Biological Therapeutics Terms to Know

Problems with Herbal Medicine

Effectiveness depends on:

• Location where grown

• Time of year harvested

• How plants were processed before administered

• What/How many other plans were included in medication

• Environmental contamination

What three tenets ushered in the Modern Era of Drug Discovery?

1. Analytical chemistry for isolating, purifying, and structural aand property determination

2. Organic synthesis for making compounds

3. Advances in our understanding of medicial sciences and mechanism for disease

What are the characteristics of health?

• Grow and develop

• Metabolize

• Maintain homeostasis

• Adapt

• Respond

• Reproduction

What are tissues? What are the four types?

A group of cells in close proximity that are organized to perform specific functions.

1. Epithelia

2. Connective

3. Muscle

   1. Nervous

What is an Organ?

A group of tissues that work together to do a particular job.

What is a disease (Definition, characteristics, biological basis)?

Medical condition that impairs the normal function of part or whole body and disrupts the body’s normal homeostatic processes.

Disease tends to have an identifiable group of signs or/and symptoms such as pain, dysfunction, distress, and social problem.

Disruption of the body’s homeostasis.

What are Extrinsic and Intrinsic Factors that cause disease?

Extrinsic:

• Physical agents (heat, radiation, force)

• Chemical agents (pollution, toxins, chemicals, carcinogens)

• Biological agents (fungus, bacteria, virus, parasites)

• Nutritional imbalance (protein, vitamin, ion deficiency)

Intrinsic:

• Genetic factors (mutation)

• Congenital factors (defects recognized at birth)

• Immunological factors (over/under immune response)

• Pscyhological factors

What are the two approaches to mechanism-based drug discovery?

1. Find a chemical (conventional/small molecular drug) to modulalte the activity of the disease-causing component and to restore homeostasis (rationale-based drug design)

   1. Find a biological therapeutic to replace or regulate the level of disease-associated component(s) to restore homeostasis

What is the Orphan Drug Act? Benefits?

Passed to encourage pharma companies to develop drugs to treat diseases which affect less than 200k people, such as Huntington’s disease, ALS, muscular dystrophy, OR more than 200k people and for which it is not expected to recover the costs of developing and marketing the drug in the US.

Benefits:

• 7 years exclusivity

• Grants to phase 2 and 3 clinicals

• Waiver of NDA/BLA fees

• Qualify for Expedited approval

• Priority review (6 vs 10 months)

What is a Drug Target?

Specific macromolecule, or biological system, with which the drug will interact

What are the steps in the Process of Drug Development?

1. Discovery & Development

2. Preclinical Research

3. Clinical Research

4. Government Review

   1. Post-Market Safety Monitoring

What are the pros and cons of the modern approach for conventional drug discovery and development?

Pros:

• rationale-based drug design and development has been proven effective and represents the best strategy for small-molecule drug discovery/development

Cons:

• Low success rate, slow and expensive

Challenge:

• How to include all factors that determine the outcome of drug treatement into the drug design and lead identification

What is Recombinant DNA?

Molcule of DNA that is created by joining segments of DNA from different sources

What is in the “toolkit” for recombinant DNA technology?

Restriction Enzymes and DNA ligase

DNA Vector

Host organisms

What are restriction enzymes?

Enzymes that recognize and cut short specific nucleotide sequences (restriction sites)

• Most restriction sites are symmetrical and palindromic

They exist in bacteria to protect cells against other organisms, cutting phosphodiester bonds in a staggered manner

• Staggered cuts = sticky ends/overhand

• Event cuts = blunt ends

What is DNA ligase?

When DNA molcule sticky ends come together, they are only able to form hydrogen bonds (not stable enough)

DNA ligase helps join the ends of DNA by reestablishing the phosphodiester bonds.

What is the most commonly used DNA vector? Why?

Bacterial plasmid vector

• Contains multicloning sites, replication origin, antibiotics selection gene

• Capable of generating high copy number

Whate is Gene Cloning by PCR?

Cloned DNA segment is synthesized by using polymerase chain reactor

1. Heated then cooled to split strands

2. Primers bind to split sequences

3. Taq polymerase extends the primers

   1. Repeat

What is cell culture?

Removal of cells from an animal or plant and subsequent cultivation in an artificial environment for scientific research.

What equipment is required for cell culture?

1. Laminar flow hood

2. Incubator

3. Inverted microscope

4. Centrifuge

5. Water Bath

6. Fridge and Freezer

7. Liquid Nitrogen Storage

8. Hemocytometer

9. Pipettes

10. Cell culture vessels

    1. Consumables (pipettes, plasticware)

What are the basic reagents for cell culture?

• Complete medium

• Buffered solution - washing cells

• Detaching agent - enzyme used to detach adherent cells from culture vessels

  • Cryoprotective agent - Agent that reduces the freezing point of media and slws the cooling rate to reduce risk of crystal formation (DMSO)

What are standard culture conditions for cells (pH, osmolarity, CO₂, temperature)?

pH = 7-7.4

Osmolarity = 280-320 mOsmol/L

CO₂ = 5-10%

Temperature = 35-37 C

What are primary cells? What are immortalized cells?

Primary:

• cells isolated directly from intact or dissociated tissues or from organ fragments and grown in a dish.

• After a primary culture has been sub-cultured once, it becomes known as a cell line.

• Finate life-span, can only be subcultured 10-20 times before senescence

Immortalized:'

• Have no limit on their lifespand and have infinite capacity to proliferate

What is adherent cell culture? What is suspension cell culture?

Adherent:

• Grow in monolayer attached to surface of cell culture plate

• When passaged, detaching agent is used to detach cells from surface

Suspension:

• do not form monolayers on surface of cell culture vessel

• Remain in suspension

• Form clumps, especially at high density

What is mammalian cell culture? Non-mammalian cell culture?

Mammalian:

• Most common

Non-mammalian:

• Plants (grown as suspension in liquid mediums or as callus on solid medium)

• Bacteria/Yeast (solid support culture like agar, large scale cultures in suspension)

• Insect cell lines

What are the four phases of cell growth?

1. Lag phase - cells acclimate to culture conditions and are not dividing

2. Log Phase - Cells are actively dividing; best for experimentation and data collection

   1. This is when cells should be sub-cultured

3. Stationary Phase - Overcrowing and cell growth slows

4. Decline Phase - Cell death predominates

What is cell confluency?

Percentage of culture vessel surface area that appears covered by a layer of cells when observed by microscopy.

What should one consider when choosing a cell line?

1. Species - species specific?

2. Functional characteristics - use appropriate cell line for experiment

3. Finite or immortalized - finite are more functionally relevent, but immortalized are easier to maintain and clone

4. Normal or transformed - transformed cells have higher growth rate and plating efficiency, but they have undergone permanent genetic change (which may impact experiment)

5. Growth conditions and characteristics

What are the characteristics of commonly used cell lines?

Species

Tissue Origin

Morphology:

• fibroblast

• epithelial

• endothelial

• neuronal

• lymphoblasts

What is cell line authentication?

Cells lines can be misidentified or contaminated with other cells, which makes it important to properly identify cells.

Authentication can be achieved by genetic profiling using polymorphic short tandem repeat (STR) loci

What four components are important to consisder when making medium for cells?

1. Basal medium - mixture of nutrients and salts

2. Glutamine - essential amino acid for cell growth

3. Animal Serum - (if included, medium is known as “complete medium”; provides growth factors and nutrients for cells)

4. Antibiotics - prevents bacterial growth (possible to exclude by using aseptic technique)

What are the Standard Cell Culture Technique Protocols?

1. Aseptic Technique

2. Primary Cell Isolation

3. Subculturing/Passaging Cells

4. Cryopreservation and Cell Reviving

5. Testing Cells for Mycoplasma Infection

6. Cell Counting

7. Cell Transfection

What safety precautions should be taken for cell culturing?

• Wear proper PPE

• Read MSDS sheets

• Decontaminate all work surfaces before and after experiments

• Clean laboratory equipment

• Avoid creation of aerosols and/or splashes

• Wash hands

• Decontamiante all infectionous materials before disposal

• Report any accidents that may result in exposure to infectious materials to appropriate personnel

  • Do not eat, drink, smoke, handle contact lenses, apply cosmetics, or store food for human consumption in the lab

What is a vaccine?

Any preparation of dead or attenuated pathogens or their products that when introduced into the body, stimulates the production of protective antibodies and/or T-cells without causing the disease, and protects the body against the same pathogens

Mimics natural immune response against an infection or invasion of an immungen

What organs are important in the immune system?

Adenoids and Tonsils: checkpoint for entrance of germs from mouth or nose

Skin and Mucous Membranes: physical barrier to prevent germ entry

Lymphoid Organs: lymph nodes and lymphatic vessel that are widely distributed in the body

Bone Marrow: the source of immune cells

Thymus: site for T cell maturation

Spleen: storage site for various immune cells

What is the function of the Lymphoid Organ?

Lymph:

• fluid and cells (lymphocytes) flowing in the lymphatic vessels

Lymph Nodes:

• Kidney shaped organs at intervals along lymphatic vessels

Lymphatic vessels:

• Vessels that link lymph nodes and collect and return interstitial fluid to blood

• Transport immune cells throughout body

• Transport lipid from intestine to blood

What is the structure of the antibody? How many types are there?

Structure:

• Contains variable region (binds to outside cells) and Constant Region (attaches itself to cell surface)

• Light chain (parallel to heavy chain on variable region)

• Heavy chain (part of main antibody structure)

• Held together by disulphide bonds

5 Immunoglobulin (Ig) Classes:

• IgM (pentamer)

  • main antibody of primary respones, monomer form acts as B cell receptor

• IgG (monomer)

  • main blood antibody of secondary responses, neutralizeds toxins, opsonization

• Secretory IgA (dimer)

  • Secreted into mucs, tears, saliva, colostrum

• IgE (monomer)

  • Antibody of allergy and antiparasitic activity

• IgD (monomer)

  • B cell receptor (IgM monomer)

What are the 3 main Immune Components?

1. Infrastructure of Immune System

2. Humoral Substances (antibodies)

3. Cellular Components

What are the Cellular Components of the Immune System? What do they do?

• Mast Cell - dilates blood vessels and induces inflammation through release of histamines and heparin; recruits macrophages and neutrophils; helps in wound healing and defense against pathogens, also uses in allergic reactions

• Macrophage - Phagocytic cell that consumes foreign pathogens and cancer cells; stimulates response of other immune cells

• NK Cells - kills tumor cells and virus-infected cells

• Dendritic Cells - presents antigens on its surface, triggers adaptive immunity

• Monocyte - differentiates into macrophages and dendritic cells in response to inflammation

• Neutrophil - first responders at the site of infection or trauma; 50-60% of all leukocytes; releases toxins that kill or inhibit bacteria and fungi; recruits other cells to site of infection

• Basophil - responsible for defense against parasites; releases histamines that cause inflammation and may be responsible for allergic reaction

• Eosinophil - Releases toxins that kill bacteria and parasites but also cause tissue damage

What is an Immunogen? What is the characteristic of the best ones?

Molecules that can induce immune response (innate or acquired0

Bests immunogens are over 10,000 MW (carbs, nucleic acids, lipids are potential immunogens)

What is an Antigen?

Molecules which can stimulate production of and binds specifical to an antibody (antibody generator)

What is a Hapten?

Small molecule which, when combined with a larger carrier such as a protein, can elicit the production of antibodies which bind specifically to it.

What is an Epitope?

Also known as an antigenic determinant, is the specific structure in an antigen that binds to a specific antibody.

What are ways pathogens can enter the body?

• Skin

• Gastrointestinal track

• Respiratory track

• Urological track

  • Eyes and ears

What are the two types of immune response? What are characteristics of each?

• Innate Immune Response

  • Firstline defense and fast

  • No memory

  • Primarly involves physical barriers, complement components, and immune cells like macrophages, neutrophils, NK cells, and dendritic cells

  • Releases proinflammatory cytokines

  • Release soluble antigens

• Adaptive Immune Response

  • Acquired imunity

  • Acquired by responding to specific pathogen/antigen

  • Involves antigen presentation, T cells, B cells

  • Produces antibodyes, cytotoxic T cells, and memory T and B cells

  • Results in long term protection

What is the process of Innate Repsonse?

1. Bacterial, viruses, or other pathogens enter wound

2. Platelets from blood release blood-clotting proteins at wound sites

3. Mast cells secrete factors that mediate vasodilation, allowing blood plasma and cells to reach injured area

4. Neutrophils secrete factors that kill and degrade pathogens

5. Neutrophils and macrophages remove pathogens by phagocytosis

6. Macrophages secrete cytokines that attract and activate immune cells to initiate tissue repair

7. Inflammatory response continues until the foreign material is eliminated and the wound is repaired

8. Dendritic cells capture antigens

What are the outcomes of the Innate Immune Response?

1. Eliminate antigen/immunogen and restore homeostasis

2. Induce inflammation, a clinical symptom defined by pain, heat, redness, and/or swelling of the area of infection. Inflammation prevents establishment and spread of pathogens and faciliate wound repair.

Chronic Inflammation: prolonged inflammation that causes tissue destruction and development of inflammatory diseases such as arthritis, allergy, asthma, autoimmune diseases, hepatitis, inflammatory bowel disease, and transplant rejection

What are the two steps of Adaptive Immune Response? What types of cells are involved?

1. Antigen presentation

2. Activation of B and T Lymphocytes (B, T Cells)

   1. B Cells

   2. T_H cells

   3. T_C cells

What is Antigen Presentation?

• Antigen Presentation Cells (APCs) are located at common entry sites of pathogens

• They express receptors that enable capture and response to antigens

• Migrate from epithelia tissues via lymphatics to lymph node

• Dendritic cells process and present the antigen to cell surface that binds to T cell receptor and activates T-cells

What are the two classes of Dendritic cells?

1. Classical Dendritic Cells (DC)

   1. Most DCs in lymphoid organs/tissues, derived from myeloid precursors

   2. Constantly sample environment

   3. Upon encountering microbes/cytokines

      1. Upregulator costimulatory molecules

      2. Produce inflammatory cytokines

      3. Mirgrate from peripheral tissue to draining lymph node

2. Plasmacytoid Dendritic Cells

   1. Rare type of immune cell; develops in bone marrow and is less than 0l4% of peripheral blood mononuclear cells

   2. Poorly phagocytic and do not sample environmental antigens

   3. Major function: secretion of Type I interferon in response to viral infections

   4. May also differentiate into cells similar as classical DC and present antigen to virus-specific T-cells

How are antigens presented?

MEdiated by major histocompatibility complex (MHC), group of cell surface proteins expressed on all nucleated cells of verterbratse.

Two types:

1. MHC Class I

2. MHC Class II

Explain the difference between MHC I and MHC II.

MHC I:

• composed of alpha(heavy)-chain in a non-covalent complex with beta2-microglobulin

• Recognized by CD8+ T Cells (Tc)

• Accomodate peptides that are 6 to 16 amino acid residues in length

• Expressed on all nuclealted cells

• Process cytosolic proteins that are protelyticall degraded in the proteasome

MHC II:

• Contain two MHC-encoded polymorphic chains, an alpha chain and a beta chain

• Recognized by CD4_ T cells (Th)

• Allows larger peptides (up to 30 amino acids) to bind

• Expressed mainly on specialized APCs

• Process extracellular proteins that are internalized into endosomes