 15% at the midfoot (35 of 41 [85.37%] vs 17 of 40 [42.50%]; P = .0001] and lower foot (37 of 41 [90.24%] vs 19 of 40 [47.50%]; P < .0001). No patients with CLI underwent major amputation in group B, compared with 4 in group A (P = .0390). No significant difference was observed in relief from rest pain or > 30% reduction in ulcer size among patients with CLI or in > 50% improvement in pain-free walking distance among patients with severe claudication.

CONCLUSIONS: Intraarterial delivery of autologous BMSCs is safe and effective in the management of severe PAD.

PMID:33248918 | DOI:10.1016/j.jvir.2020.09.003

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Randomized, Double-Blind, Placebo-Controlled Trial to Evaluate Safety and Therapeutic Efficacy of Angiogenesis Induced by Intraarterial Autologous...

Bone Marrow Stem Cells Comments Off on Randomized, Double-Blind, Placebo-Controlled Trial to Evaluate Safety and Therapeutic Efficacy of Angiogenesis Induced by Intraarterial Autologous… | December 3rd, 2020

For the first time, researchers have demonstrated how the gut microbiome the community of microorganisms living in the gut can influence the immune system in humans. Their work could lead to new treatments for immune-related conditions.

The researchers at Memorial Sloan Kettering Cancer Center in New York, NY, tracked the recovery of patients gut microbiota and immune system after bone marrow transplants (BMTs) following treatment for blood cancers.

Healthcare professionals use chemotherapy and radiation therapy to destroy cancerous blood cells in conditions such as leukemia and lymphoma. After completion of the treatment, which also kills healthy immune cells, specialists inject patients with stem cells from a donors blood or bone marrow.

These donated cells slowly restore patients ability to make their own blood cells.

However, patients have to take antibiotics in the first few weeks after the transplant because they are still vulnerable to infections. These upset the balance of their gut microbiota, killing friendly bacteria and allowing dangerous strains to thrive.

Once patients immune systems are strong enough, they can stop taking the antibiotics, which allows their gut microbiota to recover.

The researchers at Sloan Kettering used this unique opportunity to study how the microbiota affects the immune system.

The scientific community had already accepted the idea that the gut microbiota was important for the health of the human immune system, but the data they used to make that assumption came from animal studies, explains systems biologist Joao Xavier, who is co-senior author of the paper with his former postdoc Jonas Schluter.

The parallel recoveries of the immune system and the microbiota, both of which are damaged and then restored, gives us a unique opportunity to analyze the associations between these two systems, says Dr. Schluter, who is now an assistant professor at NYU Langone Health in New York, NY.

Using blood and fecal samples from more than 2,000 patients treated at the cancer center between 20032019, the researchers were able to track daily changes in their gut microbiota and the number of immune cells in their blood.

Our study shows that we can learn a lot from stool biological samples that literally would be flushed down the toilet, says Dr. Xavier. The result of collecting them is that we have a unique dataset with thousands of data points that we can use to ask questions about the dynamics of this relationship.

The researchers used a machine-learning algorithm to identify patterns in the data, which included information about patients medications and the side effects they experienced.

One of the findings was that the presence of three types of gut bacteria called Faecalibacterium, Ruminococcus 2, and Akkermansia was associated with increased blood concentrations of immune cells called neutrophils.

By contrast, two types called Rothia and Clostridium sensu stricto 1, were associated with reduced numbers of these immune cells.

Computer simulations by the researchers predicted that enriching microbiota with the three friendly genera would speed up the recovery of patients immune systems.

This research could eventually suggest ways to make BMTs safer by more closely regulating the microbiota, says co-author Marcel van den Brink.

The study appears in Nature.

Concluding their paper, the authors write:

Our demonstration that the microbiota influences systemic immunity in humans opens the door toward an exploration of potential microbiota-targeted interventions to improve immunotherapy and treatments for immune-mediated and inflammatory diseases.

A previous study found that having a greater diversity of bacterial species in the gut is associated with a better chance of survival after a stem cell transplant. This research also found that a low diversity of bacteria increased the likelihood of potentially fatal graft-versus-host disease, when the donor immune cells attack the recipients tissues.

In 2018, the Sloan Kettering researchers published results from a clinical trial in which they used fecal transplants to restore patients microbiota after treatment for blood cancer.

They used the patients own fecal matter, which had been collected and frozen before the bone marrow transplant and antibiotic treatment disrupted their gut microbiota.

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Gut bacteria can help rebuild the immune system - Medical News Today

Bone Marrow Stem Cells Comments Off on Gut bacteria can help rebuild the immune system – Medical News Today | December 3rd, 2020

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Impacts of Advancements and COVID-19 on the market.

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Market Segmentation

Some of the major companies that are covered in the report.

Regen Biopharma IncChina Cord Blood CorpCBR Systems IncEscape Therapeutics IncCryo-Save AGLonza Group LtdPluristem Therapeutics IncViaCord Inc

Note: Additional companies

Based on the type, the market is segmented into

AllogeneicAutologous

Based on the application, the market is segregated into

Peripheral Blood Stem Cells Transplant (PBSCT)Bone Marrow Transplant (BMT)Cord Blood Transplant (CBT)

Based on the geographical location, the market is segregated into

Asia Pacific: China, Japan, India, and Rest of Asia PacificEurope: Germany, the UK, France, and Rest of EuropeNorth America: The US, Mexico, and CanadaLatin America: Brazil and Rest of Latin AmericaMiddle East & Africa: GCC Countries and Rest of Middle East & Africa

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Executive Summary

Assumptions and Acronyms Used

Research Methodology

Hematopoietic Stem Cell Transplantation (HSCT) Market Overview

Hematopoietic Stem Cell Transplantation (HSCT) Supply Chain Analysis

Hematopoietic Stem Cell Transplantation (HSCT) Pricing Analysis

Global Hematopoietic Stem Cell Transplantation (HSCT) Market Analysis and Forecast by Type

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North America Hematopoietic Stem Cell Transplantation (HSCT) Market Analysis and Forecast

Latin America Hematopoietic Stem Cell Transplantation (HSCT) Market Analysis and Forecast

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Asia Pacific Hematopoietic Stem Cell Transplantation (HSCT) Market Analysis and Forecast

Asia Pacific Hematopoietic Stem Cell Transplantation (HSCT) Market Size and Volume Forecast by Application

Middle East & Africa Hematopoietic Stem Cell Transplantation (HSCT) Market Analysis and Forecast

Competition Landscape

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Trends Of Hematopoietic Stem Cell Transplantation (HSCT) Market Reviewed For 2020 With Industry Outlook To 2027 - The Market Feed

Bone Marrow Stem Cells Comments Off on Trends Of Hematopoietic Stem Cell Transplantation (HSCT) Market Reviewed For 2020 With Industry Outlook To 2027 – The Market Feed | December 3rd, 2020

(TNS) - Scientists at Bay Area universities, laboratories, biotechnology companies and drug manufacturers are fashioning drug concoctions out of blood plasma, chimpanzee viruses and cells taken from bone marrow in the race to rid the world of COVID-19.

The microbial treasure hunt is not just to find a cure which may not be possible but to control the debilitating health problems caused by the coronavirus.

Major progress has been made this year. The antiviral drug remdesivir, produced in Foster City, has improved recovery times, and the steroid dexamethasone has cut the number of deaths in severely ill patients.

What follows is a list of some of the most promising medications and vaccines with ties to the Bay Area:

Antibodies

and Immunity

Mesenchymal stem cells / UCSF and UC Davis Medical Center:

UCSF Dr. Michael Matthay is leading a study of whether a kind of stem cell found in bone marrow can help critically ill patients with severe respiratory failure, known as ARDS. Matthay hopes the stem cells can help reduce the inflammation associated with some of ARDS' most dire respiratory symptoms, and help patients' lungs recover.

In all, 120 patients are being enrolled at UCSF Medical Center, Zuckerberg San Francisco General Hospital, the UC Davis Medical Center in Sacramento and hospitals in Oregon and Texas. He said the trial, which includes a small number of ARDS patients who don't have COVID-19, should have results by summer or fall 2021. So far, 28 patients are enrolled in San Francisco.

Lambda-interferon / Stanford University:

Lambda-interferon is a manufactured version of a naturally occurring protein that had been used to treat hepatitis, and researchers hoped it would help patients in the early stages of COVID-19.

Stanford researchers completed their trial of lambda-interferon and found that it did not boost the immune system response to coronavirus infections.

"That trial did not find any difference in outcomes between the treatment and placebo," said Yvonne Maldonado, chief of pediatric infectious diseases at Lucile Packard Children's Hospital at Stanford, where 120 patients were enrolled in the trial. "It didn't work."

Antiviral drugs

Remdesivir / Gilead Sciences ( Foster City):

Remdesivir, once conceived as a potential treatment for Ebola, was approved by the Food and Drug Administration in October for use on hospitalized COVID-19 patients.

Trademarked under the name Veklury, the drug interferes with the process through which the virus replicates itself. It was one of the drugs given to President Trump and has been used regularly in hospitals under what is known as an emergency use authorization.

It was approved after three clinical trials showed hospitalized coronavirus patients who received remdesivir recovered five days faster on average than those who received a placebo. Patients who required oxygen recovered seven days faster, according to the studies.

Gilead now plans to conduct clinical trials to see how remdesivir works on pediatric patients, from newborns to teenagers, with moderate to severe COVID-19 symptoms. Remdesivir is also being studied with steroids and other drugs to see if it works better as part of a medicinal cocktail. An inhalable form of the drug is also being developed.

Favipiravir / Fujifilm Toyama Chemical ( Stanford University):

This antiviral drug, developed in 2014 by a subsidiary of the Japanese film company to treat influenza, is undergoing numerous clinical studies worldwide, including a trial involving 180 patients at Stanford University.

Stanford epidemiologists are testing favipiravir to see if it prevents the coronavirus from replicating in human cells, halts the shedding of the virus and reduces the severity of infection. Unlike remdesivir, it can be administered orally, so it can be used to treat patients early in the disease, before hospitalization is necessary.

The Stanford study has so far enrolled about 90 patients, who are given the drug within 72 hours of when they were first diagnosed with COVID-19. Half of them get a placebo. People can enroll by emailing treatcovid@stanford.edu.

Monoclonal antibodies

REGN-COV2 / Regeneron Pharmaceuticals / Stanford School of Medicine:

The REGN-COV2 cocktail is the same one Trump received, and Stanford is one of dozens of locations nationwide where clinical trials are being held. Two separate trials are under way at Stanford one for hospitalized patients, the other for outpatients. A third trial is about to begin for people who aren't sick but are in contact with carriers of the virus.

Regeneron halted testing on severely ill patients requiring high-flow oxygen or mechanical ventilation after the independent Data and Safety Monitoring Board determined that the drug was unlikely to help them.

The drug is a combination of two monoclonal antibodies lab-made clones of the antibodies produced naturally in people who have recovered from COVID-19. The antibodies bind to the virus' spike protein and block the virus' ability to enter cells.

Dr. Aruna Subramanian, professor of infectious diseases at Stanford and lead investigator for the inpatient trial, said the 21 hospitalized patients in the study receive a high dose like Trump, a lower dose or a placebo. Subramanian plans to expand the inpatient trial to 45 patients. The outpatient study has enrolled a little more than 40 of the 60 patients researchers intend to sign up.

"There's enough promising evidence that it helps people early in the infection," Subramanian said. "What we don't know is whether it helps people who are pretty sick but not critically ill."

Bamlanivimab / Eli Lilly / Stanford and UCSF:

Stanford and UCSF are testing the Eli Lilly monoclonal antibodies on outpatients after the pharmaceutical company halted trials on hospitalized COVID-19 patients because of adverse results.

Dr. Andra Blomkalns, chair of emergency medicine at Stanford and the lead in the Eli Lilly outpatient trial, said she is now enrolling older people with comorbidities like heart disease, chronic lung disease, a history of strokes and severe obesity shortly after they test positive.

The hypothesis is that the bamlanivimab monotherapy, which is very similar to the Regeneron monoclonals, might work best early in the infection. Although about 400 patients have been enrolled in the Lilly phase 3 trials nationwide, to date fewer than 10 have been enrolled at Stanford and UCSF.

Matthay, who headed up the Lilly monoclonal study with LY-CoV555 at UCSF, said the cancellation of this inpatient trial was disappointing, but "just because this one did not work, doesn't mean another one won't work for hospitalized patients."

Blomkalns said the testing criteria has been changing. She expects the outpatient trial to open soon to adolescents ages 12 and up to determine whether the drug can be used as a preventive.

Designer monoclonal antibodies / Vir Biotechnology, San Francisco:

Scientists at Vir are studying several types of monoclonal antibodies, including a type engineered to activate T cells, which can search out and destroy cells infected with the coronavirus. A study published in the journal Nature in October found that monoclonals, modified to bind with certain receptors, stimulated T cells and improved the human immune response.

"By observing and learning from our body's powerful natural defenses, we have discovered how to maximize the capacity of antibodies through the amplification of key characteristics that may enable more effective treatments for viral diseases," said Herbert Virgin, the chief scientific officer at Vir and co-author of the study.

A similarly modified monoclonal antibody, leronlimab, is being studied in coronavirus clinical trials by its Washington state drugmaker, CytoDyn, which has developed drugs to treat HIV. The company's chief medical officer is in San Francisco, and the company that does laboratory tests of leronlimab is in San Carlos.

Anti-inflammatory drugs

Colchicine / UCSF ( San Francisco and New York):

The anti-inflammatory drug commonly used to treat gout flare-ups is being studied by scientists at UCSF and New York University. The drug short-circuits inflammation by decreasing the body's production of certain proteins, and researchers hope that it will reduce lung complications and prevent deaths from COVID-19.

Preliminary results from a clinical trial found that "Colchicine can be effective in reducing systemic symptoms of COVID-19 by inhibiting inflammatory biomarkers."

Selinexor / Kaiser Permanente:

Kaiser hospitals in San Francisco, Oakland and Sacramento are studying selinexor, an anticancer drug that blocks a key protein in the cellular machinery for DNA processing. Preliminary findings during the trials indicated that low doses of selinexor helped hospitalized patients with severe COVID-19. The drug has both antiviral and anti-inflammatory properties, and it's administered orally, according to Kaiser's Dr. Jacek Skarbinski.

Vaccines

VXA-COV2-1 / Vaxart, South San Francisco:

The biotechnology company Vaxart is testing VXA-COV2-1, the only potential vaccine in pill form. It uses the genetic code of the coronavirus to trigger a defensive response in mucous membranes. The hope is that the newly fortified membranes will prevent the virus from entering the body.

"It's the only vaccine (candidate) that activates the first line of defense, which is the mucosa," said Andrei Floroiu, Vaxart's chief executive. He said intravenous vaccines kill the virus after it is inside the body, but this one stops it beforehand.

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Coronavirus Updates: The Latest Treatments and Vaccines - GovTech

Bone Marrow Stem Cells Comments Off on Coronavirus Updates: The Latest Treatments and Vaccines – GovTech | December 3rd, 2020

SOUTH SAN FRANCISCO, Calif.--(BUSINESS WIRE)--Imago BioSciences, Inc., (Imago) a clinical-stage biotechnology company developing innovative treatments for myeloid diseases, today announced the expansion of its global Phase 2b clinical study evaluating bomedemstat (IMG-7289) for the treatment of advanced myelofibrosis (MF) into Hong Kong, where the first patient has now been enrolled and dosed at the Department of Medicine, Queen Mary Hospital and the University of Hong Kong. Myelofibrosis is a rare bone marrow cancer that interferes with the production of blood cells.

In addition to Hong Kong, the Phase 2b study continues to actively enroll patients in the U.S., U.K., and E.U. The study is in the final stages of completing enrollment and continues to dose patients to evaluate safety, tolerability and efficacy.

Patients with myelofibrosis around the world are still in need of new treatment options, said Hugh Young Rienhoff, Jr. M.D., Chief Executive Officer, Imago BioSciences. We are progressing well with enrollment and are pleased to continue expanding our global Phase 2 study into new geographies like Hong Kong. We are encouraged by the signs of clinical activity and safety of bomedemstat as a treatment alternative for patients who do not benefit from the current standards of care.

Bomedemstat is an inhibitor of lysine-specific demethylase 1 (LSD1), an epigenetic regulator critical for self-renewal of malignant myeloid cells and the differentiation of myeloid progenitors. Data presented at the 25th European Hematology Association (EHA) Annual Congress in June demonstrated that the first-in-class LSD1 inhibitor was well tolerated with no dose-limiting toxicities or safety signals. Furthermore, recent data demonstrates the potential of bomedemstat as a monotherapy in intermediate-2 and high-risk patients with myelofibrosis who have become intolerant of, resistant to or are ineligible for a Janus Kinase (JAK) inhibitor.

Bomedemstat was recently granted PRIME (PRIority MEdicines) designation by the European Medicines Agency (EMA) for the treatment of MF. The EMA reviewed bomedemstat non-clinical and clinical data from the ongoing Phase 2 study. The PRIME initiative was launched by the EMA in 2016 to provide proactive and enhanced support to the developers of promising medicines with the view of accelerating their evaluation to reach patients faster.

About Bomedemstat (IMG-7289)

Bomedemstat is an orally available small molecule discovered and developed by Imago BioSciences that inhibits lysine-specific demethylase 1 (LSD1 or KDM1A), an enzyme shown to be vital in cancer stem/progenitor cells, particularly neoplastic bone marrow cells. In non-clinical studies, bomedemstat demonstrated robust in vivo anti-tumor efficacy across a range of myeloid malignancies as a single agent and in combination with other therapeutic agents. Bomedemstat is an investigational agent currently being evaluated in ongoing clinical trials (ClinicalTrials.gov Identifier: NCT03136185, NCT04262141, NCT04254978 and NCT04081220).

Bomedemstat has U.S. FDA Orphan Drug and Fast Track Designation for the treatment of myelofibrosis and essential thrombocythemia, Orphan Drug Designation for treatment of acute myeloid leukemia and PRIME designation by the European Medicines Agency for the treatment of MF.

Bomedemstat is being evaluated in two open-label Phase 2 clinical trials for the treatment of advanced myelofibrosis (MF) and essential thrombocythemia (ET), bone marrow cancers that interfere with the production of blood cells. MF patients who are resistant to a Janus Kinase (JAK) inhibitor are eligible for the study of bomedemstat. ET patients who have failed one standard of care treatment are eligible for the bomedemstat ET study.

About Imago BioSciences

Imago BioSciences is a clinical-stage biopharmaceutical company focused on discovering and developing novel therapeutics for the treatment of hematologic disorders targeting epigenetic enzymes. Imago has developed a series of compounds that inhibit LSD1, an epigenetic enzyme critical for cancer stem cell function and blood cell differentiation. Imago is advancing the clinical development of its first LSD1 inhibitor, bomedemstat, for the treatment of myeloid neoplasms. Imago BioSciences is backed by leading private, corporate, and public investors including Farallon Capital Management, LLC., funds and accounts advised by T. Rowe Price Associates, Inc., funds and accounts managed by Blackrock Advisors, LLC., Surveyor Capital (a Citadel company), Irving Investors, Kingdon Capital Management, a fund managed by Blackstone Life Sciences, Frazier Healthcare Partners, Omega Funds, Amgen Ventures, MRL Ventures Fund, HighLight Capital, Pharmaron, Greenspring Associates and Xeraya Capital. The company is based in South San Francisco, California. To learn more, visit http://www.imagobio.com, http://www.myelofibrosisclinicalstudy.com, http://www.etclinicalstudy.com and follow us on Twitter @ImagoBioRx, Facebook and LinkedIn.

Originally posted here:
Imago BioSciences Expands Phase 2 Clinical Trial of Bomedemstat (IMG-7289) for the Treatment of Myelofibrosis into Hong Kong - Business Wire

Bone Marrow Stem Cells Comments Off on Imago BioSciences Expands Phase 2 Clinical Trial of Bomedemstat (IMG-7289) for the Treatment of Myelofibrosis into Hong Kong – Business Wire | December 3rd, 2020

Brilliant news as Keisha announces daughter Aurora is back in remission Photo Keisha Pile-Gray

Amazing news has been announced today (November 30) nine-year-old Aurora Pile-Gray from Westbrook is in remission.

Aurora was diagnosed with stage 4 Burkitts Lymphoma after becoming poorly towards the end of April.

The rare cancer affects blood and bone marrow. Aurora has been undergoing chemotherapy and her family were looking at CAR-T cell therapy and an allogeneic transplant.

Earlier this year, after 4 gruelling cycles of intensive chemo, Aurora had been given the news that she was in remission, however this was short lived.

In October the family were told the cancer had in fact spread to Auroras bones and that the youngster was in need of a bone marrow transplant. The situation was also complicated by Auroras mixed ethnicity, making it that much more difficult to find a match.

Aurora is currently being treated by Royal Marsden Hospital and Great Ormond Street Hospital.

But in brilliant news Auroras mum Keisha has today revealed her daughter is now in remission.

Posting to facebook Keisha said: Aurora is officially back in remission!!

Her bone marrow assessments show no cancer cells present, and no cancer cell regeneration on new cells in both the solid and liquid part of her biopsy.

We were aware about the liquid aspirate a week ago, but weve been anxiously waiting for news on the solid part up until today!

The transplant team have also sourced an 11/12 donor match which means that Aurora will have one more round of chemotherapy and will move forward with transplant in January!

We dont know much about her donor other than her stem cells will be coming from a 36 year old female, with 2 children. We arent sure if we are allowed to get in contact before two years but just know, if you read this and its you, or of you know who it could be, we owe our entire life to you and would give you the world.

Days like today make our heart burst with pride and love and just how strong she has been throughout this whole ordeal. We are forever in awe of how she has tackled this journey and we are absolutely overjoyed that things are starting to look up!

Theres still a long way to go, but shes already come this far, we are all so over the moon, and I can barely get my words out, so for now, we are celebrating that out little lion is fighting on.

Matching bone marrow donors is a much more complex process than matching blood type. It relies on matching individual tissue type, and genetic markers that are found on most cells in the body. These markers are used by the immune system as a way to distinguish what cells are supposed to be in the body, and which arent. The markers must be as closely matched as possible between host and guest, to prevent the body rejecting the new bone marrow. Everyones tissue type is inherited, so often bone marrow donations come directly from a donor with the same ethnic background.

Theres a lack of individuals from ethnic minorities on the register, and as a whole only 2% of the entire UK population is currently signed up to become a bone marrow donor despite having a 1 in 800 chance you would be a match for someone.

Keisha added: Auroras in remission but we still have to get her through transplant and theres a 90% chance it could return within a year. Were made up, but still very apprehensive.

This month mum-of-three Keisha and St Saviours pupil Aurora were announced as joint winners, alongside Westgates Wilfred Jenkins, after our call out for Thanet heroes of 2020.

And Keishas latest update here

Find out how to join the bone marrow register:

http://www.anthonynolan.org

http://www.dkms.org

A fundraising page has been set up to help the family in case treatment abroad is needed.

Related

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Family 'over the moon' as nine-year-old Aurora confirmed as being back in remission - The Isle of Thanet News

Bone Marrow Stem Cells Comments Off on Family ‘over the moon’ as nine-year-old Aurora confirmed as being back in remission – The Isle of Thanet News | December 3rd, 2020

The mRNA technology used to create the Pfizer and Moderna vaccines for COVID is being applied to ... [+] many other medical treatments in addition to vaccines.

While the vaccines for Covid-19 seem to have been created in record time, the technology making them possible has been decades in development. The two vaccine candidates produced by Pfizer/BioNTech and Moderna are unlike any other vaccine thats come before. Should they achieve commercial success, it could usher in a new era of medical science not just for vaccines, but for cancer treatments, blood disorders, and gene therapy.

The two new vaccines are the first ever to use mRNA, which stands for messenger RNA, to generate immunity. Historically, vaccines have used dead or weakened viruses to imitate an infection, spurring the body to make antibodies against that virus without danger of getting sick. Measles, polio, and some seasonal flu shots are examples of vaccines made with whole virus particles.

Other vaccines use only certain fragments of the virus, called antigens, that provoke an immune response. To make this type of vaccine, the genetic code for the desired viral antigen molecule is put into yeast or bacteria cells. These microbes can be grown rapidly and inexpensively, and they can churn out massive quantities of antigen. Then the molecule must be purified to clinical standards so that its safe to inject into healthy people. Prevnar and Gardasil are examples of this type of vaccine.

These methods work well, but they require enormous research and development efforts. A laboratory could spend years optimizing the methods for producing one virus protein, but those methods wouldnt automatically translate to mass-producing a different protein.

For every new protein, you start over. Its a brand-new procedure every step of the way, explains immunologist Drew Weissman of the Perelman School of Medicine at the University of Pennsylvania. Weissman is one of the pioneering scientists behind the mRNA vaccine.

The way I see it, the mRNA platform is much better, its much quicker, and its cheaper, says Weissman. Thats the trilogy of what you need to improve vaccines. With mRNA, the steps are the same, no matter what virus the vaccine is targeting. This makes it easily customizable. Once an mRNA manufacturing facility is up and running, it can easily be deployed to make vaccines against any number of viral antigens.

A strand of mRNA carries the instructions for making one protein. Your cells normally make their own mRNA strands and use them as blueprints to manufacture all the proteins your body needs to function.

The vaccine slips a new strand of mRNA into the cell, like an extra page in the blueprint. This mRNA contains the instructions for making the coronavirus spike protein, and the cell reads it the same way it reads its own mRNAs, using it to build the viral protein. The immune system recognizes that protein as foreign, and starts making antibodies against it. Then, if youre exposed to the actual virus, those antibodies will be available to stop the infection. Astonishingly, in animal tests, mRNA vaccines appear to induce immunity that lasts much longer than live virus vaccines.

The beauty of mRNA is that its temporary. Your cells wont keep cranking out spike protein forever. Like an Instagram story, the mRNA fades away after a certain amount of time, because you dont need to keep making coronavirus protein forever in order to maintain the protective immunity.

Another big advantage of mRNA is that its rapidly customizable. Once scientists know the genetic sequence of a viral protein, they can make the mRNA in the lab and package it into a vaccine in a matter of weeks.

Originally envisioned as a way to deliver gene therapy, mRNA had to overcome some serious challenges before arriving at todays big moment. In 2005, Weissman and his colleague, Katalin Karik, solved one of the most difficult problems facing mRNA. In its natural form, the molecule sparks an excessive immune reaction, igniting inflammation that damages the body. To avoid this, they changed the structure of the mRNA just enough to fool the immune sentries.

Similar to DNA, RNA is made up of a series of chemical letters, a kind of code that the cell translates to make a protein. Modifying the chemical structure of one of those letters allowed the information to remain intact, and eliminated the signal that triggered the bodys immune alarms.

Before the coronavirus pandemic hit, Weissmans lab was working on vaccines for influenza, herpes, and HIV. Those will all be going into phase I clinical trials within the next year, he says. But vaccines are only the beginning of what mRNA can do.

Often in the case of genetic diseases, the problem is that a broken gene fails to produce a protein that the body needs for healthy function. The idea of gene therapy is simple: send in a healthy copy of the broken gene, which the cells can use to make the protein. Most times, researchers use viruses to deliver the gene, but viruses can cause problems of their own. Delivering mRNA to the cell without a virus circumvents some of these issues.

To ferry the mRNA into cells, it is encapsulated in a fatty coating called a lipid nanoparticle (LNP). Weissmans lab has been experimenting with ways to modify the LNP so that it can home in on certain cell types.

In sickle cell disease (SCD), a broken hemoglobin gene prevents blood cells from carrying oxygen ... [+] efficiently, and causes them to take on a rigid, sickle-shaped form.

My lab has figured out how to specifically deliver the LNP to bone marrow stem cells, Weissman says. This could lead to an inexpensive and practical cure for sickle cell anemia. An mRNA molecule can be programmed to encode the beta-hemoglobin gene, which is defective in sickle cell disease. That mRNA would be sent directly to the bone marrow cells using the specially targeted LNPs, enabling the bone marrow to produce healthy red blood cells that contain functioning beta-hemoglobin.

All that would need to be done is to give people a single intravenous injection of the mRNA LNP, and youll cure their sickle cell anemia, Weissman says. By contrast, the current FDA-approved gene-editing therapy for sickle cell requires the patients bone marrow be removed, treated, and then returned to the bodyan expensive and invasive procedure. The mRNA treatment could be simple enough to deliver in lower-income countries, where sickle cell disease impacts the health of millions of people.

An up-and-coming strategy for fighting cancer is a so-called cancer vaccine, which uses immune cells called dendritic cells (DCs). DCs perform surveillance for the immune system. When they detect something that shouldnt be there, whether its a virus, a bacteria, or even a cancer cell, the DCs chew it up, break it into its component molecules, and then show those foreign molecules to the immune cells that make antibodies.

Dendritic cells chew up viruses or other foreign bodies, and present the pieces to other immune ... [+] cells. T cells and B cells both play a role in mounting a long-lasting immunity against the pathogen.

When cancer grows slowly, though, it can slip past the DC surveillance network. To give the immune system a boost, a patients DCs are taken out and artificially loaded with tumor-specific proteins, or antigens. Back inside the body, the cells stimulate the generation of antibodies against the tumor.

Using mRNA to deliver the tumor antigen information to the DCs could provide a way to make this process easier, cheaper, and safer. BioNTech is currently conducting clinical trials on cancer vaccines for triple-negative breast cancer, metastatic melanoma, and HPV-positive head and neck cancers. Called FixVac, the vaccines include multiple tumor antigens that are frequently found across different patients. Early data published in September 2020 showed promise, suggesting that the mRNA therapy generates a lasting immune response, comparable to more expensive methods.

Karik, who is now a senior vice president at BioNTech, and Weissman both speak with an air of inevitability, as if they have only been waiting patiently for the world to catch up with their discovery. The two scientists told their stories recently at the 2nd annual mRNA Day celebration in San Diego, hosted by Trilink BioTechnologies in honor of their recently opened facility there. After hearing the tumultuous history of the technology and seeing promising new data, one attendee asked, what would you say was the turning point for mRNA therapeutics?

Karik responded simply, When people read our [2005] paper. We were waiting for somebody to respond, we did a lot of experiments, but we waited and waited. It was just too early for most people.

Weissman agreed. I think we were early, he said. It finally caught on, and it will hopefully change the world.

Full coverage and live updates on the Coronavirus

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The New Coronavirus Vaccine Is Changing The Future Of Medicine - Forbes

Bone Marrow Stem Cells Comments Off on The New Coronavirus Vaccine Is Changing The Future Of Medicine – Forbes | December 3rd, 2020

MELBOURNE, Australia and BAAR, Switzerland, Nov. 29, 2020 (GLOBE NEWSWIRE) -- Telix Pharmaceuticals Limited (ASX: TLX, Telix, the Company) announces it has entered into an agreement with Scintec Diagnostics GmbH (Scintec) to acquire TheraPharm GmbH (TheraPharm), a Swiss-German biotechnology company developing innovative diagnostic and therapeutic solutions in the field of hematology.

The acquisition of TheraPharm provides Telix with access to a portfolio of patents, technologies, production systems, clinical data and know-how in relation to the use of Molecularly Targeted Radiation (MTR) in hematology and immunology. TheraPharm is developing antibody MTR technology against CD66, a cell surface target highly expressed by neutrophils (a type of white blood cell) and tumor-infiltrating lymphocytes. As such, the technology has potentially very broad applications in the diagnosis and treatment of hematologic diseases (e.g. blood cancers), lymphoproliferative disorders and immune-mediated diseases (e.g. lupus, and multiple sclerosis). Of particular interest is the demonstrated use of the technology to safely and effectively perform bone marrow conditioning (BMC) prior to bone marrow stem cell transplant.

Telix CEO, Dr. Christian Behrenbruch stated, Telix is committed to extending and improving the lives of patients with serious diseases. As such, the acquisition of TheraPharm and its MTR assets are uniquely aligned to Telixs mission and technical strengths in antibody engineering and radiochemistry. TheraPharms technology has a significant role to play in BMC and stem cell transplantation across a broad range of blood cancers and rare diseases. The current approach to BMC employs highly toxic drugs that have a poor morbidity and mortality profile, and for which many patients are ineligible. MTR offers an excellent safety profile that may greatly expand the number of patients able to undergo life prolonging stem cell transplantation while greatly reducing the hospitalisation burden and cost associated with such procedures.

TheraPharm co-founder and Managing Director, Dr. Klaus Bosslet added, Over the past 5 years, TheraPharm, in collaboration with Dr. Kim Orchard from the University of Southampton (UK), has made excellent progress developing 90Y-besilesomab for the treatment of hematologic cancers and several related conditions including multiple myeloma, leukemia and amyloidosis. This unique asset is a logical addition to Telixs portfolio, offering a potentially rapid development path to a first commercial indication for the treatment of patients with SALA, while at the same time having potentially broad applications for stem cell transplantation in patients with more common cancers of the blood, including multiple myeloma and leukemia. We look forward to joining the Telix team in order to expedite the development of products for this under-served field.

Full transaction details, including financial terms, can be found via the Telix website and ASX portal here.

About Hematopoietic Stem Cell Transplant (HSCT)

Bone marrow conditioning (BMC) followed by hematopoietic stem cell transplantation (HSCT) is presently performed to treat patients with hematologic malignancies (blood cancers), with the objective of extending patient survival or achieving cure. HSCT is also performed for a broad range of non-cancer conditions. HSCT is preferentially performed in countries of high income (Europe >30,000, Americas >20,000, worldwide >65,000 p.a., respectively) and is growing at around 5% annually.

About Systemic Amyloid Light-Chain Amyloidosis (SALA)

SALA is a rare, but serious protein deposition disease, caused by a protein known as amyloid that is produced by abnormal plasma cells residing in the bone marrow. As amyloid accumulates in the organs of the body, organ function will eventually deteriorate, ultimately causing organ failure. SALA has an estimated prevalence of 30,000 and 45,000 in United States and Europe, respectively and while a rare disease, SALA portends a very poor prognosis, with a median survival from diagnosis of ~11 months if untreated.

The current standard of care comprises of induction therapy (typically cyclophosphamide, bortezomib, dexamethasone) plus high dose melphalan BMC, followed by HSCT. This approach is typically only accessible to a small proportion of patients (<20%) who are able to tolerate induction therapy and melphalan BMC.

About Telix Pharmaceuticals Limited

Telix is a clinical-stage biopharmaceutical company focused on the development of diagnostic and therapeutic products using Molecularly Targeted Radiation (MTR). Telix is headquartered in Melbourne, Australia with international operations in Belgium, Japan and the United States. Telix is developing a portfolio of clinical-stage oncology products that address significant unmet medical needs in prostate, kidney and brain cancer. Telix is listed on the Australian Securities Exchange (ASX: TLX). For more information visit http://www.telixpharma.com.

AboutTheraPharm GmbH

TheraPharm is a biotechnology company specialised in the research, development and manufacturing of monoclonal antibodies for targeted radiation of hematopoietic malignant and non-malignant diseases, lymphoproliferative diseases, conditioning for allogeneic stem cells as well as in diagnostics of inflammatory diseases and bone marrow metastases.

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Telix Pharmaceuticals Limited Acquires TheraPharm GmbH, Broadening Reach to Hematologic Cancers and Transplant Medicine - BioSpace

Bone Marrow Stem Cells Comments Off on Telix Pharmaceuticals Limited Acquires TheraPharm GmbH, Broadening Reach to Hematologic Cancers and Transplant Medicine – BioSpace | November 30th, 2020

Scientists at Bay Area universities, laboratories, biotechnology companies and drug manufacturers are fashioning drug concoctions out of blood plasma, chimpanzee viruses and cells taken from bone marrow in the race to rid the world of COVID-19.

The microbial treasure hunt is not just to find a cure which may not be possible but to control the debilitating health problems caused by the coronavirus.

Major progress has been made this year. The antiviral drug remdesivir, produced in Foster City, has improved recovery times, and the steroid dexamethasone has cut the number of deaths in severely ill patients.

What follows is a list of some of the most promising medications and vaccines with ties to the Bay Area:

Antibodies

and Immunity

Mesenchymal stem cells / UCSF and UC Davis Medical Center:

UCSF Dr. Michael Matthay is leading a study of whether a kind of stem cell found in bone marrow can help critically ill patients with severe respiratory failure, known as ARDS. Matthay hopes the stem cells can help reduce the inflammation associated with some of ARDS most dire respiratory symptoms, and help patients lungs recover.

In all, 120 patients are being enrolled at UCSF Medical Center, Zuckerberg San Francisco General Hospital, the UC Davis Medical Center in Sacramento and hospitals in Oregon and Texas. He said the trial, which includes a small number of ARDS patients who dont have COVID-19, should have results by summer or fall 2021. So far, 28 patients are enrolled in San Francisco.

Lambda-interferon / Stanford University:

Lambda-interferon is a manufactured version of a naturally occurring protein that had been used to treat hepatitis, and researchers hoped it would help patients in the early stages of COVID-19.

Stanford researchers completed their trial of lambda-interferon and found that it did not boost the immune system response to coronavirus infections.

That trial did not find any difference in outcomes between the treatment and placebo, said Yvonne Maldonado, chief of pediatric infectious diseases at Lucile Packard Childrens Hospital at Stanford, where 120 patients were enrolled in the trial. It didnt work.

Antiviral drugs

Remdesivir / Gilead Sciences (Foster City):

Remdesivir, once conceived as a potential treatment for Ebola, was approved by the Food and Drug Administration in October for use on hospitalized COVID-19 patients.

Trademarked under the name Veklury, the drug interferes with the process through which the virus replicates itself. It was one of the drugs given to President Trump and has been used regularly in hospitals under what is known as an emergency use authorization.

It was approved after three clinical trials showed hospitalized coronavirus patients who received remdesivir recovered five days faster on average than those who received a placebo. Patients who required oxygen recovered seven days faster, according to the studies.

Gilead now plans to conduct clinical trials to see how remdesivir works on pediatric patients, from newborns to teenagers, with moderate to severe COVID-19 symptoms. Remdesivir is also being studied with steroids and other drugs to see if it works better as part of a medicinal cocktail. An inhalable form of the drug is also being developed.

Favipiravir / Fujifilm Toyama Chemical (Stanford University):

This antiviral drug, developed in 2014 by a subsidiary of the Japanese film company to treat influenza, is undergoing numerous clinical studies worldwide, including a trial involving 180 patients at Stanford University.

Stanford epidemiologists are testing favipiravir to see if it prevents the coronavirus from replicating in human cells, halts the shedding of the virus and reduces the severity of infection. Unlike remdesivir, it can be administered orally, so it can be used to treat patients early in the disease, before hospitalization is necessary.

The Stanford study has so far enrolled about 90 patients, who are given the drug within 72 hours of when they were first diagnosed with COVID-19. Half of them get a placebo. People can enroll by emailing treatcovid@stanford.edu.

Monoclonal antibodies

REGN-COV2 / Regeneron Pharmaceuticals / Stanford School of Medicine:

The REGN-COV2 cocktail is the same one Trump received, and Stanford is one of dozens of locations nationwide where clinical trials are being held. Two separate trials are under way at Stanford one for hospitalized patients, the other for outpatients. A third trial is about to begin for people who arent sick but are in contact with carriers of the virus.

Regeneron halted testing on severely ill patients requiring high-flow oxygen or mechanical ventilation after the independent Data and Safety Monitoring Board determined that the drug was unlikely to help them.

The drug is a combination of two monoclonal antibodies lab-made clones of the antibodies produced naturally in people who have recovered from COVID-19. The antibodies bind to the virus spike protein and block the virus ability to enter cells.

Dr. Aruna Subramanian, professor of infectious diseases at Stanford and lead investigator for the inpatient trial, said the 21 hospitalized patients in the study receive a high dose like Trump, a lower dose or a placebo. Subramanian plans to expand the inpatient trial to 45 patients. The outpatient study has enrolled a little more than 40 of the 60 patients researchers intend to sign up.

Theres enough promising evidence that it helps people early in the infection, Subramanian said. What we dont know is whether it helps people who are pretty sick but not critically ill.

Bamlanivimab / Eli Lilly / Stanford and UCSF:

Stanford and UCSF are testing the Eli Lilly monoclonal antibodies on outpatients after the pharmaceutical company halted trials on hospitalized COVID-19 patients because of adverse results.

Dr. Andra Blomkalns, chair of emergency medicine at Stanford and the lead in the Eli Lilly outpatient trial, said she is now enrolling older people with comorbidities like heart disease, chronic lung disease, a history of strokes and severe obesity shortly after they test positive.

The hypothesis is that the bamlanivimab monotherapy, which is very similar to the Regeneron monoclonals, might work best early in the infection. Although about 400 patients have been enrolled in the Lilly phase 3 trials nationwide, to date fewer than 10 have been enrolled at Stanford and UCSF.

Matthay, who headed up the Lilly monoclonal study with LY-CoV555 at UCSF, said the cancellation of this inpatient trial was disappointing, but just because this one did not work, doesnt mean another one wont work for hospitalized patients.

Blomkalns said the testing criteria has been changing. She expects the outpatient trial to open soon to adolescents ages 12 and up to determine whether the drug can be used as a preventive.

Designer monoclonal antibodies / Vir Biotechnology, San Francisco:

Scientists at Vir are studying several types of monoclonal antibodies, including a type engineered to activate T cells, which can search out and destroy cells infected with the coronavirus. A study published in the journal Nature in October found that monoclonals, modified to bind with certain receptors, stimulated T cells and improved the human immune response.

By observing and learning from our bodys powerful natural defenses, we have discovered how to maximize the capacity of antibodies through the amplification of key characteristics that may enable more effective treatments for viral diseases, said Herbert Virgin, the chief scientific officer at Vir and co-author of the study.

A similarly modified monoclonal antibody, leronlimab, is being studied in coronavirus clinical trials by its Washington state drugmaker, CytoDyn, which has developed drugs to treat HIV. The companys chief medical officer is in San Francisco, and the company that does laboratory tests of leronlimab is in San Carlos.

Anti-inflammatory drugs

Colchicine / UCSF (San Francisco and New York):

The anti-inflammatory drug commonly used to treat gout flare-ups is being studied by scientists at UCSF and New York University. The drug short-circuits inflammation by decreasing the bodys production of certain proteins, and researchers hope that it will reduce lung complications and prevent deaths from COVID-19.

Preliminary results from a clinical trial found that Colchicine can be effective in reducing systemic symptoms of COVID-19 by inhibiting inflammatory biomarkers.

Selinexor / Kaiser Permanente:

Kaiser hospitals in San Francisco, Oakland and Sacramento are studying selinexor, an anticancer drug that blocks a key protein in the cellular machinery for DNA processing. Preliminary findings during the trials indicated that low doses of selinexor helped hospitalized patients with severe COVID-19. The drug has both antiviral and anti-inflammatory properties, and its administered orally, according to Kaisers Dr. Jacek Skarbinski.

Vaccines

VXA-COV2-1 / Vaxart, South San Francisco:

The biotechnology company Vaxart is testing VXA-COV2-1, the only potential vaccine in pill form. It uses the genetic code of the coronavirus to trigger a defensive response in mucous membranes. The hope is that the newly fortified membranes will prevent the virus from entering the body.

Its the only vaccine (candidate) that activates the first line of defense, which is the mucosa, said Andrei Floroiu, Vaxarts chief executive. He said intravenous vaccines kill the virus after it is inside the body, but this one stops it beforehand.

The drug, which is effective against influenza and norovirus, induced both neutralizing antibodies and T cells during coronavirus drug trials, according to preliminary trial results published in September.

VaxiPatch / Verndari (Napa and UC Davis Medical Center):

A Napa company, Verndari, is studying vaccines for COVID-19 that can be delivered using an adhesive patch. Researchers at UC Davis Medical Center in Sacramento said the patch caused an immune response in preclinical tests.

An October report in the online journal ScienceDirect touted the system, saying it could serve as a shelter in place vaccination strategy, in which vulnerable populations receive delivery at home without needing to engage an already-overtaxed health care infrastructure.

If the vaccine is proven effective and safe, patients could receive it through the mail, according to Dr. Daniel Henderson, Verndaris chief executive officer.

ChAdOx1 / AstraZeneca (UCSF, San Francisco General Hospital, Bridge HIV):

Enrollment is under way at 80 sites in the United States, including three in the Bay Area, for the phase 3 trial of AstraZenecas vaccine, developed by Oxford University from an adenovirus, which typically causes colds in chimpanzees.

At least 1,000 of the 40,000 participants in the phase 3 AstraZeneca trial will be from the Bay Area, including 500 at Sutter Healths East Bay AIDS Center in Oakland, 250 at Zuckerberg San Francisco General Hospital and another 250 at Bridge HIV San Francisco.

An interim analysis of trials in Britain and Brazil showed the vaccine was 90% effective in preventing COVID-19 in 131 patients who got a half-dose of the vaccine by mistake. The vaccine was only 62% effective in people who got a full dose, leading to major questions about the results and how the trial was conducted.

Bay Area trial leaders Dr. Annie Luetkemeyer of UCSF and Dr. Susan Buchbinder, director of Bridge HIV and a UCSF professor of medicine and epidemiology, are hoping future trial results are more clear. Thats because AstraZenecas vaccine is cheaper than those made by its rivals Pfizer and Moderna, whose vaccines were 95% and 94.5% effective in preliminary tests.

The AstraZeneca candidate can also be stored at temperatures between 36 and 46 degrees Fahrenheit, which is orders of magnitude higher than the Pfizer and Moderna vaccines. The Pfizer and Moderna vaccines must be kept at 94 degrees below zero Fahrenheit, colder than many storage facilities can manage.

Johnson & Johnson (Stanford University)

The Johnson & Johnson clinical trials have enrolled 20,000 of the 60,000 volunteers worldwide that officials expect to have signed up by Christmas. That includes 70 people at Stanford.

The vaccine is, like the AstraZeneca version, a chimpanzee adenovirus that was genetically altered so that it carries the RNA of the coronavirus spike protein. The technique inspires the body to produce antibodies that block the protein without causing people to get sick.

Phase 2 studies show that it produces a good immune response and the early results of phase 3 show that its safe, said Dr. Philip Grant, assistant professor of infectious disease at Stanford and leader of the trial.

Grant, who is enrolling about 15 people a day for the trial, said he doesnt expect results on the vaccines effectiveness until sometime in March.

Peter Fimrite is a San Francisco Chronicle staff writer. Email: pfimrite@sfchronicle.com Twitter: @pfimrite

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Coronavirus treatments and vaccines. Here are the latest developments - San Francisco Chronicle

Bone Marrow Stem Cells Comments Off on Coronavirus treatments and vaccines. Here are the latest developments – San Francisco Chronicle | November 30th, 2020

Global Research Antibodies Market: Overview

The global research antibodies market is anticipated to rise at a notable pace over the forecast period. Antibodies display exceptional physiological properties that make them sought-after for cell research.

Antibodies display other properties too. As they have the ability to attach to specific molecules, this enables specific molecules to be isolated for research. Hence, this makes for a key factor for continual research to examine the physiology and anatomy of antibodies.

The report serves to identify prevailing growth trends based on which projections made. The report constitutes most relevant data pertaining to comprehend the growth dynamics of research antibodies market. Geographical distribution of the research antibodies market and an analysis of the competitive structure are highlights of the report.

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Global Research Antibodies Market: Key Trends

Currently, pharmaceutical and biopharmaceutical companies are undertaking extensive R&D activities to introduce novel products. These pursuits involve widespread use of antibodies because of their exceptional physiological properties. Therefore, research on antibodies receives a boost for their use in secondary cell research.

Pharmaceutical giants are also making hefty investments for advancement of antibodies research.

Increasing incidence of chronic diseases and life-threatening diseases such as cancer has led to extensive initiatives for advanced therapeutics. Pharmaceutical and biotechnology companies are making efforts in terms of upgrading their R&D capability and pumping money. These efforts are aimed to develop advanced therapeutics as well as personalized medicine for a gamut of chronic and fatal diseases. These factors collectively bode well for research antibodies market.

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At present, stem cell research is finding keen interest of researchers and geneticists. Several studies support the efficacy of stem cell for blood cancers, blood and bone marrow diseases, immune disorders. Lately, stem cells from the umbilical cord and stem cells from the blood stream have been used to treat rare blood related diseases. Due to the dependency an antibodies for stem cell research, researchers are involved to isolate different antibodies molecules. This is aiding growth of research antibodies market.

Lastly, novel use of antibodies that are receiving acceptance of accredited bodies is serving to boost the research antibodies market. For example, in a new development, FDA approved the clinical application of DNA-encoded monoclonal antibody therapy as a prevention against Zika virus.

Global Research Antibodies Market: Regional Analysis

The global research antibodies market is spread across North America, Asia Pacific, Europe, Latin America, and the Middle East & Africa. Among them, North America holds supremacy in the overall market. The region being home to large biotechnology and biopharmaceutical companies, along with immense government aid for research are key factors behind exceptional growth of North America antibodies market.

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Stringent regulations in place for manufacturers of antibodies and due adherence to these regulations accounts for high quality products from the region. This further pushes the North America research antibodies market.

On the other hand, Asia Pacific is emerging as a key region for research antibodies. Increasing R&D for antibodies and adoption of novel techniques for the production of antibodies is serving to fuel the region.

Global Research Antibodies Market: Competitive Outlook

Prominent players in the global research antibodies market include Abcam plc, Agilent Technologies, and Thermo Fisher Scientific Inc.

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Research Antibodies Market is Driven by Increasing Incidence of Chronic Diseases and Life-threatening Diseases - Cheshire Media

Bone Marrow Stem Cells Comments Off on Research Antibodies Market is Driven by Increasing Incidence of Chronic Diseases and Life-threatening Diseases – Cheshire Media | November 30th, 2020

A one-year-old boy has been diagnosed with a condition so rare only one in a million people suffer from it.

Max Gardner was diagnosed with aplastic anaemia - a condition that means the bone marrow and stem cells do not produce enough blood cells and is fatal if untreated.

He was diagnosed after his parents, Connor Gardner and Rachel Nicholson, who live in Hebburn, became alarmed by significant bruising and rashes all over his body.

The couple took him to South Tyneside District Hospital, where he was incorrectly diagnosed with immune thrombocytopenic purpura, a condition which a child will grow out of.

However, as Maxs condition worsened, he ended up at the Royal Victoria Infirmary in Newcastle, where doctors conducted tests which showed he had the much rarer aplastic anaemia.

Connor said: He looked like he was a child abuse victim; we were really worried about what people would think, as he was covered in bruises.

We took him to the RVI for further tests, and they realised that maybe the condition was worse. Initially, we thought he would be diagnosed with leukaemia, but the consultant told us that it was aplastic anaemia after a bone marrow biopsy, which was administered under anaesthetic.

They told us about the condition, and that the outcome could lead to death if Max was to catch any type of sickness bug, as his immune system was non-existent.

We got our emotions out after we got the diagnosis we had a cry but we knew that we needed to be there for Max and help him get better.

The only way to cure aplastic anaemia fully is with a bone marrow transplant, and both Connor, 29, and Rachel, 27, were tested to see if they were matches.

Fortunately, Rachel was a near-perfect match, a very rare scenario.

Connor said: Usually they would use siblings for the transplant but Max does not have any. There is about a 25% chance that me or Rachel would be a match, and then there is about a 1% chance that it would be a 9/10 match.

The condition that Max has affects one in a million people, so it is very unfortunate for Max to have had this condition, but it is lucky that his mother has been a near-perfect match.

Chemotherapy is the next stage before you have the transplant, but that can lead to wiping out fertility, so we agreed to a new trial that would give Max the best chance of being able to have children of his own when he grows up.

They take a biopsy of one of his testicles and they store it for future; it is the best chance he has of having a child when he is older if he is infertile.

The family now have to shield for two weeks, before Max and his mother head back to hospital and onto the transplant ward, where he will spend the next two months.

Fortunately, Rachel can stay with Max during this time, but Connor can only see his son at specific visiting hours and has to isolate, so that the risk of spreading any illness is at a minimum.

He said: Max starts his chemotherapy on December 10, which takes place over five days, and during that time Rachel will be getting treatment so that the hospital can help harvest her bone marrow.

Then, when she goes to give the transplant, she will be there for four hours while the machine separates the bone marrow before it is given to Max.

Then he gets a bone marrow transplant, which is very similar to a normal blood transfusion."

Connor and Rachel have set up a fundraising page to help pay for the added costs of not working and to help them support them through this tough time.

He said: We have been overwhelmed with the support that people have given us and the GoFundMe page has been a great way for people to give us time.

I have been taken back by the generosity of total strangers.

Connor stressed the importance of raising awareness for bone marrow transplants, and had his fiance not been a very rare match, they would likely have had to wait for a match on the donor register.

I think it is important to raise awareness of the Anthony Nolan page. We have been lucky enough to get a donor for Max through his mam, but there are lots of people out there who have not been so lucky and are waiting for a donor.

We have met a little girl who is eight years old and she hasnt got a match yet, so we are just hoping that people will join the donor list as it may save someones life.

You can donate to the fundraising campaign by visiting here.

Continued here:
Family's heartache after Hebburn boy diagnosed with one in a million condition - Chronicle Live

Bone Marrow Stem Cells Comments Off on Family’s heartache after Hebburn boy diagnosed with one in a million condition – Chronicle Live | November 30th, 2020

The U.S. Food and Drug Administration (FDA) has approved naxitamab* (naxitamab-gqgk; Danyelza; Y-mAbs Therapeutics), a humanized form of the mouse antibody 3F8, in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), for the treatment of pediatric patients 1 year of age and older and adult patients with relapsed or refractory high-risk neuroblastoma in the bone or bone marrow who have demonstrated a partial response (PR), minor response (mR), or stable disease (SD) to prior therapy.[1]

A rare diseaseNeuroblastoma is a heterogeneous pediatric neoplasm that arises in the sympathetic nervous system. The disease is the most common extra-cranial solid tumor in infants and children, representing between 8%-10% of all childhood tumors. Overall, neuroblastoma accounts for approximately 15% of all cancer-related deaths in children. [1]

The clinical behavior of neuroblastoma is highly variable, with some tumors being easily treatable, resulting in near-uniform survival. The majority of tumors are, however, very aggressive, with a high risk of death. [2] Age, stage, and amplification of the MYCN oncogene are the most validated prognostic markers.[2]

The incidence of neuroblastoma is 10.2 cases per million children under 15 years of age. [3] In the United States, nearly 700 new cases are reported each year. While 90% of cases are diagnosed before the age of 5, approximately 30% of patients are diagnosed within the first year. The median age of diagnosis is 22 months. [4]

Neuroblastoma develops in very early forms of nerve cells that are usually found in a developing baby, which explains why children as young as newborns can develop this cancer.

The disease rarely presents in adolescence and adulthood, but outcomes are much poorer in this age group. There does not appear to be an increased prevalence among races, but there is a slight predilection for males (1.2:1).[4]

Neuroblastoma develops in a part of the peripheral nervous system called the sympathetic nervous system. Since some of the sympathetic nervous system cells are concentrated in the adrenal glands, which sit above the kidneys, neuroblastoma often starts growing there. Tumors typically begin in the belly, neck, chest, pelvis, or adrenal glands and can spread to other parts of the body, including the bones.

All patients are staged based on the International Neuroblastoma Staging System Committee (INSS) system, ranging from stage 1 through stage 4S. Based on this staging system, patients with stage 4 disease diagnosed after one year of age are classified in the high-risk category, where the neuroblastoma tumor cells have already metastasized to other sites in the body, such as the bone or bone marrow.

Essentially all patients who have tumors with many copies, or amplification, of the MYCN oncogene also have high-risk disease, even if they do not have evidence of the tumor having spread.

Although children with a family history of neuroblastoma may have a higher risk for developing this disease, this factor accounts for only 1-2 % of all cases of neuroblastoma. The vast majority of children who develop the tumor, do not have a family history of the same.

Mechanism of actionIn simple terms, naxitamab, conceived and developed by physician-scientist Nai-Kong Cheung, M.D., Ph.D., a medical oncologist at Memorial Sloan Kettering ** who heads the organizations neuroblastoma program, detects neuroblastoma cells that have survived chemo- or radiation therapy by attaching to GD2, a ganglioside that is ubiquitously expressed in the plasma membrane of neuroblastoma and is shed into the circulation, after which the patients own immune system, especially white blood cells, can destroy the malignant neuroblastoma cells. [5]

In the late 1980s, investigators at Memorial Sloan Kettering started using 3F8 in combination with surgery and chemotherapy to treat patients diagnosed with neuroblastoma. The investigational treatment significantly improved cure rates for pediatric patients with high-risk disease.

Later, in 2007, Cheung and colleagues began developing a humanized form of 3F8 called Hu3F8. In August 2011 the researchers started a phase I study of Hu3F8 (NCT01419834). The study was designed to investigate the best and safest dose to give to patients.

Accelerated approval The new indication of naxitamab + GM-CSF is approved under accelerated approval regulation based on overall response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefits in a confirmatory trial.

Naxitamab is a humanized, monoclonal antibody that targets the ganglioside GD2, which is highly expressed in various neuroectoderm-derived tumors and sarcomas. The drug is administered to patients three times per week in an outpatient setting and the treatment is repeated every four weeks. The product has received Priority Review, Orphan Drug, Breakthrough Therapy, and Rare Pediatric Disease designations from the FDA.

Much needed treatmentOver the last decades, the development of novel treatments for pediatric cancers has been successful. For example, the five-year survival rates for children diagnosed with cancer in the late 1980s approaches 70%. For some types of localized embryonal tumors, including retinoblastoma and Wilms tumor, the cure rates approach or exceed 90%.

However, for every two children who survive today, one child still succumbs to their disease. And for some childhood cancers, such as neuroblastoma and certain types of brain cancer, the prognosis remains poor. Hence, despite the observed successes, there remained a major unmet medical need remains patients diagnosed with neuroblastoma. The development and subsequent approval of naxitamab may be one much-needed treatment options for these patients. [6]

[The approval represents a major milestone] for children living with refractory/relapsed high-risk neuroblastoma, noted Thomas Gad, founder, Chairman, and President of Y-mAbs Therapeutics, whose own daughters neuroblastoma was successfully treated with 3F8 at Memorial Sloan Kettering more than a decade ago.

In 2015, Memorial Sloan Kettering licensed Hu3F8 to Y-mAbs Therapeutics tpo expand the clinical trial and development program and manufacturing of naxitamab.

Its very exciting to see this treatment go from being an experimental therapy used at my daughters bedside to now being FDA approved, Gad added.

We believe that naxitamab in combination with GM-CSF is a much-needed treatment for patients with relapsed/refractory high-risk neuroblastoma in the bone or bone marrow who have historically not had approved treatments available. This approval of Y-mAbs first BLA represents a key step in working towards our mission of becoming a world leader in developing better and safer antibody-based oncology products addressing unmet pediatric and adult medical needs, said Claus Moller, Y-mAbs Therapeutics Chief Executive Officer.

Clinical trialsThe FDA approval of naxitamab is supported by clinical evidence from two pivotal studies in patients with high-risk neuroblastoma with refractory or relapsed disease.

In these clinical studies, naxitamab appears to be well tolerated with few discontinuations of treatment. The observed treatment-related adverse events were clinically manageable.

The efficacy of naxitamab in combination with GM-CSF was evaluated in two open-label, single-arm trials in patients with high-risk neuroblastoma with refractory or relapsed disease in the bone or bone marrow.

Both trials included patients with relapsed or refractory neuroblastoma in the bone marrow or bone. Participating patients received a 3 mg/kg of naxitamab intravenously on days one, three, and five of each four-week cycle, in addition to GM-CSF subcutaneously, or under the skin, at varying doses throughout the cycle. Patients were allowed to receive preplanned radiation in specific areas based on which trial they were enrolled in.

Efficacy outcomes included overall response rate (ORR) according to the revised International Neuroblastoma Response Criteria (INRC), as determined by independent pathology and imaging review and confirmed by at least one subsequent assessment. An additional efficacy outcome measure was the duration of response (DOR).

Study 201In the first study (Study 201; NCT03363373), a multicenter open-label, single-arm trial. researchers evaluated the combination of naxitamab in combination with GM-CSF in a subpopulation of patients who had refractory or relapsed high-risk neuroblastoma in the bone or bone marrow and demonstrated a partial response, minor response, or stable disease to prior therapy. Patients with progressive disease were excluded.

Of the 22 patients included in the efficacy analysis, 64% had refractory disease and 36% had relapsed disease. The median age was 5 years (range 3 to 10 years), 59% were male; 45% were White, 50% were Asian and 5% were Black.

MYCN amplification was present in 14% of patients and 86% of patients were International Neuroblastoma Staging System (INSS) stage 4 at the time of diagnosis. Disease sites included 59% in the bone only, 9% in bone marrow only, and 32% in both. Prior therapies included surgery (91%), chemotherapy (95%), radiation (36%), autologous stem cell transplant (ASCT) (18%), and anti-GD2 antibody treatment (18%).

Study 12-230The second study (Study 12-230; NCT01757626), a single-center, open-label, single-arm clinical trial, included a subpopulation of patients who had relapsed or refractory high-risk neuroblastoma in bone or bone marrow and demonstrated a partial response, minor response, or stable disease to prior therapy. In this study patients with progressive disease were excluded.

Participating patients received at least one systemic therapy to treat disease outside of the bone or bone marrow prior to enrollment. They were required to have received at least one dose of naxitamab at a dose of 3 mg/kg or greater per infusion and have evaluable disease at baseline according to independent review per the revised INRC. Radiation to non-target bony lesions and soft tissue lesions was permitted at the investigators discretion (assessment of response excluded sites that received radiation).

Of the 38 patients included in the efficacy analysis, 55% had relapsed neuroblastoma and 45% had refractory disease; 50% were male, the median age was 5 years (range 2 to 23 years), 74% were White, 8% Asian and 5% were Black, 5% Native American/American Indian/Alaska Native, 3% other races and 5% was not available. MYCN-amplification was present in 16% of patients and most patients were International Neuroblastoma Staging System (INSS) stage 4 (95%).

Fifty percent (50%) of patients had disease involvement in the bone only, 11% only in bone marrow, and 39% in both. Prior therapies included surgery (100%), chemotherapy (100%), radiation (47%), autologous stem cell transplant (ASCT) (42%), and anti-GD2 antibody treatment (58%)

Adverse eventsThe most common adverse reactions (incidence 25% in either trial) in patients receiving naxitamab were infusion-related reactions, pain, tachycardia, vomiting, cough, nausea, diarrhea, decreased appetite, hypertension, fatigue, erythema multiforme, peripheral neuropathy, urticaria, pyrexia, headache, injection site reaction, edema, anxiety, localized edema, and irritability.

The most common Grade 3 or 4 laboratory abnormalities (5% in either trial) were decreased lymphocytes, decreased neutrophils, decreased hemoglobin, decreased platelet count, decreased potassium, increased alanine aminotransferase, decreased glucose, decreased calcium, decreased albumin, decreased sodium, and decreased phosphate.

Boxed warningThe prescribing information for naxitamab contains a Boxed Warning which states that the drug can cause serious infusion-related reactions and neurotoxicity, including severe neuropathic pain, transverse myelitis, and reversible posterior leukoencephalopathy syndrome (RPLS). Hence, to mitigate these risks, patients should receive premedication prior to each naxitamab infusion and be closely monitored during and for at least two hours following completion of each infusion.

Note* Also known as humanized 3F8 or Hu3F8,** Researchers at Memorial Sloan Kettering Cancer Center (MSK) developed naxitamab, which is exclusively licensed by MSK to Y-mAbs. As a result of this licensing arrangement, MSK has institutional financial interests related to the compound and Y-mAbs.

Clinical trialsHumanized 3F8 Monoclonal Antibody (Hu3F8) in Patients With High-Risk Neuroblastoma and GD2-Positive Tumors NCT01419834Humanized 3F8 Monoclonal Antibody (Hu3F8) When Combined With Interleukin-2 in Patients With High-Risk Neuroblastoma and GD2-positive Solid Tumors NCT01662804Humanized Anti-GD2 Antibody Hu3F8 and Allogeneic Natural Killer Cells for High-Risk Neuroblastoma NCT02650648Study of the Safety and Efficacy of Humanized 3F8 Bispecific Antibody (Hu3F8-BsAb) in Patients With Relapsed/Refractory Neuroblastoma, Osteosarcoma and Other Solid Tumor Cancers NCT03860207Combination Therapy of Antibody Hu3F8 With Granulocyte- Macrophage Colony Stimulating Factor (GM-CSF) in Patients With Relapsed/Refractory High-Risk Neuroblastoma NCT01757626Naxitamab for High-Risk Neuroblastoma Patients With Primary Refractory Disease or Incomplete Response to Salvage Treatment in Bone and/or Bone Marrow NCT03363373

Highlights of prescription informationNaxitamab (naxitamab-gqgk; Danyelza; Y-mAbs Therapeutics) [Prescribing Information]

Reference[1] Park JR, Eggert A, Caron H. Neuroblastoma: biology, prognosis, and treatment. Hematol Oncol Clin North Am. 2010 Feb;24(1):65-86. doi: 10.1016/j.hoc.2009.11.011. PMID: 20113896.[2] Modak S, Cheung NK. Neuroblastoma: Therapeutic strategies for a clinical enigma. Cancer Treat Rev. 2010 Jun;36(4):307-17. doi: 10.1016/j.ctrv.2010.02.006. Epub 2010 Mar 12. PMID: 20227189.[3] Maris JM. Recent advances in neuroblastoma. N Engl J Med. 2010 Jun 10;362(23):2202-11. doi: 10.1056/NEJMra0804577. PMID: 20558371; PMCID: PMC3306838.[4] Esiashvili N, Anderson C, Katzenstein HM. Neuroblastoma. Curr Probl Cancer. 2009 Nov-Dec;33(6):333-60. doi: 10.1016/j.currproblcancer.2009.12.001. PMID: 20172369.[5] Balis FM, Busch CM, Desai AV, Hibbitts E, Naranjo A, Bagatell R, Irwin M, Fox E. The ganglioside GD2 as a circulating tumor biomarker for neuroblastoma. Pediatr Blood Cancer. 2020 Jan;67(1):e28031. doi: 10.1002/pbc.28031. Epub 2019 Oct 14. PMID: 31612589.[6] Balis FM. The Challenge of Developing New Therapies for Childhood Cancers. Oncologist. 1997;2(1):I-II. PMID: 10388032.

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US FDA Approves Naxitamab for the Treatment of Neuroblastoma - OncoZine

Bone Marrow Stem Cells Comments Off on US FDA Approves Naxitamab for the Treatment of Neuroblastoma – OncoZine | November 28th, 2020

INTRODUCTION

The myeloproliferative neoplasms are a family of clonal blood disorders characterized by overproduction of platelets [essential thrombocythemia (ET)], overproduction of red blood cells [polycythemia vera (PV)], or bone marrow fibrosis [myelofibrosis (MF)]. The genetic bases for these diseases have largely been described: Mutations in JAK2 are found in 99% of PV and 50 to 60% of ET and MF cases, while frameshift mutations in CALR are responsible for 25 to 40% of cases of ET and MF (13). Frameshift mutants of calreticulin (CALR) have a novel C terminus that acts as a rogue ligand for the thrombopoietin receptor, MPL, and activates Janus kinasesignal transducer and activator of transcription (JAK-STAT) signaling (4, 5). We recently described the generation of a mouse model of mutant CALR-driven ET that faithfully recapitulates the key phenotypes of the human disease, namely, increased numbers of cells throughout the megakaryocytic (MK) lineage, particularly platelets (6).

Hematopoiesis is classically modeled as a stepwise process beginning with a multipotent hematopoietic stem cell (HSC), which is functionally defined by its capability to reconstitute multilineage hematopoiesis when transplanted into a myeloablated recipient (7). This HSC then transits through a series of intermediate stages with increasing lineage restriction to terminally differentiated blood cells (8, 9). However, newly popularized single-cell technologies such as single-cell RNA sequencing (scRNAseq) have reshaped our understanding of hematopoiesis and suggest that cells travel through a continuum of differentiation rather than a series of rigidly defined stages (10, 11). In a recent demonstration of the power of scRNAseq to untangle complex differentiation processes, it was used to interrogate the transcriptomes of hematopoietic stem and progenitor cells (HSPCs) to identify novel intermediate populations within erythropoiesis, which could then be isolated and characterized via fluorescence-activated cell sorting (FACS) strategies (12).

While HSCs are traditionally defined to be capable of reconstituting all blood lineages in transplantation experiments, there is an increasing body of evidence that some cells within the immunophenotypic HSC compartment already exhibit some lineage bias or restriction (1315). Studies in mice have shown that MK and erythroid lineages may branch off before other myeloid and lymphoid lineages (1618), and lineage tracing studies have shown the MK lineage to be the earliest generated from HSCs (1923). A transposon-based lineage tracing strategy showed some tags to be shared between long-term HSCs (LT-HSCs) and megakaryocyte progenitors (MkPs) but not multipotent progenitors (MPPs), indicative of a direct pathway linking HSCs and MK bypassing MPP (19). We therefore asked whether our mouse model of mutant CALR-driven ET could allow us to interrogate the differences in the hematopoietic landscapes between wild-type (WT) and disease model mice, with a particular focus on MK trajectories.

We generated scRNAseq data from FACS-sorted HSPCs [Lin Sca1+ cKit+ (LSK) and Lin Sca1 cKit+ (LK) populations] from a pair of WT and CALR DEL (knock-in of del52 allele) homozygous (HOM) littermate mice. After quality control, we retained 11,098 WT (5959 LSK and 5139 LK) and 15,547 HOM (7732 LSK and 7815 LK) cells for downstream analysis. We began by defining highly variable genes, which we used to perform principal component analysis (PCA) and generate a k = 7 nearest-neighbor graph. Cells were then assigned to clusters by mapping onto a previously published dataset of 44,082 LK cells (24), with manual annotation of clusters (fig. S1A). Cells from all major blood lineages can be seen and separate into distinct trajectories. To determine which cells were over- or underrepresented in the CALR DEL HOM mouse, we compared relative numbers of cells from each genotype. The most notable changes in relative cell abundance were increased numbers of cells in the HSC and MK clusters (fig. S1B), consistent with the increased platelet phenotype of our ET mouse model (6). We repeated the analysis on a second pair of WT and CALR DEL HOM littermate mice, in this case retaining 3451 WT (972 LSK and 2479 LK) and 12,372 HOM (4548 LSK and 7824 LK) cells for downstream analysis after quality control, and again observed an increase in cells in the HSC and MK clusters (fig. S1C).

To better understand the subgroups of cells within stem/progenitor cells, we chose to use partition-based graph abstraction (PAGA) (25) to visualize our data. This method generates a graph in which each node represents a group of closely related cells and edge weights correspond to the strength of connection between two nodes. We again compared relative abundances between WT and CALR DEL HOM mice and colored the nodes so red nodes are enriched in CALR mice, while blue nodes are underrepresented. We observed that the fine cluster that was most overrepresented in CALR DEL HOM mice (marked with an arrow) fell between the HSC and MK clusters in both repeats (Fig. 1A and fig. S1D). We plotted the expression of the MK markers Cd9, Itga2b (CD41), Mpl, Pf4, and VWF in our PAGA and hypothesized two MK trajectories, as indicated by the green and blue arrows (fig. S1E). As the fine cluster most overrepresented in CALR DEL HOM mice would be an intermediate on one of these trajectories (green arrow), we further hypothesized that these cells would be of particular relevance in the disease setting of mutant CALR-driven ET and thus aimed to further study them.

(A) PAGA of scRNAseq data from WT and CALR DEL HOM mice. Red nodes represent those present at increased abundance in CALR DEL HOM mice, while blue nodes represent those at reduced abundance. The most highly enriched node is noted with an arrow. (B) RNA expression of the flow cytometry markers CD48, EPCR (Procr), and CD150 (Slamf1) plotted on PAGA graphs from (A). Cells within our node of interest (marked with an arrow) are CD48, EPCR, and CD150+. (C) Representative plots of SLAM cells from WT and CALR DEL HOM mice. CALR DEL HOM mice show higher numbers of both ESLAMs (Lin CD48 CD150+ CD45+ EPCR+) and pMKPs (Lin CD48 CD150+ CD45+ EPCR). FITC, fluorescein isothiocyanate; PE, phycoerythrin. (D) Quantification of bone marrow frequency of pMKPs in WT and CALR DEL HOM mice. The frequency of pMKPs within live bone marrow mononuclear cells (BMMNCs) is significantly increased in CALR DEL HOM mice (WT, n = 3, 0.00029 0.00008; HOM, n = 3, 0.0025 0.0008; *P = 0.042).

We examined the expression of a series of genes typically used to FACS isolate different hematopoietic populations and found this fine cluster to be CD48, EPCR (Procr), and CD150+ (Slamf1) (Fig. 1B). We designed an immunophenotypic scheme to identify and isolate cells from this fine cluster, defining them to be Lin, CD150+, CD48, EPCR, and CD45+. On the basis of our subsequent characterization of these cells, we eventually termed them proliferative MkPs or pMKPs. Consistent with our transcriptomic data, when comparing WT mice to CALR mutant mice, we found an increase in the frequency of pMKPs in CALR DEL HOM mice as assayed by flow cytometry (Fig. 1, C and D). We also found that pMKPs were expanded in CALR DEL HET mice, albeit to a lesser extent than observed in CALR DEL HOM mice (fig. S1F).

To characterize pMKPs, we FACS-sorted single ESLAM (EPCR+ SLAM) HSCs (Lin CD45+ CD48 CD150+ EPCR+) (26), pMKPs (Lin CD45+ CD48 CD150+ EPCR), and MkPs (Lin Sca1 cKit+ CD41+ CD150+) (27) (fig. S2A) from WT mice into individual wells of a 96-well plate and observed them every day for 4 days. We analyzed our sort data and observed that in pMKPs, markers traditionally used to define MkPs were Sca1/lo/mid, cKit+, and CD41mid/+ (fig. S2B). pMKPs were additionally CD9+ and MPL+ (fig. S2C). On each day, we classified each well with surviving cell(s) into one of four categories, using cell size as a proxy for megakaryopoiesis (2830): (i) exactly one large cell, presumed to be a megakaryocyte; (ii) multiple large cells; (iii) mixed expansion, with both large and small cells; and (iv) expansion with only small cells (Fig. 2A). To verify that larger cells represented MK cells, using cells from day 4 ESLAM, pMKP, and MkP colonies, we quantified average CD41 intensity via immunofluorescence and classified cells as small or large via bright-field microscopy, using a small/large dichotomy assessed via bright-field microscopy to match the classification scheme used in Fig. 2A. Here, we confirmed that large cells have significantly higher CD41 staining, supporting their identification as MK (fig. S2D). In some cases, particularly large cells within mixed colonies showed very high CD41 staining and membrane extensions that resembled proplatelets (representative picture is shown in fig. S2E). Furthermore, we sorted pMKPs from VWF (von Willebrand factor)green fluorescent proteinpositive (GFP+) mice and found that large cells had a very bright VWF-GFP signal, supporting their identification as MK. Smaller cells in these clones had a much dimmer VWF-GFP signal, suggesting that they likely represent more immature cells that have not progressed as far through megakaryopoiesis (fig. S2F).

(A) Representative pictures of in vitro culture output of single ESLAMs, pMKPs, and MkPs into four categories: 1 MK, >1 MK, mixed, or proliferation only. (B) Classification of in vitro culture output of single ESLAMs, pMKPs, and MkPs at day 4 after FACS isolation. ESLAMs almost exclusively proliferated without producing megakaryocytes, while MkPs almost exclusively produced MKs, usually producing only a single MK. pMKPs showed a strong MK bias but were more likely to proliferate than were MkPs. ESLAMs, n = 306 wells from five experiments; pMKPs, n = 291 wells from six experiments; MkPs, n = 235 wells from five experiments. Chi-square test, ****P < 0.0001. (C) Timing of megakaryopoiesis in ESLAMs, pMKPs, and MkPs. Individual cells were observed for 4 days after sort, and the first date on which cell(s) showed signs of megakaryopoiesis was noted. MkPs were faster to begin megakaryopoiesis than were pMKPs (at day 2, MkPs: 89.5 0.7%; pMKPs: 50 6%; *P = 0.02). ESLAMs, n = 5; pMKPs, n = 6; MkPs, n = 5. (D) Log2-transformed cell counts of megakaryocytes from pMKPs and MkPs after 4 days of culture. Each point represents the average value from one of four separate experiments. Average of four experiments: pMKP, 1.12; MkP, 0.412, *P = 0.0295. (E) Histogram of the minimum number of cell divisions for 103 pMKPs and 158 MkPs that produced only megakaryocytes after 4 days of culture across four experiments. Chi-square test, ***P = 0.0001.

The vast majority of ESLAMs showed expansion with only small cells at day 4, consistent with being highly primitive HSCs with considerable proliferative potential, but not yet producing megakaryocytes. Similarly, as predicted for MkPs, more than 95% of wells showed exclusively production of MKs at day 4, with the majority producing only one MK. This lack of in vitro proliferation for single MkPs is consistent with previously published results, where 75% of MkPs did not divide and none produced more than 10 MKs (31). pMKPs exhibited an intermediate phenotype: While approximately 90% of wells showed production of some MKs, they were much more likely to produce multiple MK than were MkPs. In particular, pMKPs frequently proliferated into mixed colonies with both large and small cells, a behavior that was rarely seen for either ESLAMs or MkPs (Fig. 2B). Kinetic analysis showed that MkPs were faster to begin megakaryopoiesis than were pMKPs (Fig. 2C), and when considering only wells that produced only MKs, pMKPs produced more MKs than did MkPs (Fig. 2, D and E). pMKPs maintained their MK bias even when incubated under pro-erythroid or pro-myeloid conditions (fig. S3A). Culturing cells with thrombopoietin (THPO) increased the proportion of pMKPs that formed colonies with multiple MKs while reducing the number of mixed colonies (fig. S3B). To verify that our observed MK bias is not simply due to culture conditions supporting only megakaryopoiesis, we cultured ESLAMs under the same conditions for 10 days followed by flow cytometric analysis and observed multilineage differentiation (fig. S3C).

To examine the extent of overlap between our pMKPs and traditionally defined MkPs, we stained bone marrow with a panel incorporating all necessary markers and index sorted single pMKPs and MkPs. On the basis of index sort values, 97% of MkPs were CD45+, 50% were EPCR, and only 2% were CD48; when taken together, fewer than 1% of immunophenotypic MkPs also fell within the pMKP gate (fig. S3D); thus, pMKPs and MkPs can be FACS-separated on the basis of CD48 and EPCR. In contrast, we found that an average of 51% of pMKPs were also immunophenotypically MkPs (CD41+ Sca1 cKit+) (fig. S3E). As we observed a partial overlap between pMKPs and MkPs, we used our index sort data to assign each pMKP an overlap score based on the levels of CD41, Sca1, and cKit: 1/3 if only one marker overlapped, 2/3 if two overlapped, and 3/3 for pMKPs that also fall within the MkP immunophenotypic gate. No pMKPs had an overlap score of 0/3. We used the same classification scheme as in Fig. 2B and found that lower overlap scores correlated to a more proliferative, less MK-restricted phenotype: The pMKPs that are least similar to MkPs are the most proliferative and the least restricted to the MK lineage, although they still display a strong preference for MK production (fig. S3F). pMKPs with the lowest overlap score took the longest to enter megakaryopoiesis (fig. S3G). Together, our data indicate that pMKPs represent a group of cells with an MK bias and an increased proliferative potential as compared to traditionally defined MkPs.

We next determined whether pMKPs were capable of producing platelets in vivo. We made use of CD45.2 VWF-GFP donor mice and cKit W41/W41 CD45.1 recipient mice, which allowed us to track platelets (via VWF-GFP) and nucleated cells (by CD45.1/CD45.2 staining) (Fig. 3A). We FACS-sorted ESLAMs, pMKPs, and MkPs from VWF-GFP donor mice and transplanted 30, 60, or 120 cells per recipient into sublethally irradiated W41 mice along with 250,000 spleen MNCs (mononuclear cells) (SPMNCs) as helper cells and assayed peripheral blood chimerism every week for 4 weeks and at 16 weeks. We did not sort on VWF-GFP+ at this stage, but flow cytometry analysis showed that ESLAMs, pMKPs, and MkPs were all highly enriched for VWF-GFP expression when compared to total bone marrow (fig. S4A). We also transplanted one mouse per cohort with 250,000 SPMNCs alone to serve as a negative control to help with gating to avoid false positives. Representative gating strategies are shown in fig. S4 (B and C). As expected, ESLAMs were able to generate relatively high levels of platelets at all three cell doses, starting with a very low level at week 1 and increasing over the course of 4 weeks and continuing up to 16 weeks (although one recipient of 30 ESLAMs was lost to follow-up before the 16-week time point). pMKPs and MkPs were only able to reconstitute platelets at a very low level (1/105 to 1/104), even at the highest cell dose (Fig. 3, B to D and summarized in E). Low levels of donor-derived platelets were detected in 10 of 12 pMKP recipients and 8 of 13 MkP recipients within the first 4 weeks; extended observation up to 16 weeks showed that few recipients continued to produce VWF-GFP+ platelets, although all 3 pMKP recipients at the highest dose still showed VWF-GFP+ platelets. ESLAMs successfully produced CD11b+ myeloid cells in 10 of 10 recipients across varying cell doses, while pMKPs and MkPs only produced CD11b+ cells at a low level in 3 of 12 and 2 of 10 recipients, respectively (fig. S4, D to F and summarized in G). Therefore, we concluded that while pMKPs and MkPs have limited capabilities in a transplantation experiment, they both show an MK bias, in agreement with their in vitro behaviors. These low levels of reconstitution suggest that pMKPs and MkPs do not divide considerably in vivo, again similar to in vitro data.

(A) Schematic of VWF-GFP+ transplantation strategy. ESLAMs, pMKPs, and MkPs were sorted from VWF-GFP+, CD45.2 donor mice and transplanted into sublethally irradiated cKit W41/W41 CD45.1 recipients. PB, peripheral blood. (B) Platelet reconstitution from 30 donor cells. (C) Platelet reconstitution from 60 donor cells. (D) Platelet reconstitution from 120 donor cells. (E) Table summarizing numbers of mice with successful platelet production from ESLAMs, pMKPs, and MkPs. Here, transplanted cells were defined to have produced platelets if platelets were observed at a level of at least 1 in 105 at one or more time points within the first 4 weeks after transplantation.

Our single-cell transcriptomic analysis showed pMKPs to be an intermediate stage on an MK trajectory maintaining CD48 negativity (Fig. 1B and green arrow in fig. S1E), which suggests that they bypass the traditional MPP2 pathway (blue arrow in fig. S1E). We therefore asked whether we could show production of pMKPs from HSCs in an MPP2-independent manner by making use of a mouse model allowing inducible depletion of HSPCs. In this model, Tal1-Cre/ERT mice are crossed with R26DTA mice, wherein treatment with tamoxifen leads to specific expression of diphtheria toxin in HSCs and primitive progenitors and hence suicidal depletion of these early populations (Fig. 4A) (32). Within 6 weeks after HSC depletion, very few LT-HSCs remain, but levels of MPPs, committed progenitors, and mature blood cells are only slightly lower than in control animals (32). We reasoned that if pMKPs arise directly from HSCs, they should be depleted to a similar extent as HSCs, while if they arise from an MPP pathway, they should be depleted to a similar extent as MPPs (i.e., to a lesser extent than HSCs).

(A) Schematic of DTA (diphtheria toxin fragment A) HSC depletion model experiment. Tal1-CreERT/R26DTA mice were treated with four doses of tamoxifen at 0.1 mg/g to induce suicidal depletion of HSCs and then euthanized after 6 weeks for bone marrow (BM) analysis. (B) Frequencies of stem and progenitor cells with or without stem cell depletion. Cell populations that were significantly diminished by suicidal depletion of HSCs include ESLAMs (Cre, 17.1 10.8/105 BMMNC; Cre+, 4.3 2.0/105 BMMNC; *P = 0.012), LTHSCs (LSK CD48 CD150+) (Cre, 15 12/105 BMMNC; Cre+, 3.6 1.7/105 BMMNC; *P = 0.031), pMKPs (Cre, 13.0 7.6/105 BMMNC; Cre+, 4.1/105 BMMNC; *P = 0.013), and MkPs (Cre, 44.2 26.4/105 BMMNC; Cre+, 21.4 6.1/105 BMMNC; *P = 0.046); Cre, n = 8 and Cre+, n = 10. MPP2 (Cre, 25.1 29.1/105 BMMNC; Cre+, 13.3 3.6/105 BMMNC; P = 0.48) and preMegE (Cre, 90.0 62.9/105 BMMNC; Cre+, 73.9 29.6/105 BMMNC; P = 0.66) populations were depleted to lesser extents that did not reach statistical significance; Cre n = 4 and Cre+ n = 6. ns, not significant.

We compared mice carrying either no Cre or Tal1-Cre/ERT after treatment with tamoxifen to induce specific depletion of HSCs. We observed a depletion of approximately 75% in the numbers of HSCs [whether using ESLAM markers or LT-HSC (LSK CD48 CD150+) markers] and a 68% reduction in the numbers of pMKPs in HSC-depleted mice. By contrast, there was no significant reduction in MPP2 or preMegE populations, while MkPs were reduced by approximately 51% (Fig. 4B). Consistent with previously published results, we observed no statistically significant reduction in other multipotent populations, including MPP3 and MPP4 (33), and committed progenitor populations, including CFU-E (erythroid colony-forming units), pCFU-E, pGM (pre-granulocyte/macrophage), and GMP (granulocyte/monocyte progenitors) (fig. S5) (27). We noted that one Cre mouse was an outlier, with noticeably higher frequencies of almost all progenitor populations, and tested removing this outlier to ensure our conclusions were not unduly relying on this mouse. With the outlier removed, we calculated reductions of 68% in ESLAMs (P = 0.0001), 60% in pMKPs (P = 1.5 105), and an increase of 24% in MPP2 (P = 0.50). Our analysis is therefore robust to the removal of this outlier and demonstrates that the reduction in pMKP levels correlates more closely to that of ESLAMs than that of MPP2. Together, these data support a model in which pMKPs are produced from HSCs in an MPP2-independent manner and MkPs can be generated from pMKPs or via MPP2, accounting for their intermediate level of reduction.

After characterizing the pMKP population in WT mice, we next asked whether there were qualitative differences between WT and CALR DEL HOM cells along the MK trajectory and not solely a quantitative difference. To do so, we sorted single ESLAMs, pMKPs, and MkPs from WT and CALR DEL HOM mice and monitored their in vitro behavior over 4 days. While very few WT ESLAMs showed any MKs within the first 4 days after sort, a higher proportion of CALR DEL HOM ESLAMs showed MKs within mixed colonies (Fig. 5A). CALR DEL HOM pMKPs showed similar proportions of wells in each category (Fig. 5B), while CALR DEL HOM MkPs were more likely to form multiple MKs and less likely to form a single MK (Fig. 5C). To assess the statistical significance of these differences, using a Fishers exact or chi-square test required consolidation of our data into fewer categories, as some categories contained values that were too low (for example, for day 4 ESLAMs, the categories 1 MK and >1 MK were 0 in both WT and HOM). We thus consolidated ESLAM data into two categoriesno MK and MK (Fig. 5D)and pMKP and MkP data into three categories1 MK, >1 MK, and mixed + prolif only (Fig. 5, E and F). This showed that CALR DEL HOM ESLAMs were significantly more likely to form MKs (Fig. 5D). CALR DEL HOM pMKPs showed no statistically significant difference, suggesting no change in their MK bias or proliferative behavior compared to WT pMKPs (Fig. 5E). CALR DEL HOM MkPs were significantly more proliferative than were WT MkPs (Fig. 5F). We also extended our observation of ESLAM clones to day 7 and observed an even stronger increase in the production of megakaryocytes from CALR DEL HOM ESLAMs, an increase noted both in wells producing mixed clones and in those producing MK-only clones (Fig. 5, G and H).

(A) Classification of in vitro culture output of single ESLAMs from WT and CALR DEL HOM mice at day 4, using the classification scheme as in Fig. 2A. WT, n = 223; HOM, n = 225. (B) Classification of in vitro culture output of single pMKPs from WT and CALR DEL HOM mice at day 4; WT, n = 117; HOM, n = 161. Chi-square test P = 0.9201. (C) Classification of in vitro culture output of single MkPs from WT and CALR DEL HOM mice at day 4; WT, n = 136; HOM, n = 152. (D) Reclassification of data from (A) into two categories (MK or no MK) for a Fishers exact test, *P = 0.0191. (E) Reclassification of data from (B) into three categories (1 MK, >1 MK, and mixed + prolif only) for a chi-square test, P = 0.8183. (F) Reclassification of data from (C) into three categories (1 MK, >1 MK, and mixed + prolif only) for a chi-square test, **P = 0.0069. (G) Classification of in vitro culture output of single ESLAMs at day 7; WT, n = 136; HOM, n = 152. (H) Reclassification of data from (G) into two categories (MK or no MK) for a Fishers exact test, **P = 0.0014. (I) pMKPs as a proportion of live cells generated from in vitro culture of WT and CALR DEL HOM ESLAMs, assessed at day 3. WT, 0.062 0.015; HOM, 0.193 0.036, *P = 0.0135, n = 3 independent mice.

We also considered log2-transformed cell counts from those wells with exclusively megakaryocytes (i.e., 1 MK and >1 MK). In some cases, we observed the death of a cell or cells over our 4-day observation period; to account for cell death, we used the maximum number of cells observed over these 4 days. Mann-Whitney U tests showed no significant difference for pMKPs but a significant increase in MK production from CALR DEL HOM MkPs (fig. S6, A and B). Similarly, calculations of the minimum number of divisions required to produce the observed number of MKs found no difference for pMKPs but a significant shift to more divisions from CALR DEL HOM MkPs (fig. S6, C and D). We also cultured ESLAMs in vitro and assayed for the production of pMKPs, finding that CALR DEL HOM ESLAMs gave rise to significantly more pMKPs than did their WT counterparts (Fig. 5I). Together, we conclude that CALR DEL is acting at multiple stages of megakaryopoiesis, promoting an MK bias from the earliest HSC compartments and increased proliferation at both HSC and MkP levels. While pMKPs are increased in number in CALR DEL HOM mice, these cells do not show altered proliferation or MK bias in vitro.

Last, we made use of our scRNAseq data to compare gene expression between WT and CALR DEL HOM cells along the MK trajectory. We considered cells within 2 of the 13 clusters defined by our transcriptomic data (HSC and MK; fig. S1A) and 1 fine cluster (pMKP; arrow in Fig. 1A) (Fig. 6, A to C). As the pMKP fine cluster had fewer cells (24 in WT and 247 in CALR DEL HOM) than the larger HSC and MK clusters, we were only able to confidently call a small number of differentially expressed genes (DEGs) within this cluster. We performed Ingenuity Pathway Analysis (IPA) to determine which biological pathways and upstream regulators were most affected in the HSC and MK clusters; the small numbers of DEGs in pMKPs resulted in no statistically significant hits via IPA. The most affected canonical pathways fell into three broad groups: cell cycle (in blue), unfolded protein response (gold), and cholesterol biosynthesis (green) (Fig. 6, D and E). Full lists of canonical pathways, P values, and z scores are available in tables S1 (HSC) and S2 (MK). Genes contributing to these three pathways are highlighted in the same colors in Fig. 6, A to C; we note that pMKPs also show up-regulation of several UPR (unfolded protein response)associated genessuch as Hspa5, Pdia3, and Pdia6in addition to two known STAT targets (Ifitm2 and Socs2).

(A to C) Volcano plots showing DEGs between WT and CALR DEL HOM cluster 3 (HSC) (A), pMKP fine cluster (B), and cluster 11 (MK) (C). Genes within certain representative Gene Ontology (GO) terms are colored: regulation of cholesterol biosynthetic process (GO:0045540) (green), response to ER stress (GO:0034976) (gold), and regulation of mitotic cell cycle (GO:0007346) (blue). Other DEGs are colored in red. (D and E) Bar graphs showing z scores for up-regulated canonical pathways in cluster 3 (HSC) (C) and cluster 11 (MK) (D), filtered by P < 0.01 and z score of >1 or <1. Bars are highlighted in green for cholesterol biosynthesis, gold for ER stress/unfolded protein response, or blue for cell cycle. (F) Upstream regulator analysis. Hits were filtered by P < 0.01. Bar graph showing the 10 most up-regulated and 10 most down-regulated predicted upstream regulators, when comparing WT and CALR DEL HOM cluster 3 (HSC) (blue) and cluster 11 (MK) (red), as measured by combining the z scores from WT and MK analyses.

While cell cycle and UPR have previously been described as up-regulated in human CD34+ cells with CALR mutation (34), the discovery of cholesterol biosynthesis was somewhat unexpected. However, this aligned with the predicted significant activation of the lipid and cholesterol biosynthetic transcriptional machinery controlled by the sterol regulatory elementbinding proteins (SREBPs; SREBF1 and SREBF2) and the SREBF chaperone (SCAP) and their inhibitor insulin-induced gene 1 (INSIG1) (Fig. 6F). Moreover, as discussed further below, a role for cholesterol biosynthesis in a proliferative, platelet-biased blood disorder is biologically plausible. Upstream regulator analysis also pointed to activation of ERN1 (Ire1) and Xbp1, two constituents of UPR, as well as STAT5 (table S3), which is consistent with previous demonstrations that mutant CALR acts via STAT signaling (4, 3537). We additionally observed other previously undescribed signaling processes to be predicted to be activated, including drivers of proliferation such as CSF2 [granulocyte-macrophage colony-stimulating factor (GM-CSF)] and hepatocyte growth factor (HGF), or repressed, like the known tumor suppressors TP53 and let-7.

Single-cell transcriptomic approaches have allowed detailed examinations of differentiation landscapes in both normal and perturbed hematopoiesis without a requirement to initially define populations based on a set of cell surface markers. We therefore used single-cell transcriptomics to investigate our recently generated mutant CALR-driven mouse model of ET and found an expected increase in both HSCs and MK lineage cells. We also found an increase in a previously unknown group of cells, here termed pMKPs, linking HSCs with the MK lineage. In vitro, pMKPs displayed behaviors intermediate to those of HSCs and MkPs: Similarly to HSCs, they had some proliferative potential, but similarly to MkPs, they were almost exclusively restricted to the MK lineage. In transplantations, pMKPs and MkPs showed similar behavior: They both transiently produced platelets at a low level. We hypothesize that while pMKPs are more proliferative than MkPs in vitro, neither population is capable of sufficient proliferation to significantly contribute to platelet production in the transplant setting. While this manuscript was in preparation, another group described separating SLAM (Lin CD48 CD150+) cells based on EPCR and CD34, finding that EPCR SLAM cells performed poorly in transplants and showed gene expression profiles (high Gata1, Vwf, and Itga2b) indicative of MK bias (38), results that are broadly consistent with our own.

Our characterization of pMKPs accords well with an increasing understanding that at least a portion of megakaryopoiesis occurs via an early branch point directly from HSCs. While the standard model of hematopoiesis shows megakaryocytes subsequent to MPP2, lineage tracing experiments have shown that some MkPs are generated in an MPP2-independent way (19). Furthermore, in vivo labeling of the most primitive HSCs showed that within 1 week of label induction in LT-HSCs, label can be seen in MK lineages but no other, indicating that the HSC-to-MK pathway can be noticeably faster than pathways producing other lineages (22). Our results suggest that pMKPs are likely to arise independently of the MPP2 stage, as suicidal depletion of the earliest HSPCs reduces pMKPs to a much greater extent than MPP2s. It is therefore tempting to speculate that our pMKP sort scheme may isolate intermediate cells on this shorter, faster bypass trajectory. A recent study of JAK2 V617F-driven MF in humans attributed increased megakaryopoiesis to the expansion of both traditional MkPs and a novel MkP-like population, suggesting that cells that may be analogous to our pMKPs are relevant in human disease (30).

We also investigated an outstanding question about at which stages mutant CALR acts to drive a platelet phenotype. Mutant CALR has been demonstrated to increase the number of immunophenotypic HSCs and MkPs (6), and we also saw an expansion in the number of pMKPs. When considering the behavior of cells individually, it is clear that mutant CALR acts from the stem cell compartment: CALR DEL HOM HSCs were more proliferative and faster to produce megakaryocytes than were their WT counterparts. Mutant CALR did not show a strong effect on the proliferation or MK bias of pMKPs at the level of a single cell but drove an increase in proliferation of MkPs and thus the number of megakaryocytes produced. We therefore concluded that mutant CALR drives platelet bias and proliferation at multiple stages of megakaryopoiesis, although this effect is strongest within HSCs.

Last, we used our single-cell transcriptomic data to ask which biological pathways were most differentially regulated in our CALR DEL HOM mice. Mutant CALR was associated with an up-regulation of the unfolded protein response, as would be expected for cells with impaired chaperone activity and as has been seen in human patient cells (34). In addition, mutant CALR cells showed an increase in cell cycle genes, again consistent with observations from human patient cells (34) and in agreement with our in vitro data, which showed that mutant CALR HSCs and MkPs were more proliferative. We also found up-regulation of cholesterol biosynthesis pathway genes in mutant CALR hematopoietic cells. While cholesterol biosynthesis is broadly increased across numerous cancers (39), including hematological cancers (40), CALR has also been directly linked to cholesterol biosynthesis. CALR/ mouse embryonic fibroblasts show impaired endoplasmic reticulum (ER) Ca2+ levels, leading to overactivation of SREBPs, which then up-regulate cholesterol and triacylglycerol biosynthesis genes (41). As mutant CALR lacks its Ca2+-binding domain, it is possible that CALR DEL HOM cells phenocopy knockout cells with respect to ER Ca2+ stores, thus leading to the observed overactive transcription of cholesterol biosynthesis genes. While megakaryocytes derived from human patient samples have been shown to have increased store-operated Ca2+ entry due to the perturbation of a complex between STIM1, ERp57, and CALR (42), none of our differentially activated pathways from IPA pointed to altered cytoplasmic Ca2+ signaling in the stem and progenitor populations tested. This may reflect differences between progenitor and mature cells. Mice with impaired cholesterol efflux have more proliferative HSCs (43) and an increase in MkP proliferation and an ET-like phenotype (44), suggesting that there may be a previously unknown link between the CALR DEL mutation, cholesterol metabolism, proliferation of MkPs, and thus the overproduction of platelets. While cholesterol biosynthesis was the most prominent novel target found in our transcriptomic analysis, it was by no means alone. IPA upstream regulator analysis predicted an up-regulation of interleukin-5 (IL-5), GM-CSF, and HGFall with known roles in hematopoiesisin addition to several unexpected results, such as TBX2, a transcription factor that has not been studied in hematopoiesis. Upstream regulators predicted to be decreased include the tumor suppressor TP53; let-7, a microRNA with a role in the self-renewal of fetal HSCs (45); and KDM5B (Jarid1b), a histone methylase required for HSC self-renewal (46).

Overall, our study has characterized a previously undescribed MK trajectory implicated in the progression of ET. We find that pMKPs are an intermediate stage within one pathway of megakaryopoiesis and hypothesize that they may be situated within the MPP2-independent MK shortcut. Last, our analysis confirmed that JAK-STAT signaling, unfolded protein response, and cell cycle are all increased by the presence of mutant CALR and found up-regulation of cholesterol biosynthesis, in addition to numerous other potential upstream regulators. Functional validation of these biological pathways and upstream regulators may represent promising avenues of future research to better understand mutant CALR-driven disease and in the development of therapeutic strategies.

The objectives of the study were to generate transcriptomic data from our CALR mouse model of ET and to use these data to determine how the hematopoietic landscape is affected by the CALR DEL mutation. All mouse procedures were performed in strict accordance with the U.K. Home Office regulations for animal research under project license 70/8406.

Bone marrow cells were harvested from the femurs, tibia, and iliac crests of mice. Bones were crushed in a mortar and pestle in phosphate-buffered saline (PBS) and 2% fetal bovine serum (FBS) and 5 mM EDTA and then filtered through a 70-m filter to obtain a suspension of bone marrow cells. The suspension was incubated with an equal volume of ammonium chloride solution (STEMCELL Technologies, Vancouver, Canada) for 10 min on ice to lyse erythrocytes, followed by centrifugation for 5 min at 350g. The cell pellet was resuspended in PBS and 2% FBS and 5 mM EDTA, filtered again through a 70-m filter, and centrifuged again for 5 min at 350g. For cell sorting experiments, bone marrow mononuclear cell suspensions were immunomagnetically depleted of lineage (Lin)positive cells (EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit, catalog no. 19856, STEMCELL Technologies). For staining, cells were incubated with the indicated antibodies for 40 min on ice; see attached tables for catalog information and concentrations used (table S4). Flow cytometry was performed on BD LSRFortessa analyzers, and flow cytometric sorting was performed on BD Influx 4 and 5 cell sorters (BD Biosciences, San Jose, USA). Flow data were analyzed using FlowJo software (Tree Star, Ashland, USA).

For 10x Chromium (10x Genomics, Pleasanton, CA) experiments, Lin c-Kit+ (LK) and Lin Sca1+ cKit+ (LSK) cells were sort purified as described above and processed according to the manufacturers protocol. Sample demultiplexing, barcodes processing, and gene counting were performed using the count commands from the Cell Ranger v1.3 pipeline (https://support.10xgenomics.com/single-cell-gene-expression/software/overview/welcome). After Cell Ranger processing, each sample (LK and LSK for WT and CALR HOM DEL) was filtered for potential doublets by simulating synthetic doublets from pairs of scRNAseq profiles and assigning scores based on a k nearest-neighbor classifier on PCA-transformed data. The 1 and 4.5% of cells with the highest doublets scores from each LSK or LK sample were removed from further analysis, respectively. Cells with >10% of unique molecular identifier (UMI) counts mapping to mitochondrial genes, expressing fewer than 500 genes, or with a total number of UMI counts further than 3 SDs from the mean were excluded. After quality control, 11,098 WT (5139 LK and 5959 LSK) and 15,547 HOM (7815 LK and 7732 LSK) cells were retained for downstream analysis from our first repeat. For our second repeat, 3451 WT (2479 LK and 972 LSK) and 12,372 HOM (7824 LK and 4548 LSK) cells were retained for downstream analysis. These cells were then normalized to the same total count. All scRNAseq data were analyzed using the Scanpy Python Module (47).

To assign cell type identities to WT and CALR samples, a previously published landscape of 45,000 WT LK and LSK hematopoietic progenitors (24) was used as a reference for cell type annotation. This reference was clustered using Louvain clustering, resulting in 13 clusters. LK + LSK samples were joined for each genotype (WT and CALR DEL HOM) and projected into the PCA space of this reference dataset. Nearest neighbors were calculated between the two datasets based on Euclidean distance in the top 50 PCA components. Cells were assigned to the same cluster to which the majority of their 15 nearest neighbors in the reference belonged.

A force-directed graph visualization of the 45,000 cell reference dataset was calculated by first constructing a k = 7 nearest-neighbor graph from the data, which was then used as input for the ForceAtlas2 algorithm as implemented in Gephi 0.9.1 (https://gephi.org). In the ForceAtlas2 algorithm, all cells are pushed away from each other, with the nearest-neighbor connections pulling them back together to segregate separate trajectories.

A fine-resolution clustering of the reference dataset was calculated using the Louvain algorithm, resulting in 63 clusters. These were used as input for a PAGA analysis of the reference dataset using the Scanpy Python Module with default parameters. The results of the PAGA analysis were visualized by using the nodes and their edge weights as input into the ForceAtlas2 algorithm for calculating force-directed graphs as implemented in Gephi 0.9.1. For visualization, only connections with edge weights of >0.3 were shown.

To visualize gene expression of the PAGA graph, the mean normalized expression of all cells belonging to each node was calculated and displayed on a per-node basis.

To calculate differential abundances, votes were given out from each WT LK and CALR LK cell to their k-nearest neighbors in the reference dataset, with k chosen such that the total number of votes given out by each sample was the same. For each cell in the reference dataset, the difference between the number of votes received from the WT and CALR HOM samples was calculated. This difference acts as a proxy for the differential abundance of WT and CALR HOM cells for the region of the LK landscape in which the reference cell is located. This differential abundance proxy could then be visualized either on the reference landscape itself or on the PAGA graph calculated using the reference landscape. In the latter case, each node of the PAGA graph was colored by the mean differential abundance of all cells belonging to that node.

After flow sorting, cells were cultured in StemSpan SFEM (serum-free expansion medium) (STEMCELL Technologies) supplemented with 10% FBS (STEMCELL Technologies), 1% penicillin/streptomycin (Sigma-Aldrich), 1% l-glutamine (Sigma-Aldrich), stem cell factor (SCF; 250 ng/ml), IL-3 (10 ng/ml), and IL-6 (10 ng/ml; STEMCELL Technologies), with or without thrombopoietin (100 ng/ml; STEMCELL Technologies), in round-bottom 96-well plates (Corning, Corning, USA). For pro-erythroid conditions, cells were cultured as above but with the following cytokines: SCF (250 ng/ml), THPO (thrombopoietin) (50 ng/ml), EPO (erythropoietin) (5 U/ml), IL-3 (20 ng/ml), and Flt3L (50 ng/ml). For pro-myeloid conditions, cells were cultured as above but with the following cytokines: SCF (250 ng/ml), THPO (50 ng/ml), granulocyte colony-stimulating factor (50 ng/ml), IL-3 (20 ng/ml), Flt3L (50 ng/ml), and GM-CSF (50 ng/ml).

At 1, 2, 3, 4, and, in some cases, 7 days after flow sorting, single cellderived clones were visually inspected. Wells with surviving cells were classified into one of four categories: (i) exactly one enlarged cell, presumed to be a megakaryocyte; (ii) multiple enlarged cells; (iii) mixed expansion, with both small and enlarged cells; and (iv) expansion with only small cells. In some cases, the experimenter was blinded to the identity of the cell population initially sorted into the well he/she was inspecting and the genotype of the mouse.

For immunofluorescence, cells were allowed to adhere to the surface of poly-l-lysinecoated slides for 30 min at 37C (Poly-Prep Slides, Sigma-Aldrich). Cells were then fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS overnight at 4C, permeabilized with 0.25% Triton X-100 (Sigma-Aldrich) in PBS for 10 min at room temperature, and blocked with 1% bovine serum albumin (Sigma-Aldrich) for 1 hour at room temperature. Cells were stained with CD41 Alexa Fluor 488 (BioLegend, catalog no. 133908) overnight and mounted with 4,6-diamidino-2-phenylindole (DAPI) (VECTASHIELD Mounting Medium with DAPI, Vector Laboratories Inc., Burlingame, USA; catalog no. H-1500). Pictures were acquired on LSM-710 and LSM-780 confocal microscopes (Zeiss) and analyzed using ZEN software (Zeiss). For quantification of immunofluorescence, cells were cultured on CD44-coated glass-bottom plates for immobilization (48), followed by fixation and staining as above. Pictures were acquired on a Leica DMI4000 microscope (Leica), and CD41 intensity and cell size were quantified using Fiji software.

FACS-sorted cells from VWF-GFP+ donors were injected into the tail veins of W41/W41 (CD45.1) recipient that had been sublethally irradiated with 1 400 centigrays with 250,000 spleen cells as helpers. Peripheral blood was analyzed 1, 2, 3, 4, and 16 weeks after transplant for all cohorts.

Differential expression analysis was performed between WT (LK + LSK) and CALR DEL HOM (LK + LSK) clusters using the Wilcoxon rank sum test on all genes that passed initial quality control (typically approximately 15,000). A Benjamini-Hochberg correction was applied to correct for multiple testing. Genes with an adjusted P value of <0.05 and a fold change of >1.5 between genotypes were marked as differentially expressed. The original normalized counts were used in all cases.

DEGs were studied using IPA (Qiagen). We imputed the whole transcriptome in IPA and then filtered for analysis only statistically significant (adjusted P < 0.01) items with a log2FC > 0.3785 or log2FC < 0.3785. Pathways and upstream regulator networks showing relationships and interactions experimentally confirmed between DEGs and others that functionally interact with them were generated and ranked in terms of significance of participating genes (P < 0.05) and activation status (z score).

Data were analyzed, and graphs were generated in Microsoft Excel (Microsoft) and GraphPad PRISM (GraphPad, La Jolla, USA). Data are presented as means SD. Unless otherwise stated, statistical tests were unpaired Students t tests. P values are as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Acknowledgments: We would like to acknowledge J. Aungier, T. Hamilton, D. Pask, and R. Sneade for invaluable technical assistance; R. Schulte, C. Cossetti, and G. Grondys-Kotarba at the CIMR Flow Cytometry Core Facility for assistance with cell sorting; and S. Loughran, T. Klampfl, and E. Laurenti for valuable discussions. Funding: Work in the Gttgens laboratory is supported by the Medical Research Council (MR/M008975/1), Wellcome (206328/Z/17/Z), Blood Cancer UK (18002), and Cancer Research UK (RG83389, jointly with A.R.G.). Work in the Green laboratory is supported by Wellcome (RG74909), WBH Foundation (RG91681), and Cancer Research UK (RG83389, jointly with B.G.). Author contributions: D.P. and H.J.P. designed and conducted experiments with assistance from J.L. S.W. and H.P.B. performed bioinformatic analyses. M.V. performed IPA with supervision from A.V.-P. A.G. provided DTA mice. D.P. analyzed data and wrote the manuscript with input from H.J.P. and J.L. and supervision from B.G. and A.R.G. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. We have deposited scRNAseq data in the NCBI Gene Expression Omnibus (GEO) database with accession number GSE160466. Additional data related to this paper may be requested from the authors.

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The stem/progenitor landscape is reshaped in a mouse model of essential thrombocythemia and causes excess megakaryocyte production - Science Advances

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