Therapeutic antibodies
Biosimilars → highly similar replicates of therapeutic antibodies (biologic) that already
received market approval. Improve patients access to biological therapy and addresses
concerns of escalating costs of health care.
- Must be comparable to originator reference in structure, function, animal toxicity,
pharmacokinetics, pharmacodynamics, clinical safety, immunogenicity, and
effectiveness
- Produced, formulated, and administered is same regimen as originator.
Discovery & Clinical trials Licensing
development
Therapeutic In vitro & in vivo Phase 1 to 3 for safety, efficacy, FDA and EMA with clinical and
Toxicological studies and dosing non-clinical data
Biosimilar Comparability Phase 1 to compare ADME. Phase Analytical, preclinical, clinical
In vivo studies 3 for efficacy and immunogenicity data to prove bio-similarity
Monoclonal antibodies:
- Chimeric → foreign-derived
amino acids for entire variable
domain of heavy and light chains.
- Humanized → CDR amino acid grafted from murine to human antibody
Classical hybridoma technology
Antibody secreting cells from spleen
of immunized animals fused with
immortalized non-antibody secreting
cells, resulting in cells that divide in
certain conditions.
Immunization of animal with antigen
→ animal is immunized with antigen.
Purity is crucial for adequate
immune response, contamination
should be considered.
- Haptens (small or weakly immunogenic antigens) cannot trigger immune response,
therefore are conjugated with immunogenic carrier protein.
Removal of spleen and fusion with myeloma → spleen is aseptically removed and
splenocytes containing antibody-producing B cells are fused with myeloma, producing
hybridoma cells.
1
, - Myeloma cells lack hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT)
enzyme, used in salvage synthesis of nucleic acids. Preventing them to grow in
certain culture media.
Hybridoma culture → Hybridoma cells are cultured in 96-well plate in presence of
hypoxanthine-amino-thymidine (HAT) selection medium. HAT eliminates unfused myeloma
cells since they lack HGPRT, unfused B cells die naturally.
Hybridoma screening → Antibody-producing hybridoma cells are screened via ELISA,
identifying and picking only specific antibody producing hybridomas.
Hybridoma expansion → Hybridoma cells are expanded in bigger wells to be maintained
and provide sufficient cells for cryopreservation and supernatants for investigation.
- Advantages → high specificity and affinity, long-term production.
- Disadvantages → time-consuming, animal immunization, murine antibodies.
Recombinant technology
Advanced and flexible method
for generating monoclonal
antibodies, in which antibody
genes are cloned and expressed
in host cells.
Can be used for therapeutics, imaging and diagnostics, protein-protein interactions, and
cell profiling.
Isolation of antibody genes → Antibody-producing B cells are collected from immunized
animal or human. Genes encoding variable region are amplified with PCR and cloned to
expression vectors.
Antibody engineering → Modifications like humanization and affinity maturation to
antibody. Single-chain variable fragments (scFv), Fab fragments, and bispecific antibodies
can be engineered in recombinant DNA.
Expression in host cell → Cloned genes transferred to mammalian cell line (CHO, HEK293)
or into bacteria or yeast. Host expressed antibody as recombinant protein.
Production and purification → Antibody can be produced via bioreactors.
- Advantages → Fully human or humanized, scalable production, genetic
modification for improved function.
- Disadvantages → More complex compared to hybridoma, high costs.
2
,Phage display (PDT)
Recombinant technology
which involves expression
of protein on surface of
phages via fusion with
phage coat protein and
genetic sequence that is
packaged withing.
Antibody library construction → genes for antibody fragments, such as scFc or Fab, are
cloned to bacteriophage vectors, creating huge diversity of antibody variants.
Display on phage coat protein → phage expressed antibody fragment on surface, allowing
to interact with target antigen.
Biopanning (selection) → phage library is exposed to target antigen on surface, phages with
high-affinity are retained, while weak or non-binders are washed away.
Amplification and re-selection → bound phages are amplified in bacteria and subjected to
multiple rounds of selection, improving affinity.
Cloning and production → genes with high-affinity are sequenced and cloned into
mammalian cells for large-scale production.
- Advantages → no animal immunization, fully human antibodies, rapid selection.
- Disadvantages → difficult, full-length functional antibody not always generated,
high-throughput screening.
Antibody library
Collection of antibody variants which are synthetically generated from immune sources,
serve as starting point for selecting high-affinity antibodies.
- Naïve library → created from non-immunized individuals
- Immune library → created from B ells of immunized individuals
- Synthetic library → designed by introducing mutations into antibody genes
- Semi-synthetic library (combinatorial) → mixed different heavy
and light chains for new combinations.
Alternatives:
- Bispecific antibodies → recognize two different epitopes
- Antibody-drug conjugates (ADCs) → antibody, linker, payload (warhead), cytotoxic
chemical that reduce severity of side effects by targeting their payload to tumor site.
3
, Omalizumab
Humanized (mouse) IgG1κ antibody of and CHO cell
line binding to IgE.
- Neutralizes free serum IgE, reducing its
surface levels on FcɛRI-expressing cells,
including the mast cells and basophils.
- Effector cells lose ability to bind allergen and
undergo IgE-dependent activation.
- Without IgE crosslinking, mast cells do not
degranulate, preventing release of histamine, leukotrienes, and cytokines causing
allergy symptoms.
Modifications → extending half-life (YTE mutation), affinity maturation, dual-targeting.
Evolocumab
Human IgG2 antibody targeting PCSK9.
- Adjunct to LDL cholesterol reducing therapies.
- Inhibits LDL receptor recycling.
Modifications → Fc engineering to improve interaction, subcutaneous administration.
Infliximab
Chimeric IgG1 antibody (human/murine).
- Binds soluble and transmembrane forms of TNF-α with high affinity to disrupt the
pro-inflammatory cascade signaling.
- Blocking leads to downregulation of local and systemic pro-inflammatory cytokines,
reduction of lymphocyte and leukocyte migration, induction of apoptosis of TNF-
producing cells, increased levels of NF-kBi, and reduction of endothelial cell
adhesion molecules and acute phase proteins.
Modifications → humanizing, improve CDC or ADCC, remove fucose.
Trastuzumab
Humanized IgG1κ produced in CHO cell lines binding HER2.
- Blocks cleavage of HER2, binds HER2 and activates NK-cells.
Modifications → radio conjugated antibody, optimize NK killing, bispecific.
4
Biosimilars → highly similar replicates of therapeutic antibodies (biologic) that already
received market approval. Improve patients access to biological therapy and addresses
concerns of escalating costs of health care.
- Must be comparable to originator reference in structure, function, animal toxicity,
pharmacokinetics, pharmacodynamics, clinical safety, immunogenicity, and
effectiveness
- Produced, formulated, and administered is same regimen as originator.
Discovery & Clinical trials Licensing
development
Therapeutic In vitro & in vivo Phase 1 to 3 for safety, efficacy, FDA and EMA with clinical and
Toxicological studies and dosing non-clinical data
Biosimilar Comparability Phase 1 to compare ADME. Phase Analytical, preclinical, clinical
In vivo studies 3 for efficacy and immunogenicity data to prove bio-similarity
Monoclonal antibodies:
- Chimeric → foreign-derived
amino acids for entire variable
domain of heavy and light chains.
- Humanized → CDR amino acid grafted from murine to human antibody
Classical hybridoma technology
Antibody secreting cells from spleen
of immunized animals fused with
immortalized non-antibody secreting
cells, resulting in cells that divide in
certain conditions.
Immunization of animal with antigen
→ animal is immunized with antigen.
Purity is crucial for adequate
immune response, contamination
should be considered.
- Haptens (small or weakly immunogenic antigens) cannot trigger immune response,
therefore are conjugated with immunogenic carrier protein.
Removal of spleen and fusion with myeloma → spleen is aseptically removed and
splenocytes containing antibody-producing B cells are fused with myeloma, producing
hybridoma cells.
1
, - Myeloma cells lack hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT)
enzyme, used in salvage synthesis of nucleic acids. Preventing them to grow in
certain culture media.
Hybridoma culture → Hybridoma cells are cultured in 96-well plate in presence of
hypoxanthine-amino-thymidine (HAT) selection medium. HAT eliminates unfused myeloma
cells since they lack HGPRT, unfused B cells die naturally.
Hybridoma screening → Antibody-producing hybridoma cells are screened via ELISA,
identifying and picking only specific antibody producing hybridomas.
Hybridoma expansion → Hybridoma cells are expanded in bigger wells to be maintained
and provide sufficient cells for cryopreservation and supernatants for investigation.
- Advantages → high specificity and affinity, long-term production.
- Disadvantages → time-consuming, animal immunization, murine antibodies.
Recombinant technology
Advanced and flexible method
for generating monoclonal
antibodies, in which antibody
genes are cloned and expressed
in host cells.
Can be used for therapeutics, imaging and diagnostics, protein-protein interactions, and
cell profiling.
Isolation of antibody genes → Antibody-producing B cells are collected from immunized
animal or human. Genes encoding variable region are amplified with PCR and cloned to
expression vectors.
Antibody engineering → Modifications like humanization and affinity maturation to
antibody. Single-chain variable fragments (scFv), Fab fragments, and bispecific antibodies
can be engineered in recombinant DNA.
Expression in host cell → Cloned genes transferred to mammalian cell line (CHO, HEK293)
or into bacteria or yeast. Host expressed antibody as recombinant protein.
Production and purification → Antibody can be produced via bioreactors.
- Advantages → Fully human or humanized, scalable production, genetic
modification for improved function.
- Disadvantages → More complex compared to hybridoma, high costs.
2
,Phage display (PDT)
Recombinant technology
which involves expression
of protein on surface of
phages via fusion with
phage coat protein and
genetic sequence that is
packaged withing.
Antibody library construction → genes for antibody fragments, such as scFc or Fab, are
cloned to bacteriophage vectors, creating huge diversity of antibody variants.
Display on phage coat protein → phage expressed antibody fragment on surface, allowing
to interact with target antigen.
Biopanning (selection) → phage library is exposed to target antigen on surface, phages with
high-affinity are retained, while weak or non-binders are washed away.
Amplification and re-selection → bound phages are amplified in bacteria and subjected to
multiple rounds of selection, improving affinity.
Cloning and production → genes with high-affinity are sequenced and cloned into
mammalian cells for large-scale production.
- Advantages → no animal immunization, fully human antibodies, rapid selection.
- Disadvantages → difficult, full-length functional antibody not always generated,
high-throughput screening.
Antibody library
Collection of antibody variants which are synthetically generated from immune sources,
serve as starting point for selecting high-affinity antibodies.
- Naïve library → created from non-immunized individuals
- Immune library → created from B ells of immunized individuals
- Synthetic library → designed by introducing mutations into antibody genes
- Semi-synthetic library (combinatorial) → mixed different heavy
and light chains for new combinations.
Alternatives:
- Bispecific antibodies → recognize two different epitopes
- Antibody-drug conjugates (ADCs) → antibody, linker, payload (warhead), cytotoxic
chemical that reduce severity of side effects by targeting their payload to tumor site.
3
, Omalizumab
Humanized (mouse) IgG1κ antibody of and CHO cell
line binding to IgE.
- Neutralizes free serum IgE, reducing its
surface levels on FcɛRI-expressing cells,
including the mast cells and basophils.
- Effector cells lose ability to bind allergen and
undergo IgE-dependent activation.
- Without IgE crosslinking, mast cells do not
degranulate, preventing release of histamine, leukotrienes, and cytokines causing
allergy symptoms.
Modifications → extending half-life (YTE mutation), affinity maturation, dual-targeting.
Evolocumab
Human IgG2 antibody targeting PCSK9.
- Adjunct to LDL cholesterol reducing therapies.
- Inhibits LDL receptor recycling.
Modifications → Fc engineering to improve interaction, subcutaneous administration.
Infliximab
Chimeric IgG1 antibody (human/murine).
- Binds soluble and transmembrane forms of TNF-α with high affinity to disrupt the
pro-inflammatory cascade signaling.
- Blocking leads to downregulation of local and systemic pro-inflammatory cytokines,
reduction of lymphocyte and leukocyte migration, induction of apoptosis of TNF-
producing cells, increased levels of NF-kBi, and reduction of endothelial cell
adhesion molecules and acute phase proteins.
Modifications → humanizing, improve CDC or ADCC, remove fucose.
Trastuzumab
Humanized IgG1κ produced in CHO cell lines binding HER2.
- Blocks cleavage of HER2, binds HER2 and activates NK-cells.
Modifications → radio conjugated antibody, optimize NK killing, bispecific.
4
