Lorna (Biologics and biosimilars)

Question 1

Biologic drugs and biosimilars

Answer 1

• Large molecules made using living cells (usually by genetically modifying cells.
• They are big and very complex molecules, often 200 to 1000 times the size of more common small molecule drugs.
• Examples are insulin and vaccines
  FDA definition: “blood derived products, vaccines, in vivo diagnostic allergenic products, immunoglobulin products, products containing cells or microorganisms, and most protein products applicable to the prevention, treatment, or cure of a disease or condition of human beings”
• Biosimilars are less costly imitations of biologics but they are different from generics in that they are not exact copies- e.g. neurofen and own brand

Question 2

Biologic products

Answer 2

• There is a wide range of products such as vaccines, blood and blood components, somatic cells, gene therapy, tissues, and recombinant therapeutic proteins
• Can be composed of sugars, proteins, or nucleic acids or complex combinations of these. They may also be living entities such as cells or tissues
• Isolated from a variety of natural sources- human, animal, or microorganism- and may be produced by biotechnology methods and other technologies

Products:
- Monoclonal antibodies
- Proteins intended for therapeutic use, including cytokines, enzymes
- Immunomodulators (non-vaccine and non-allergenic products intended to treat disease by inhibiting or modifying a pre-existing immune response)
- Cellular products, including products composed of human, bacterial, or animal cells
- Gene therapy products including nucleic acids, viruses, or genetically engineered microorganisms that mediate their effect by transcription or translation
- Vaccines
- Allergenic extracts used for the diagnosis and treatment of allergenic diseases and allergen patch tests
- Antitoxins, antivenins, and vemons
- Blood, blood components, plasma derived products

Question 3

What do biologics have to offer?

Answer 3

• Ability to bind with high specificity (and highly selective to their targets) and affinity to a wide variety of molecules
• Dramatically improved patient outcomes e.g. in rheumatoid arthritis and cancer
  (Humans are biological so work better with biologics).

Question 4

Differences between biologics and ‘conventional’ drugs

Answer 4

Small drug molecules
- Chemically synthesised
- Low molecular weight
- Structure is known, easily identifies or characterised
- Less sensitive to environment
- Absorption across biological membranes is generally satisfactory
- Not necessary to use aseptic principles from initial manufacturing steps
- Variable specificity, may not be very high

Biological products
- Biotechnology products
- High molecular weight
- Structure is complex, not easily identified or characterised
- Very sensitive to environment (acid, heat, enzymatic degradation)
- Very poorly absorbed across biological membranes (have to be injected, too big to move across membrane, oral would be broken down by stomach)
- Susceptible to microbial contamination so necessary to use aseptic principles from initial manufacturing steps
- Target specificity very high (they are designed to bind to other biologics)
- Must be involved in more complex testing, Have to prove it works every time not just that it contains a certain atom (like in conventional drugs)

Question 5

Examples of biologics

Answer 5

• Insulin
• Erythropoietin (EPO) (used for treatment of anaemia resulting from kidney disease)
• Human growth hormone (used for treatment of growth hormone deficiency)
• Pegfilgrastim (used to reduce the change of infection in people with certain cancers receiving chemotherapy)
• Etanercept (used in rheumatoid arthritis)

Question 6

Categories of biologics

Answer 6

• Hormone- a substance produced by one tissue and conveyed by the bloodstream to another to effect physiological activity, such as growth or metabolism
• Interferons- proteins that are normally produced by cells in response to viral infection and other stimuli
• Interleukins- a large group of cytokine proteins. Most are involved in directing other immune cells to divide and differentiate
• Growth factor- a substance such as vitamin B12 or an interleukin that promoted growth, especially cellular growth
• Monoclonal antibodies- a single species of immunoglobulin molecules produced by culturing a single clone of a hybridoma cell. MAbs recognise only one chemical structure.
• Polypeptides- peptides containing ten or more amino acids
• Proteins- naturally occurring and synthetic polypeptides having molecular weights greater than about 10,000
• Vaccine- an agent containing antigens produced from killed, attenuated, or live pathogenic microorganisms, synthetic peptides, or by recombinant organisms. Used for stimulating the immune system of the recipient to produce specific antibodies providing active immunity and/or passive immunity in the progeny.

Question 7

Antibodies

Answer 7

Antibody- immune system related proteins (immunoglobulins)
- Large Y-shaped proteins
- Two pairs of polypeptide chains, each pair containing a heavy and a light chain of different sizes
- Produced by the B lymphocyte and recruited by the immune system to identify and neutralise foreign objects, e.g. bacteria and viruses
- The binding activity of IgG molecules is generated by the variable domains of the heavy and light chains (the variable region has 3 complementarity determining regions (CDRs) and these 3 loops change, not the whole beta sheet)

Question 8

Monoclonal antibodies (MAbs)

Answer 8

• Monoclonal antibodies are antibodies which have been artificially produced against a specific antigen.
• Extremely specific binding to target antigens
  Monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell

Question 9

MAbs mechanism of action

Answer 9

Function by three principal modes of action
1) block the action of specific molecules
2) target specific cells, or
3) function as signalling molecules

Question 10

Blocking activity of therapeutic MAbs

Answer 10

Achieved by preventing growth factors, cytokines, or other soluble mediators reaching their target receptors. This can be accomplished either by the antibody binding to the factor itself or to its receptor.
e.g. Infliximab, Etanercept, and Adalimumab all block TNFalpha, and are indicated for a number of inflammatory conditions including rheumatoid arthritis, psoriasis, and Crohn’s disease

Question 11

Targeting action of therapeutic MAbs

Answer 11

Involves directing antibodies towards specific populations of cells and is a versatile approach
Antibodies can be engineered to carry effector moieties such as drugs, enzymes, toxins, radionuclides, cytokines, or even DNA molecules to the target cells, where the attached moiety can then exert its effect.
e.g. antibody-drug conjugates

Question 12

Function as signalling molecules

Answer 12

The natural effector functions of antibodies are associated with binding to Fc receptors or binding to complement proteins and inducing antibody-dependant cellar toxicity or complement dependant cytotoxicity.
(basically- get antibody to bind to something you want to destroy and the binding will trigger the activation pathway)

Question 13

Example biologics- antibody-drug conjugates

Answer 13

• Three component system consisting of a potent agent, a linker and a monoclonal antibody
• The antibody binds to specific markers (antigens or receptors) at the surface of the cancer cell
• The conjugates is then internalised by the cancer cell
• The linker is degraded and the active drug released
• Antibody-drug conjugates enable the delivery of the attached drug selectively to cells that display the antibody’s target on their surfaces (less side effects and toxicity)
• This selectivity and targeted delivery reduces the side effects of the attached drugs which increases the number of drugs we can use as the side effects won’t be experienced (cytotoxic drugs for cancer treatment)

Question 14

Production of therapeutic MAbs

Answer 14

• Complex- involves many steps
• Involves animals and cell culture
• The mouse hybridoma technology was an important step in the development of antibody technology and paved the way for the emergence of therapeutic monoclonal antibodies
• The procedure yields a cell line capable of producing one type of antibody protein for a long period. A tumour from this ‘immortal’ cell line is called a hybridoma

Question 15

Hybridoma technology

Answer 15

1- Induce the production of B lymphocyte producing the antibody of interest in an organism - usually done by injecting a ‘foreign’ protein in mice

2- Extract B cells from the spleen of the mouse and add to a culture of myeloma cells (cancer cells). This results in the formation of hybridomas- cells formed by the fusion of a B cells and a myeloma cell

3- Screen for the hybridomas

4- Screen for desired antibody production. Initial collection of B cells is heterogeneous i.e. they do not all produce the same antibody. Therefore, hybridoma population also does not produce a single antibody.

5- Once sure that a certain hybridoma is producing the right antibody, that hybridoma can be cultured indefinitely and monoclonal antibodies harvested

Question 16

Problems with murine MAbs

Answer 16

• Initial treatments using monoclonal antibodies produced using the hybridoma technology were associated with problems
• During repeat administration to humans in clinical trials, it was observed that the half-life decreased and the products became less effective with each injection
• This is due to the immunogenicity of murine proteins in humans and the rapid development of a human anti-murine antibody (HAMA) response in patients
• HAMA response neutralised the effectiveness of the murine antibodies and resulted in their rapid clearance from the body

Question 17

Overcoming HAMA response

Answer 17

The solution was to make recombinant antibodies using antibody engineering

Question 18

Antibody engineering

Answer 18

• The first MAbs were murine molecules and were recognised as foreign when injected into patients leading to problems.
• Developments in molecular biology made it possible in the early 90s to clone the genes of IgG molecules and as a result the genes of MAbs of interest could be cloned in eukaryotic expression vectors
• In this way, recombinant versions of any MAb could be obtained from diverse cell lines in a reproducible fashion
• Initial major application of antibody engineering was the possibility to create chimeric antibodies
• Antibodies are well conserved through evolution so possible to create chimeras by fusing murine variable domains (responsible for the binding activity) with human constant domains leading to ‘chimeric antibodies’, which are 70% human and considerably less immunogenic in humans.
• further development of antibody engineering made it possible to decrease even further the murine part of MAbs, using an approach called complementary determining region grafting leading to ‘humanised’ antibodies, which are 85-90% human and are even less immunogenic than chimeric antibodies

Question 19

Human Hybridoma Technology
(fully human MAbs)

Answer 19

An in vitro process based on the immortalisation of B-cells from infected patients (can isolate B-cells from bone marrow)
- The B-cells harvested can be immortalised by fusion with Epstein-Barr Virus (EBV) in the presence of an oligonucleotide
- Immortalisation can also be performed with a human myeloma cell line
- The hybridomas can then be screened for specific antibodies

Question 20

Transgenic Mouse Technology
(fully human MAbs)

Answer 20

An in vivo process based on the immunisation of a transgenic mouse.
- The mouse has been genetically engineered and bred for the expression of human Ig’s.
- The B cells harvested after immunisation can be immortalised by fusion with a myeloma cell line
- The hybridomas can then be screened for specific antibodies

(Mouse hybridomas but producing human antibodies because the mouse produces human Ig’s.)
Not everyone has the same sequence so there is still some immunogenicity but only a small amount.

Question 21

Chinese hamster ovary (CHO)

Answer 21

• CHO cells are currently the predominant host used to produce therapeutic proteins.
• The process for development of a stable line starts with expression vector construction and transfection.
• After being transfected with plasmin bearing the antibody of interest genes, cells are screened for antibody production.

Question 22

Summary of biologics production

Answer 22

Producing biologic products is more complex than production of relatively small molecules such as aspirin or penicillin. It requires:
- Genetic engineering
- Cell line development
- Upstream processing
- Downstream processing
- Formulation
- Quality control
- Regulation and registration

Question 23

Key challenges with biologics

Answer 23

• Production
• Price
• Administration
• Immunogenicity
• Formulation and stability
• Biosimilars

Question 24

Biosimilars

Answer 24

A biosimilar product is a biological product that is approved based on a showing that it is highly similar to an FDA-approved biological product, known as a reference product, and has no clinically meaningful differences in terms of safety and effectiveness from the reference product.

Only minor differences in clinically inactive components are allowed in biosimilar products.

Biosimilars are marketed after the patent for the branded product has expired.

Although they might be regarded as ‘generic’ medicines, they are not chemically identical, so the process for approval of licenses for marketing is not the same as it is for generic drugs.

Question 25

How similar are biosimilars? And why aren’t they identical?

Answer 25

Biologics are complex products produced in living systems. Because of this similar but not exact copies can be made.

• Due to their complex makeup biologics are highly sensitive to manufacturing and handling conditions.
• Biologics and biosimilars are produced in living cells in a multi-step process: initially a basic protein is translated from DNA, then post-translational modifications, including changes and additions, are made to that basic protein structure.
• The impact of post-translational modifications can be significant.
• Similarly, differences in biological systems (e.g. type of cell and environments) used to manufacture biosimilars may cause different types and levels of modifications, which in turn may affect the quality, safety, or effectiveness of the product.
• So, whereas a generic can be identical to a small molecule reference product, biosimilars cannot and are not required to be exactly like the biologic reference product.
• Given the expected differences, regulatory authorities have outlined requirements to demonstrate similarity. Biosimilar manufacturers will generally need to generate lab, non-clinical, and clinical data to show that the product provides the same therapeutic benefit and risks to the patient as the reference product.

Question 26

Biobetters

Answer 26

• Biobetters are drugs in the same class as existing, approved biologics but are not identical to the original.
• They are engineered with improvements.
• Unlike biosimilars, biobetters are considered completely new and benefit from patent and market protection.
• E.g. Rituxan (Rituximan)

Question 27

Summary of biologics, biosimilars, and biobetters

Answer 27

Biologic drugs are big, complex, and sensitive molecules, with complicated production processes. They offer unparalleled target specificity and therapeutic potential. There are issues with delivery, stability and cost.

Biosimilars are less costly imitations of biologics. They are different from generics in that they are not exact copies and cannot be patented.

Biobetters are drugs in the same class as existing approved biologics. They are not identical to the original and are engineered with improvements. They can be patented as they are considered a new drug.