BIOCHEMISTRY, BIOMEDICINE & PHARMACEUTICS

Human Pan T cell Isolation The human Pan T cells may be isolated from mononuclear cells using an indirect immunomagnetic method that utilizes antibodies against the cell surface marker for monocytes, neutrophils, eosinophils, B cells, stem cells, dendritic cells, NK cells, granulocytes, or erythroid.   The cocktail includes antibodies against cells markers CD14, CD16, CD19, CD34, CD36, CD56, CD123, CD235a.  General Protocol for Pan T cell Isolation (Miltenyl Biotec) Pan T Cell Biotin-Antibody Cocktail, human: Cocktail of biotin-conjugated monoclonal antibodies against CD14, CD15, CD16, CD19, CD34, CD36, CD56, CD123, and CD235a (GlycophorinA). MicroBeads conjugated to monoclonal anti-biotin antibody (isotype: mouse IgG1) and monoclonal anti-CD61 antibody (isotype: mouse IgG1). Prepare cells and determine cell numbers. Resuspend cell pellet in 40 μL of buffer per 10⁷ total cells. Add 10 μL of Pan T Cell Biotin-Antibody Cocktail per 10⁷ total cells. Mix well and incubate for...

BLINCYTO® (blinatumomab): Bispecific CD19-Directed CD3 T-cell Engager   BLINCYTO is a bispecific CD19-directed CD3 T-cell engager indicated for the treatment of adults and children with:  • B-cell precursor acute lymphoblastic leukemia (ALL) in first or second complete remission with minimal residual disease (MRD) greater than or equal to 0.1%. This indication is approved under accelerated approval based on MRD response rate and hematological relapse-free survival. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.  • Relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL).  Mechanism of Action: Blinatumomab is a bispecific CD19-directed CD3 T-cell engager that binds to CD19 expressed on the surface of cells of B-lineage origin and CD3 expressed on the surface of T cells. It activates endogenous T cells by connecting CD3 in the T-cell receptor (TCR) c...

Cytokine Release Syndrome Cytokine Release Syndrome (CRS) is a supraphysiological response following any immune therapy that results in the activation or engagement of endogenous or infused T cells and or other immune effector cells. Symptoms can be progressive, must include fever at the onset, and may include hypotension, capillary leak, hypoxia, and end-organ dysfunction.  Classification/Stages of CRS  Grade 1 CRS:  Grade 1 CRS is defined as fever (> 38 degrees celsius) with or without constitutional symptoms of CRS include myalgia, arthralgia, and malaise with the coincidence of fever.  Grade 2 CRS:  Grade 2 CRS is defined as fever (>38 degrees celsius) with hypotension not requiring vasopressin and or hypoxia requiring the use of a low-flow nasal canula or blow-by. Grade 3 CRS: Grade 3 CRS is defined as fever (>38 degrees celsius)  with hypotension requiring 1 vasopressor with or without vasopressin and hypoxia requiring high flow nasal ca...

The enzyme-linked immunospot (ELISpot) assays are widely used for studying cellular immunology. It provides both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient-derived peripheral blood mononuclear cells to detect the antigenic restriction of T-cell responses for infectious diseases. This technology is has emerged as the gold standard for identifying T cell-mediated response against viral capsid protein and transgenes.  Principle of ELIspot Basic Principle A species-specific monoclonal antibody for IFN-γ has been pre-coated onto a PVDF (polyvinylidene difluoride)-backed microplate. Appropriately stimulated PBMCs/ T cells are pipetted into the wells and the microplate is placed into a humidified 37 °C CO2 incubator for an optimized period of time. During this incubation period, the immobilized antibody in the immediate vicinity of the secreting cells binds secreted IFN-γ. After washin...

  Etranacogene dezaparvovec For Treatment of Hemophilia B (Highlights) Data from the HOPE-B pivotal study showed that participants continued to demonstrate durable, sustained increases in Factor IX (FIX) activity at 52-weeks post-infusion with a mean FIX activity of 41.5 percent of normal, as measured by a one-stage APTT-based clotting assay, compared to a mean FIX activity of 39.0 percent of normal at 26-weeks of follow-up. There continued to be no clinically significant correlation between pre-existing neutralizing antibodies to AAV5 (NAbs) and FIX activity in patients with NAb titers up to 678.2, a range expected to include more than 93 percent of the general population.  During the 52-week period, a single dose of etranacogene dezaparvovec significantly reduced the annualized rate of bleeding requiring treatment by 80 percent from a prospectively collected 3.39 at baseline to 0.68 bleeding episodes per year (p-value <0.0001). The annualized rate of spontaneous bleeding ...

HPN536 is a novel, MSLN targeted, trispecific, T-cell–activating protein construct that can potently redirect T cells to lyse tumor cells and was remarkably well tolerated in nonhuman primates at single doses up to 10 mg/kg, which is far above the expected therapeutic dose level. Harpoon Therapeutics are conduction "A Phase 1/2a Open-label, Multicenter, Dose Escalation and Dose Expansion Study of the Safety, Tolerability, and Pharmacokinetics of HPN536 in Patients With Advanced Cancers Associated With Mesothelin Expression Who Have Failed Standard Available Therapy" The objective of the trial is to -assess safety and tolerability at increasing dose levels  - Pharmacokinetic and pharmacodynamic data  ‐ Evaluate preliminary anti-tumor activity Dosing has occurred across 10 fixed-dose cohorts of 6 to 560ng/kg and three step dose cohort up to 1200ng/kg, with total enrollment of 60 patients. Tumor types treated include late-stage ovarian (47%), pancreatic (40%) and peritoneal ...

HPN424 is a prostate-specific membrane antigen (PSMA)- targeting T cell engager, engineered with three binding domains: • PSMA (for tumor binding) • Albumin (for half-life extension) • CD3 (for T cell engagement) HPN424 is constructed as a small, globular protein (~50kDa) to enable efficient solid tumor penetration with prolonged half-life and excellent stability. HPN424 is designed to bind monovalently to CD3 and PSMA, minimizing non-specific T-cell activation. Harpoon Therapeutics is currently conducting a "Phase 1/2a Open-label, Multicenter, Dose Escalation and Dose Expansion Study of the Safety, Tolerability, and Pharmacokinetics of HPN424 in Patients With Advanced Prostate Cancer Refractory to Androgen Therapy" As of May 31, 2021, the data cutoff date for the Company’s HPN424 presentation, the Company updated the ASCO interim data to describe newly enrolled patients and additional follow-up on existing patients already enrolled in the 300 ng/kg step dose cohort. The...

NAGLAZYME (galsulfase) injection for intravenous use NAGLAZYME is a formulation of galsulfase, which is a purified human enzyme that is produced by recombinant DNA technology in a Chinese hamster ovary cell line. Galsulfase (glycosaminoglycan N– acetylgalactosamine 4-sulfatase, EC 3.1.6.12) is a lysosomal enzyme that catalyzes the cleavage of the sulfate ester from terminal N–acetylgalactosamine 4-sulfate residues of glycosaminoglycans (GAG), chondroitin 4- sulfate and dermatan sulfate. Galsulfase is a glycoprotein with a molecular weight of approximately 56 kDa. The recombinant protein consists of 495 amino acids and possesses six asparagine-linked glycosylation sites, four of which carry a bis-mannose–6–phosphate residue for specific cellular recognition. Post-translational modification of Cys53 produces the catalytic amino acid residue, C alpha-formylglycine, which is required for enzyme activity.   NAGLAZYME is intended for intravenous infusion and is supplied as a sterile...

  Clinical Stage Pipeline Transthyretin amyloidosis (ATTR) Hereditary angioedema (HAE) Sickle Cell Disease Acute Myeloid Leukemia Transthyretin amyloidosis (ATTR) Transthyretin amyloidosis is a slowly progressive condition characterized by the buildup of abnormal deposits of a protein called amyloid (amyloidosis) in the body's organs and tissues. These protein deposits most frequently occur in the peripheral nervous system, which is made up of nerves connecting the brain and spinal cord to muscles and sensory cells that detect sensations such as touch, pain, heat, and sound. Protein deposits in these nerves result in a loss of sensation in the extremities (peripheral neuropathy) Gene Defect: Mutations in the TTR gene cause transthyretin amyloidosis. The TTR gene encodes a protein called transthyretin that transports vitamin A and thyroid hormone. The disease is inherited in autosomal dominant manner. Treatment Approach: NTLA 2001 is a  systemically delivered investiga...

LUMIZYME (alglucosidase alfa) for injection, for intravenous use Pompe disease (acid maltase deficiency, glycogen storage disease type II, GSD II, glycogenosis type II) is an inherited disorder of glycogen metabolism caused by the absence or marked deficiency of the lysosomal enzyme GAA. LUMIZYME provides an exogenous source of GAA. Binding to mannose-6-phosphate receptors on the cell surface has been shown to occur via carbohydrate groups on the GAA molecule, after which it is internalized and transported into lysosomes, where it undergoes proteolytic cleavage that results in increased enzymatic activity. It then exerts enzymatic activity in cleaving glycogen.  Initial U.S. Approval: 2010 INDICATIONS AND USAGE LUMIZYME (alglucosidase alfa) is a lysosomal glycogen-specific enzyme indicated for patients 8 years and older with late (non-infantile) onset Pompe disease (GAA deficiency) who do not have evidence of cardiac hypertrophy. The safety and efficacy of LUMIZYME have no...

ONPATTRO (patisiran)  small interfering RNA for Treatment of Polyneuropathy of Hereditary Transthyretin-Mediated Amyloidosis ONPATTRO contains patisiran, a double-stranded small interfering ribonucleic acid (siRNA), formulated as a lipid complex for delivery to hepatocytes. Patisiran specifically binds to a genetically conserved sequence in the 3’ untranslated region (3’UTR) of mutant and wild-type transthyretin (TTR) messenger RNA (mRNA). The molecular formula of patisiran sodium is C412 H480 N148 Na40 O290 P40 and the molecular weight is 14304 Da.  CLINICAL PHARMACOLOGY Mechanism of Action  Patisiran is a double-stranded siRNA that causes degradation of mutant and wild-type TTR mRNA through RNA interference, which results in a reduction of serum TTR protein and TTR protein deposits in tissues.  Pharmacodynamics  The pharmacodynamic effects of ONPATTRO were evaluated in hATTR amyloidosis patients treated with 0.3 mg/kg ONPATTRO via intravenous infusion once eve...

  About Harpoon Therapeutics Harpoon Therapeutics is a clinical-stage immunotherapy company developing a novel class of T cell engagers that harness the power of the body’s immune system to treat patients suffering from cancer and other diseases. T cell engagers are engineered proteins that direct a patient’s own T cells to kill target cells that express specific proteins, or antigens, carried by the target cells. Using its proprietary Tri-specific T cell Activating Construct (TriTAC ® ) platform, Harpoon is developing a pipeline of novel TriTACs initially focused on the treatment of solid tumors and hematologic malignancies. HPN424 targets PSMA and is in a Phase 1/2a trial for metastatic castration-resistant prostate cancer. HPN536 targets mesothelin and is in a Phase 1/2a trial for cancers expressing mesothelin, initially focused on ovarian and pancreatic cancers. HPN217 targets BCMA and is in a Phase 1/2 trial for relapsed, refractory multiple myeloma. HPN328 targets D...

  About Cullinan Oncology Cullinan Oncology is a biopharmaceutical company that strives to deliver results for our various stakeholders through disciplined capital allocation, decisive action, prudent risk-taking and creative business development. The company seeks to drive shareholder returns by focusing on the patient. The Company’s strategy is to build a diversified pipeline of targeted and immuno-oncology therapeutic candidates that are uncorrelated across multiple dimensions, with a focus on assets that it believes have novel technology, employ differentiated mechanisms, are in a more advanced stage of development than competing candidates, or have a combination of these attributes   About CLN-049 CLN-049 is a humanized bispecific antibody being developed for relapsed/refractory AML. CLN-049 is designed to simultaneously bind to FLT3 on target leukemic cells and to CD3 on T cells, triggering the T cells to kill the targeted cancer cells via their intrinsic cytol...

    About Autolus Therapeutics plc Autolus is a clinical-stage biopharmaceutical company developing next-generation, programmed T cell therapies for the treatment of cancer. Using a broad suite of proprietary and modular T cell programming technologies, the company is engineering precisely targeted, controlled and highly active T cell therapies that are designed to better recognize cancer cells, break down their defense mechanisms and eliminate these cells. Autolus has a pipeline of product candidates in development for the treatment of hematological malignancies and solid tumors.  Pipeline AUTO1 / AUTO1-AL1 – Adult Acute Lymphoblastic Leukemia (ALL) and Pediatric ALL AUTO1 is a CD19 CAR T cell investigational therapy designed to overcome the limitations in safety - while maintaining similar levels of efficacy - compared to current CD19 CAR T cell therapies. Designed to have a fast target binding off-rate to minimize excessive activation of the programmed T cells, AU...

  Werewolf Therapeutics, Inc. Werewolf Therapeutics, Inc. is an innovative biopharmaceutical company pioneering the development of therapeutics engineered to stimulate the body’s immune system for the treatment of cancer. They are leveraging our proprietary PREDATOR™ platform to design conditionally activated molecules that stimulate both adaptive and innate immunity with the goal of addressing the limitation of conventional proinflammatory immune therapies. Our INDUKINE™ molecules are intended to remain inactive in peripheral tissue yet activate selectively in the tumor microenvironment. The most advanced product candidates, WTX-124 and WTX-330, are systemically delivered, conditionally activated Interleukin-2 (IL-2) and Interleukin-12 (IL-12) INDUKINE molecules for the treatment of solid tumors. We are continuing preclinical studies for both WTX-124 and WTX-330 and expect to advance each candidate in multiple tumor types as a single agent and in combination with an immune checkpo...

PALYNZIQ (pegvaliase-pqpz) injection, for subcutaneous use  Pegvaliase-pqpz is a phenylalanine-metabolizing enzyme that is composed of recombinant phenylalanine ammonia lyase (rAvPAL) conjugated to N-hydroxysuccinimide (NHS)-methoxypolyethylene glycol (PEG). rAvPAL is manufactured in Escherichia coli bacteria transformed with a plasmid containing the phenylalanine ammonia lyase (PAL) gene derived from Anabaena variabilis.  rAvPAL is a homotetrameric protein with a molecular weight of 62 kD per monomer. To produce pegvaliase-pqpz, an average of nine (9) 20 kD PEG molecules are covalently bound (or conjugated) to each monomer of rAvPAL. The total molecular weight of pegvaliase-pqpz (rAvPAL-PEG) is approximately 1000 kD. Palynziq (pegvaliase-pqpz) injection, intended for subcutaneous injection, is a clear to slightly opalescent, colorless to pale yellow, sterile, preservative-free solution and is formulated at pH 6.6 to 7.4.  Palynziq is provided in a single-dose prefilled s...

Vector Persistence, Integration, and Reactivation and Genome Modification After Administration of Gene Therapy: Assessing Long Term Risks Gene Therapy(GT) that modifies the host genome necessitates the mutation analysis, off-target effect, and viral vector integration into the host genome. The integrating vectors (gammaretrovirus, lentivirus,, herpesvirus capable of latency-reactivation, and genome editing products (TALEN, CRISPR) introduces the risk for delayed adverse effects. These gene therapy products pose risk by  1) introducing the permanent change in the host genome;  2) the potential for off-target genome modifications that can lead to aberrant gene expression, chromosomal translocation, induce malignancies, etc.;  3) the risk for insertional mutagenesis when integrating vectors are used to deliver the gene delivery or, genome editing components, and the associated risk of tumorigenicity Long Read Sequencing Technology for Study Vector Integration Long-...

Nextera XT DNA Sample Preparation Kit  enable the researchers to prepare sequence-ready libraries for small genomes, PCR amplicons, and plasmid in 90 minutes. The low amount (1ng) of input DNA makes this method amenable to precious samples available in limited quantities. The prepared library is compatible with all Illumina Sequencing  technologies.  Advantages of Nextera XT Kit include Fastest and Easiet Sample Prep Workflow Innovative Sample Normalization Flexible Multiplexing Standard Operation Procedure For Preparation of DNA Library Tagmentation Genomic DNA include the fragmentation and tagging of DNA with adapter sequence Amplification of  libraries using limited cycle PCR that adds i7 and i2 adapters and sequences required for sequencing cluster generation Clean up libraries using single sided bead purification to purify amplified beads Library Quality check  Normalization of library quantity  Diluting Library  for the sequencing system...