Index

Biotech and traditional drugs ........................................................................................................ 3

Antibodies as drugs................................................................................................................... 3
Structure of antibodies .............................................................................................................. 4
Antigen-antibody recognition ..................................................................................................... 5
Supramolecular structures of immunoglobulins ......................................................................... 5
Classes of immunoglobulins and their main features .................................................................. 5
Isotypes, allotypes and idiotypes ............................................................................................... 6
Light chain rearrangement ......................................................................................................... 6
Heavy chain rearrangement ....................................................................................................... 6
CDR variability .......................................................................................................................... 6
Interactions between antibody and binding site .......................................................................... 7
Transcriptional regulation of immunoglobulin genes .................................................................... 7
Affinity maturation .................................................................................................................... 7
Class switch ............................................................................................................................. 8
Pipeline of drug development from lab to market ............................................................................ 8
Evolution of guidelines .............................................................................................................. 9
Antibodies as therapeutic agents ............................................................................................... 9
Therapeutic antibodies sources ............................................................................................... 10
Hybridoma technology......................................................................................................... 12
Transgenic mice technology ................................................................................................. 12
Examples of antibody production from human cells ............................................................... 12
Antibody formats used as therapeutics ................................................................................. 13
Example of lead compound selection and optimization................................................................. 19
Antibiotic resistance ............................................................................................................... 19
Identification and optimization of new antimicrobials peptides .................................................. 19
Function of M-33 ................................................................................................................. 21
Antimicrobial activity in vitro ................................................................................................ 21
Antimicrobial activity in vivo ................................................................................................. 22
Immunomodulatory and anti-inflammatory activity in vitro ..................................................... 23
Immunomodulatory and anti-inflammatory activity in vivo ..................................................... 23
Pre-clinical stages ............................................................................................................... 23
Nanocarriers as drug delivery systems .................................................................................. 24
Applications of biosensors ...................................................................................................... 25
1
 Sepsis ................................................................................................................................ 25
Cosmetic applications ............................................................................................................ 26
Bevacizumab ............................................................................................................................. 27
General characteristics ........................................................................................................... 27
Colorectal cancer ................................................................................................................... 27
Mechanism of action ............................................................................................................... 27
Therapeutic indications ........................................................................................................... 27
Tumor angiogenesis ................................................................................................................ 28
Mode of administration and dosage.......................................................................................... 28
LUCENTIS............................................................................................................................... 28
Encoded combinatorial libraries: from ligand discovery to targeted therapeutics ............................ 29

2
 Antibodies and peptides for drug discovery

08/03/2024
General information about the course
PowerPoint presentation, about 20 minutes about a specific drug (preclinical trial, clinical trials etc…) only for
people attending all lectures.

22nd March is seminar

Biotech and traditional drugs

• Small molecular weight molecules (on the left of the slide)
o We can produce large amounts of these molecules with a low cost (ketoprofen, Acetylsalicylic
acid, Paracetamol, Penicillin)

• On the right side of the slide, we have the RNA molecule of the Pfizer vaccine for COVID, monoclonal
antibodies, fusion recombinant proteins and recombinant proteins (such as insulin), which are much
larger (thousands of atoms). They are not produced by chemical synthesis, but with recombinant
procedures. For this, we need specific facilities for cell cultures, for fermentation etc… The cost of
production is very high, because they are difficult to be isolated (various years of research and production
is laborious)

o Biological market analysis by drug classes: 30-35% of the market is made up of monoclonal
antibodies

Antibodies as drugs

• Antibodies as drug are isolated using

o Hybridoma technology
o Phage Display: large libraries to isolate antibodies for specific target
o Transgenic mouse technology
o Antibody production from human cells

• In the human being, there are immune organs for development of immune system. Antibodies are
generally derived from stem cells in the bone marrow, but also from
o Thymus

3
 o Spleen
o Tonsils and adenoids
o Lymph glands
o Etc…

• We can divide the immune system in vertebrates in

o Humoral immunity → Antibody production → B lymphocyte (specifically, plasma cells)

o Cell-mediated immunity → Antigen presenting cells and cell killing → T lymphocytes

• Production of antibodies
o B lymphocyte is the evolution of stem cells in
the bone marrow (change in DNA)
o When the antibody gets is touch with the
antigen, B cells proliferate and are
transformed in plasma cells, which get into
the blood circulation (in the picture, we can
see the rough ER that is very prominent
because of the high protein synthesis)

• B-lymphocyte activation
o After antigen selection, B cells proliferate and
maturate in clones (cells that are all the
same). Some clones become plasma cells, for production of antibodies, while others become
memory cells and stay in the blood for memory
o Antibodies derived from a mono-clone are called monoclonal antibodies, because they
recognize the same epitope of an antigen

Structure of antibodies

Heavy chains are larger and light chains. The two heavy
chains and the two light chains are exactly identical in the
same antibody. The two heavy chains are bonded by disulfide
bonds, while light chains and heavy chains are linked by one
disulfide bond.

At the N terminal end, we have VH (Variable Heavy domain),

which contain part of the binding site (it is different because
it has to recognize antigens); the second domain we have
CH1, that vary only between classes of antibodies (it’s the
same among all antibodies of one class); CH2 can have
linked carbohydrates (we can have five different sequences
of the heavy chains). Heavy chains corresponds to the
immunoglobulin class (isotypes): IgG, IgM, IgA, IgE, IgD.
For the light chain, we have a variable domain, and a
constant domain. The two isotype of light chains don’t define
the isotype of the antibody.

Antigen recognition is a binding between amino acids of CDRs and the epitope present on the antigen itself.
Antibodies are able to bind the antigen (without eliminating it) and are able to trigger the processes for eliminating
the pathogen. The binding sites are at the N terminal domains, and contain CDR regions (3 in the light and 3 in
the heavy chain) which form a binding pocket for the antigen. These binding sites are identical (avidity). The Fc
fragment (crystallizable fragment) …

CDRs are not usually positioned near each other, but are separated by framework regions.

4
Antigen-antibody recognition
Antigens can be parasites, bacteria, viruses, cancer cells etc… which have to be destroyed. The epitope is the
part of the antigen which is specifically recognized by the Ab binding site.

Supramolecular structures of immunoglobulins

• IgM are produced in a pentameric structure, in

which 5 antibodies are linked together. In terms of
energy, this molecule is very hard to produce: the
effector function, though, is potentiated because it
can bind more than two epitopes at once. The first
time we get in touch with an antigen, we produce IgM,
but these antibodies have very low affinity (strength of
binding). Low affinity is compensated by the high
avidity (we have more binding sites with low affinity).
This is the base of selection of antibodies drugs (if the
affinity is not good, the drug is not useful)

• IgA are produced in a dimeric structure, because

they are presence in the mucosa, which comes in
contact with many antigens (thus we need high avidity antibodies)

• IgG, IgE, IgD are monomeric

IgG molecules have higher affinity for antigens in respect to IgM. IgD are produced at the same moment as IgM
but are not circulating antibodies.

Classes of immunoglobulins and their main features

• The most important function of IgM is complement activation

• IgG most important activity is binding to macrophages with the Fc domain. They also cross the placenta
(only two subclasses, but useful to protect the fetus)
• IgA can bind to macrophages and activate alternative pathways
• IgE are used to protect against parasites, mainly in wounds: they bind to mast cells instead of
macrophages, which possess histamines that is a vasodilator. This is aimed at the destruction of the
antigen through use of different molecules. When the body commits a mistake in identifying some
antigens as allergens (such as pollen), this massive release of histamine causes the allergic reaction
5
Isotypes, allotypes and idiotypes

• Allotypic differences
• Idiotypic differences
• Isotypic differences

Light chain rearrangement

Human antibody repertoire is about 109 different molecules. Human genome comprises 25000 genes, roughly.
How is it possible to produce such a high number (in the order of billions) of different antibodies with a limited
gene diversity?

• L is the leader protein

• All the V fragments are not the same
• V3 and J2 are joined together in the B cell
• In the primary RNA, the cassette and the C region are divided by an intron, which is spliced during
maturation of mRNA
• All combinations of V and J fragments are possible, so we can produce large binding sites

Heavy chain rearrangement

In the heavy chain, we also have D1 and D2. During the first recombination step, a D locus is joined to a J locus,
and then the cassette is joined to a V locus. The C locus contains also all the genes for the various isotypes (the
first one for example is the one for IgM, so we produce IgM first).

All living being are focused on saving energy. This rearrangement is completely random, but the system of control
of rearrangement is very strict, so that the final structure of the antibody is functioning.

When the enzymes cut and rebound the DNA, they can do that only when the eptamer and nonamer sequences
are opposite to each other (for light chain). Enzymes recognize the links between the eptamers and nonamers.
For heavy chains the situation is a bit different, but the concept is the same.

CDR variability

6
The CDR regions are always in contact with the antigens, so they are the most variables (the variability in the FR
regions is not as high). The most variable region is the CDR3, which is the most central out of all the CDR
molecules. CDR1 and CDR2 are already present in the genome, while the CDR3 is formed when a V, D and J
fragments join together (the variability is given by the fact that this process gives rise to mutations,
rearrangements etc… which causes the high variability of the CDR3 region).

Interactions between antibody and binding site

• H bonds
• Electrostatic bonds
• Wan der Walls (less important, but still present)
• Hydrophobic interactions

There are no covalent bonds between antibody and binding sites, because these interactions would require
energy.

Transcriptional regulation of immunoglobulin genes

All other promoters are not active because they are very far away from the enhancer.

Affinity maturation

7
Point mutations occur randomly in the antibody sequence (no difference between CDR or framework regions).
The first time we get in touch with an antigen, we express IgM antibodies, which have low affinity (but high avidity),
so they are not very good for eliminating the pathogen (that’s why we have symptoms of the disease). When we
get in touch with the same antigen the second time, we don’t suffer any symptoms (we don’t realize that we have
been infected) because of the presence of memory cells. This happens because, in the meantime between first
and second contact with the antigen, the DNA undergoes changes because of point mutations (both in the
constant regions, CDRs and framework): some of the CDRs mutations improve the binding to the antigen for
future recognition. If the virus mutates (for example, flu or COVID-19 mutate frequently), the immunoglobulins
may not recognize the new variant anymore.

Class switch
All the cassette, including V, D, J and the
enhancer are moved just before Cγ. This causes
the most abundant production of
immunoglobulins because of the production
from plasma cells.

11/03/2024
Pipeline of drug development from lab to market

The process of developing a drug is very long

8
 • Research is done by universities, public or private research groups, academic spin-offs and so on. The
production of new molecules depends on our objective: for example, if our drug is against cancer cells,
we first identify the target (target identification); then we try to find the lead compound (lead generation)
against the target we found previously; then we need to optimize the lead compound (in terms of toxicity,
solubility etc…). This whole phase takes up to 6 years and it costs up to 5 million euros (usually found by
financing from bigger companies)

• Development increases costs a lot (up to 7 million euros), because the drug has to be produced in large
quantities (we would need fermentation systems, for example, if we talk about a biotechnological drug)
for animal testing, usually on rats (mice are too small), dogs or monkeys; we also need the evaluation of
toxicity and efficacy of the molecule (evaluation of the pharmaco-toxicology), excretion, secretion and
tissue target. All of this is used for evaluation before human clinical trials (toxicology evaluation is
essential before clinical trials)

• After the approval of pre-clinical phases, we pass to clinical trials (human experimentation), which
involves companies, but also health institutions (doctors, nurses) and regulatory organizations for
patents and administration. Clinical trials are divided into three main phases (approval of each phase
comes from the EMA or FDA)

o Phase I, in healthy volunteers (not having the disease the drug is tested against), to test the drug
with the dosage discovered in the previous trials

o Phase II, in patients with the specific disease

o Phase III, with a larger number of patients (to re-evaluate the efficacy and all the
pharmacological aspects)

These trials are usually done by comparing two groups: one of the groups is treated with the drug, while
the other is treated with the placebo. Clinical trials are the bottleneck of drug experimentation and
development: most drugs do not pass the clinical trials.

• If the final approval arrives from the FDA or EMA, the company is allowed to launch and market the
product

What happened during COVID-19 pandemic, the FDA/EMA were called to the pharmaceutical companies to
evaluate the process.

Evolution of guidelines
The guidelines for drug development are in continue evolution. In July 2017, the EMA released a revised guideline
titled “Strategies to identify and mitigate risks for first-in-human and early clinical trials with investigational
medicinal products” (EMEA/CHMP/SWP/28367/07 Rev 1), following an incident that resulted in one death and
serious neurologic outcomes in healthy subjects during a phase I multiple ascending-dose (MAD) in France. The
tragedy prompted industry sponsors to learn from this rare event and to re-examine their processes.

Antibodies as therapeutic agents

At the end of the 19th century, nobody new what antibodies were, or what the
immune response is. Then, someone discovered that, in the sera of vertebrates,
there was something that could fight diseases. Sera of horses, immunized with
toxins or microorganisms, were used to protect patients from diseases (for example
bacterial infections, toxins etc…). One of the most important examples was the
anthrax antitoxin, or the diphtheria antitoxin discovered by Emil Adolf von Behring.
After a prolonged period of repeated administrations of animal antisera, however,
this practice becomes toxic to humans.

Paul Ehrlich (which was awarded the Nobel Prize in 1908) was the first man in the
world that suggested the concept of receptors (which we now know as antibodies), secreted by cells of the
9
immune system in response to foreign antigens. He foresaw their use as magic bullets, to specifically attack a
wide variety of diseases.

How antibodies work

• Blocking, by ligand/receptor antagonist

• Signaling

• ADCC – Antibody-Dependent Cell-mediated

Cytotoxicity
A type of immune reaction in which a target cell or
microbe is coated with antibodies and killed by certain
types of white blood cells. The white blood cells bind
to the antibodies and release substances that kill the
target cells or microbes. Also called ADCC and
antibody-dependent cell-mediated cytotoxicity.

• CDC
• Targeting, which is the delivery of a payload (like a
toxic compound) that kills the cell

Therapeutic antibodies sources

10
 Hybridoma technology (1970) Phage Display (1980)

In this process, antibody-producing B lymphocytes Phage display is a molecular biology technique by

are isolated from the spleen of mice after immunizing which phage genomes are modified in such a way that
the mice with specific antigen and are fused with the coat proteins of assembled virions are fused to
immortal myeloma cell lines (a cancer of B cells) to other proteins or peptides of interest (of any origin),
form hybrid cells, called hybridoma cell lines. These displaying them thus to the external milieu. This
hybridoma cells are cultured in a lab to produce technology is based on the construction of large
monoclonal antibodies, against a specific antigen. libraries of antibodies binding sites.
Examples of antibodies obtained with this technique
are Orthoclone, Zevalin or Bexxar. Antibodies fragment obtained are of human origin
and are to be modified in fully human
The antibodies are of animal origin. Modified murine immunoglobulin. Examples of these fragments are
genes can be used for humanization or chimerization. Humira (2002, it recognizes TNF-α for rheumatoid
Humanized antibodies are for example Avastin, arthritis), Simponi, Benlysta, Cyramza and many
Herceptin, Rituxan, Synagis. others.

Transgenic mouse technology (2000s) Antibody production from human cells (2010s)

Antibodies of human origin. The first hybridoma-
derived human mAb isolated from transgenic animals
is panitumumab. It was approved in 2006 for the
treatment of EGFR expressing metastatic colorectal
cancer with disease progression.
Antibodies from human cells. Examples are
Casirivimab in combination with Imdevimab
(Ronapreve, Regeneron Roche); Regdanvimab
(Regkirona, Celltrion Healthcare); sotrovimab
(Xevudy, GSK). These are the only fully approved anti
Sars Cov-2 antibodies used for COVID19

11
Hybridoma technology
Mice are immunized with antigen A and given an intravenous booster
immunization three days before they are killed, to produce a large population
of spleen cells secreting specific antibody. Spleen cells die after a few days in
culture. To produce a continuous source of antibody they are fused with
immortal myeloma cells by using polyethylene glycol (PEG) to produce a
hybrid cell line called a hybridoma. The myeloma cells are selected
beforehand to ensure that they are not secreting antibody themselves and
that they are sensitive to the hypoxanthine-aminopterin-thymidine (HAT)
medium that is used to select hybrid cells because they lack the enzyme
hypoxanthine: guanine phosphoribosyl transferase (HGPRT). The HGPRT
gene contributed by the spleen cell allows hybrid cells to survive in the HAT
medium, and only hybrid cells can grow continuously in culture because of
the malignant potential contributed by the myeloma cells. Therefore, unfused
myeloma cells and unfused spleen cells die in the HAT medium, as shown
here by cells with dark, irregular nuclei. Individual hybridomas are then
screened for antibody production, and cells that make antibody of the desired
specificity are cloned by growing them up from a single antibody-producing
cell. The cloned hybridoma cells are grown in bulk culture to produce large
amounts of antibody. As each hybridoma is descended from a single cell, all
the cells of a hybridoma cell line make the same antibody molecule, which is
thus called a monoclonal antibody.

Transgenic mice technology

Amgen’s Xenomouse technology provides an effective system by which fully human mAbs by which fully human
mAbs can be readily produced. The XenoMouse strains are generated by introducing human immunoglobulin
genes into mice engineered to lack functional mouse immunoglobulin genes. Ultimately, the result is the
generation of antibodies that have no mouse protein sequences and are fully human. The mouse embryonic stem
cell contains murine genes. The XenoMouse is then used in the hybridoma technique to produce fully human
mAbs.
13/03/2024
Examples of antibody production from human cells

12
chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://www.cell.com/cell/pdf/S0092-8674(21)00224-
5.pdf

Antibody formats used as therapeutics

• Chimeric and Humanized are from antibody engineering technology

o Chimeric antibodies have the binding sites formed by murine parts
o Humanized antibodies are completely formed by human parts, while the CDR are murine

• Fully human antibodies are obtained by phage display, transgenic mice and human cells, without using
humans

Approach for the development of therapeutic antibodies

Approaches for the development of therapeutic antibodies. a The hybridoma technique starts by
immunization of mice with desired antigens to trigger an immune response. Harvested splenocytes are fused
with myeloma cells to produce hybridoma cells that persistently secrete antibodies. After the screening,
selected leads are used to generate chimeric or humanized antibodies. b | Phage display technology uses
human libraries to select antigens of interest. After several rounds of panning, immunopositive phage clones
are screened by ELISA; then DNA sequences are analysed. c | Transgenic mouse are used to obtain fully human
antibodies following a similar procedure to the mouse hybridoma technique

List of monoclonal antibodies approved for clinical use

• 7 murine
• 10 chimeric
• 22 humanized
• 16 human
• 6 fusion proteins

Most are fusion proteins, with parts of antibodies fused with other proteins. The first humanized (from hybridoma
technology with murine genes) antibody was produced in 1997, while the first human was produced in 2002.

13
Antibody fragments
By modifying genes of the immunoglobulins, we can obtain

• Fab, which contains antigen binding site

• Fc, which has the effector function
o Pepsin and papain can be used but the process is difficult, because of purification
• Fab2
• Fv, fragment variable, which contains only the binding site variable region
o No covalent bonds are present because we have VL and VH so we only have electrostatic
interactions
• scFv (single chain Fv), which has stretch of 15-20 amino acids that allow the formation of the binding site
between VL and VH (flexibility)
o Differences between scFv and immunoglobulin
▪ Absence of the Fc fragment, so the scFv can bind the antigen and segregate it, but cannot
induce a response from the immune system
▪ Molecular weight is much smaller (most important difference)
▪ Half-life of scFv is much less (usually hours)
• Diabody has a smaller link
• Small immunoprotein (SIP)
• Fusion proteins
o scFv fused with cytokine IL-2, which is used for activation of the immune response (useful against
cancer cells)
o scFv fused with Pseudomonas Exotoxin A
o Fc fragment with receptor
o Peptibody, which has a small peptide able to interact with something

Bispecific antibodies

14
 Antibody formats and biodistribution. a | Schematic illustration of various recombinant antibody formats.
From left: full IgG, Small Immunoprotein (SIP), Fragment Antigen Binding (Fab), Diabody and scFv. b |
Biodistribution profile for different mAb formats: tumour (solid lines) and blood levels (dashed lines) plotted
vs. hour after injection (h) for IgG (blue), diabody (black) and scFv (red) in tumour-bearing mice.

The half-life of a molecule is the time it takes for the concentration of that substance to fall to half of its initial
value. Short half life depends on many factors

• Presence of disulfide bonds

Short half-life molecules have to be administered more frequently. A single scFV has the shortest half-life and
are completely filtered away by the kidneys. Diabodies are completely gone after 72 hours, while scFv are present
only for the first hours.

Phage Display Technology

The technology was invented in the 80s by George Smith (Nobel prize 2018). It is possible to display antibody
fragments or peptides or enzymes on the surface of bacteriophages, constructing large repertoires of ligands to
be selected against specific targets. This technique is also used for the identification of new antibodies.

• M13 bacteriophage infects the sex pilum between two bacteria

• In this technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing
the phage to "display" the protein on its outside while containing the gene for the protein on its inside,
resulting in a connection between genotype and phenotype

• Library dimension is the number of phages present in a library

• Library diversity is given by the number of different ligands present in a library

• Naïve and synthetic libraries containing antibody fragments

15
 o Naïve libraries: libraries from circulating lymphocytes of immunized or non-immunized
individuals (we need to design primers for the DNA of B lymphocytes, and then the DNA is
shuffled to generate a sort of artificial immune system)

▪ Can we produce antibodies against self-antigens? No

o Synthetic libraries: synthetic genes constructed in laboratory starting from germ-line genes

▪ The CDR3 is formed by the recombination of genes, so it is not present in these stem cells,
and it can be added artificially (random amino acid sequences); we can build completely
artificial human antibodies (which are not present in nature)

▪ Can we produce antibodies against self-antigens) Yes, because the DNA is not taken from
circulating antibodies and the cells have not undergone any development

▪ Used for selection against cancer markers

It is not possible to build a full immunoglobulin on a phage particle.

The selection happens using a column with antigens. Phages displaying a specific antigen remain attached to
the column. Another method is using streptavidin and biotin.

After the third cycle of selection, we can identify the positive clones by ELISA test. We can then construct the full
human IgG.

Parameters to be evaluated for antibodies as lead compounds

• Specificity → Specificity is the capability of an antibody to bind selectively with a single epitope. If an
antibody binds more than one epitope we can talk about cross-reaction and the antibody is consequently
with poor specificity. Specificity is independent of affinity

• Affinity → Affinity is the strength used by the antibody to bind the antigen. It is a thermodynamic
expression of the primary interaction of a single epitope with a single binding site and, in principle, the
concept should be used only for a monovalent antigen binding to a single Fab or scFv fragment (“intrinsic
affinity”)

o The affinity is the main problem with monoclonal antibodies → maturation process, by mutating
some amino acids

o Are specificity and affinity related?

o Affinity measurement is possible in terms of energy

• Avidity → In the case of multivalent antigens and antibodies, the antibody-binding capacity should be
expressed by the term “avidity” (also reported as “functional affinity”), which incorporates the role played
by antigen and antibody valency in the observed reaction

Understanding affinity constants: will the patient survive?

16
Kd is the dissociation constant (affinity) of the antibody. The concentration of the Ab is less than the Kd, so the
patient dies. If the affinity is low, even if we use a load of Ab, the toxin will not be bound.

15/03/2024

Antibody affinity maturation

Affinity maturation is the most important lead optimization procedure for monoclonal antibodies. This happens
also in vivo, because the first time we get in touch with an antigen we have the primary immune response: the
primary immune response is so weak that we have symptoms of the disease. The second time we get in touch
with the same antigen, the secondary immune response which is based on IgG (they are more and have higher
affinity). The higher affinity of the IgG class of antibodies is due to the rise of random point mutations: some of
these can generate higher affinity to the antigen.

The process in vitro happens after we select a phage (which is comparable to a primary immune response in
vivo): we can mutate the amino acids in the variable parts of VL and VH (CDR and framework); the CDR are the
most important, and we don’t need random mutations, but we can act on specific sites.

Reformatting antibody fragments

17
Mimicking the immune system
Phage display can mimic the natural immune system. The target is completely random.

Some of the B cells remain in circulation: some become plasma cells, some have random mutations which
improve the affinity of the antibody they produce.

For phage display, we obtain V-genes (we might need only 1 and apply mutations). Infection of E.coli cells by the
selected phage (primary immune response) cause expression of antibody fragments.

Phage display of peptides

18
Adding a cysteine at the end of the sequence and one at the beginning of the sequence, we can form disulfide
bridge (which is a covalent bond) and can form a cyclic structure of the protein.

18/03/2024

Example of lead compound selection and optimization

Targets identification, lead generation and lead optimization can be done in universities and small research
centers, because the costs are low.

Setlance focuses its activity on 3 research area at different stages of development

• Antimicrobial activity of SET-M33 peptide and its backup molecule against infectious diseases
• Medical device for the selective removal of bacterial toxins from bloodstream of patients
• Cosmetic applications of antimicrobial compounds

Antibiotic resistance
Antibiotics are very potent, but unfortunately are often misused: for example, we use antibiotics when we don’t
know if the disease we have is actually caused by bacteria. Since antibiotic resistance is caused by random
mutations, antibiotics should be used only after antibiograms. A failure to address the problem of antibiotic
resistance could result in 10 million deaths by 1050, costing around £66 trillion. Antibiotics are drugs that are
used for a very short period, not used for chronic diseases (like chemo drugs or drugs for autoimmune diseases)
thus they are not useful for financial gain: small research centers are the most important centers for the
resolution of this problem, because big corporations are not interested due to the low monetary gain.

Identification and optimization of new antimicrobials peptides

Antimicrobial peptides are not as potent and as specific as antibiotics.

19
The molecule with the sequence QKKIRVRLSA was selected from a phage library and has a typical amphipathic
structure, because it is made up of hydrophilic and hydrophobic (non-polar) amino acids. This molecule was
able to kill bacteria very easily and in a short period of time (even though this sequence was not present in nature,
it had the same mode of action of antimicrobial peptides).

Is this a good lead compound? Actually no. After obtaining the peptide from a second mode of preparation, it
was found that there was a modification of some amino acids.

Tetra branched peptides are made up of several peptides linked together by other smaller peptides. Peptides are
very unstable molecules, so when they are used in a biological matrix they are degraded very quickly (and quite
unuseful to be used as drugs). So it was found that Multiple Antigen Peptides (MAPs) acquire high resistance to
protease and peptidase activity making these molecules good candidates for in vivo use.

The peptides was called M-33, which has different formed. One of them has already finished the development
process and is ready to be used in clinical trials. It is used against G- MDR infections (E.coli, Pseudomonas
aeruginosa etc…). The same molecule with D amino acids is sued against G+ in sepsis and pneumonia.

20
Function of M-33
SET-M33 showed concentration-dependent bactericidal
activity against colistin-susceptible and resistant isolates of
P. aeruginosa and K. pneumoniae. Scanning and
transmission microscopy studies showed that SET-M33
generated cell blisters, blebs, membrane stacks and deep
craters in K. pneumoniae and P. aeruginosa cells. NMR
analysis and CD spectra in the presence of sodium dodecyl
sulfate micelles showed a transition from an unstructured
state to a stable α-helix, driving the peptide to arrange itself
on the surface of micelles.

SET-M33 kills Gram-negative bacteria after an initial

interaction with bacterial LPS (the peptide is positive, so it
interacts with the negative parts of the LPS). The molecule
becomes then embedded in the outer membrane surface,
thereby impairing cell function. This activity of SET-M33, in
contrast to other similar antimicrobial peptides such as
colistin, does not generate resistant mutants after 24h of
exposure, non-specific interactions or toxicity against
eukaryotic cell membranes, suggesting that SET-M33 is a
promising new option for the treatment of Gram-negative
antibiotic-resistant infections.

The cytoplasm leaks from the cell after interaction between

LPS and SET-M33; water goes inside the cell, which causes
the explosion of the cell. The discovery of the mechanism of
action is very important for creating a dossier for a new drug.

Antimicrobial activity in vitro

21
Polymyxin B was used as a reference (because it is a naturally occurring peptide). But what is the amount of
peptide needed to perform these experiments? A rather small amount, which is doable for a small amount. For
antimicrobial activity in vivo, the production needs to be scaled up (because we need grams).

Antimicrobial activity in vivo

The end of these analysis is the statistical analysis of the results. The results can be significant, or not. When
they are not significant, it means that the results obtained are maybe due to chance and not to the effect of the
molecule itself.

22
Immunomodulatory and anti-inflammatory activity in vitro
Since most antimicrobials peptides are part of the innate immune system, they also have anti-inflammatory
activity. It was found that the expression of pro-inflammatory cytokines and enzymes are strongly inhibited. The
expression of cytokines was studied by mRNA expression (by converting mRNA into cDNA). A decrease in the
band intensity indicates a lower expression (or an inhibition) of a protein. All of this was compared to a control.

Immunomodulatory and anti-inflammatory activity in vivo

Mice were challenged with LPS from Pseudomonas by nebulization. After 8 hours, BAL was collected, and they
concentration of cytokine was analyzed.

Pre-clinical stages

• Biodistribution and plasma clearance: the molecules was conjugated with a radioactive isotope. The
plasma clearance is the persistence of the molecule in the blood, which is quite rapid (the drug is not
present in the blood after about 2 hours). This means that the molecule is not eliminated by the body, but
it’s present in the organs (as displayed by the other graphs)

o The %ID in the thyroid is high because of the iodine conjugated to the peptide
o In the liver, after about 15 minutes, there is about 15% of ID; the graph for urine is complementary
to the one for the liver → the most important information is that excretion is mainly through the
kidneys and urine; we also have a small excretion from feces
23
 • Toxicity in vivo: international guidelines for drug development require the evaluation of toxicity in two
animal species including a rodent (usually rats, because of their size) species and a non-rodent species
(monkey or dog); these experiments require specific facilities. The parameters evaluated in rats and dogs
are

o Dose Ranging Finding (DRF)

o Neurological Toxicity (by studying behavioral aspects)
o Respiratory Function
o Cardio Performance
o Clinical Laboratory Investigations (such as cholesterol, glucose levels etc…)
o Bioanalytics
o Toxicokinetics

We obtain the NOAEL, or the No-Observed-Adverse-Effect Level. M33 was given by infusion in a 4-week
daily administration of 1 hour and animals were checked for macroscopic and clinical signs, and the
NOAEL resulted < 1mg/Kg. This information is useful when comparing it with efficacy: if the efficacy is
below, then the therapeutic window is positive and the drug can be used in clinical trials (with a starting
dose which is much lower than the NOAEL)

Nanocarriers as drug delivery systems

Nanocarriers are used to extend the therapeutic window of the antimicrobial peptide. Nanocarriers have a range
of pros

• Well-tolerated
• Non-immunogenic
• Biocompatibility
• Better drug pharmacokinetics and Biodistribution
• Controlled release
• Prolonged residence time
• Lower frequency of administration
• protection from enzyme (i.e. Protease, b-lactamase)
• Biofilm penetration
• Decrease of toxicity
• Avoid mucus interaction

SET-33 was encapsulated using PLGA-based nanoparticles.

PLGA-based nanoparticles (NPs) containing SET-M33
peptide is prepared by a modified emulsion/solvent
diffusion technique at 4% theoretical loading (4mg of M33
per 100 mg of NP). Formulation was prepared adding poly
(lactic-co-glycolic acid) (PLGA) conjugated with
polyethylene glycol (PEG) at two different molecular weight
(2000 Da and 5000 Da) to the organic phase containing
PLGA.

It was found that the inhibition of bacterial growth is

significant in a dose-dependent manner, in comparison with
the controls (only bacterial an NPs unloaded).

The anti-biofilm activity of SET-M33 in nanoparticles was

also tested. We can’t see differences between the free and
encapsulated peptide. The histograms represent the % of
bacterial biomass measured at the times indicated in the
internal legend after incubation of free SET-M33 or NPs with
bacterial biofilms.

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The encapsulated peptides do not result toxic

19/03/2024
Applications of biosensors

• Medical industry
• Drug discovery
• Plant studies
• Fermentation industries
• Defense and fighting bioterrorism
• Cell and metabolism
• Quality of food

They are based of layers of materials (like metals) where a biological ligand is bound. When this happens, the
metal is excited, and it emits a signal. They can be used in medical devices, such as the pregnancy test: binding
to the antibody produces a colored band.

Sepsis
Sepsis is a life-threatening condition that arises when the body responds to an infection injuring its own tissues
and organs. It may lead to shock, multiple organ failure, and death, especially if not recognized early and treated
promptly. Chemicals released into the bloodstream fight an infection by triggering inflammatory responses
(cytokine storm) throughout the body. There is no specific therapeutic opportunity for sepsis.

The two main toxins produced by bacteria in sepsis are lipopolysaccharide and lipoteichoic acid

• LPS, or endotoxin, the major constituents of the outer membrane of G- bacteria, is the major bacterial
product responsible for the clinical syndrome of sepsis. LPS binds to the host receptor Toll-like receptor
4 triggering an inflammatory response

• Lipoteichoic acid (LTA), …

According to 2016 data, sepsis was the most expensive condition treated, accounting for $17 billion, or 6.2% of
the aggregate costs for all hospitalizations. Around 27 million people develop sepsis, and 8 million people die
from sepsis globally each year, causing sepsis to be the leading cause of hospital deaths.

The most important course of action for the treatment of sepsis is the early administration of the right antibiotic;
administration of fluids, colloids and vasopressors (for blood pressure). In the last 15-20 years, very different
medical devices are in use in ICUs for the blood purification of patients, in terms of removing sepsis-associated
25
mediators or endotoxins, through hemofiltration or plasmapheresis practices. Toraymixin and Alteco take
advantage of peptides. Cytosorb and Seraph are based on polymer beads to absorb non-specifically cytokines
or sepsis mediators. No one of the commercially available devices is reported to selectively remove LPS, LTA,
living bacteria and different cytokines at the same moment.

SET-M33 was used in this type of application: the idea was the production of a new cartridge with a biocompatible
matrix containing SET-M33 peptide. The antibody has to be covalently bound, because the peptide could detach
from the matrix. The peptide was synthesized with a thiol functional group, for the peptide conjugation ot a
biocompatible matrix based on agarose beads.

Ex vivo experiments were conducted with mice (very difficult because of the small size): CD1 mice were injected
with 0.05 mg/kg LPS from E.coli. Blood was drawn after 4 hours and processed in the medical device for LPS
removal. Y axis indicated the amount of endotoxins units per ml.

After all the testing

• Large scale peptide synthesis using GMP production for preclinical and clinical evaluations
• Animal model of sepsis and extracorporeal blood filtration
• Clinical evaluation

Cosmetic applications
Acts on keratinocytes: after 24h, the model of the would was completely closed. The cells, in the presence of the
peptide, stimulate movement of cells (not growth or proliferation, because it could result in cancer).

SET-M33 stimulates the collagen biosynthesis and it seems to be potential candidate for the prevention of …

26
 Bevacizumab
General characteristics
• Trade name: Avastin
• MAB: Monoclonal AntiBody
• ZU: humanized
• Production: it is produced in a Chinese Hamster Ovary mammalian cell expression system in a nutrient
medium containing the antibiotic gentamicin and has a molecular weight of approximately 149 kDa
• Half-life: about 20 days

Colorectal cancer
CRC is the third most commonly diagnosed cancer in both men and women in the United States and the second
leading overall cause of cancer death. Adenocarcinoma, which accounts for approximately 95% of CRC tumors,
begins in the lining of the colon or rectum, and is preceded by colorectal polyps (adenomas; abnormal tissue
growth).

The 5-year survival rate of people with localized stage colorectal cancer is 90%. About 39% of patients are
diagnosed at this early stage. If the cancer has spread to surrounding tissues or organs or to the regional lymph
nodes, the 5-year survival rate is 71%. If the cancer has spread t distant part of the body, the 5-year survival rate
in 14%.

Mechanism of action

• Recombination Humanized monoclonal antibody that is 93% human and 7% murine (CDR)
• It inhibits all active isoforms of VEGF by neutralizing the ability of VEGF to bind its receptor on endothelial
cells
• This inhibition of VEGF makes the tumor vessel regress and inhibit the new formation of a vascular
reticulum

Therapeutic indications
Avastin is approved for

• Metastatic colorectal cancer (main one)

• Advanced non-squamous non-small cell lung cancer
• Advanced cervical cancer
• Platinum resistant ovarian cancer
• Breast cancer
• Renal cell carcinoma
• Pancreatic cancer
• Sotf-tissue sarcoma

In 2011, the US FDA notified Genentech that was revoking the approval of Avastin’s indication for the treatment
of metastatic breast cancer in the US. Mabs must be administered with other chemotherapeutic drugs.

Side effects include

• Most serious but uncommon

o GI perforation
o Wound that don’t heal
o Serious bleeding
o Abonrmal passage in the body fistulas
o Severe high blood pressure
o Kidney problems
o Infusion reactions
o Stroke or heart problems

27
 • Most common
o Perineuria
o Nose bleeding
o Rectal bleeding
o Back pain
o Dry skin

Tumor angiogenesis
Angiogenesis is the formation of new blood vessels from preexisting vasculature. It is necessary for tumor growth
and metastasis. The receptor for angiogenesis are

• VEGF family, in mammalians

• …

Signaling is activated by VEGFR-2

• PI-3 Kinase/Akt pathway

• Classic Ras-dependent signaling cascade
• MAP kinase through ERK-1 and ERK-2

Mode of administration and dosage

• Pharmaceutic form: concentrate solution for infusion

• Administration: initial dose with endovenous infusion of 90 minutes. If well-tolerated is possible to make
a second infusion in 60 minutes
• Overdose: the higher dose tested in a man is of 20 mg/kg of body weight (associated with severe
migraine)

LUCENTIS
Ranibizumab is a humanized antibody fragment (Fab) was made by Novartis. It was approved in 2006 by FDA for
Wet Age-Related Macular Degeneration (AMD), a condition in which new abnormal blood vessels leak fluids and
blood leading to loss of vision. Ranibizumab was initially derived from the humanized anti-VEGF variant known
as MB1.6: it was modified to take only the Fab fragment. Affinity was improved because of loss of avidity complex
(we only have one binding site).

Ranibizumab has a half life of 3 days and it is delivered by intravitreal injection, 2 mg in dose.

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 Encoded combinatorial libraries: from ligand discovery to targeted therapeutics
The fascination of a molecular targeting of diseases
The observation that certain chemicals could specifically stain tissue structures inspired the vision or targeting
disease with selective medications is called chemotherapy.

• He discovered Mast cells

• He spent most time staining tissues

Vascular permeability: not all tumors have the same

Concentration of antigens
BBB is broken in brain cancer

If we have ultra-high binding affinity we can afford to give less; potent signaling cascade. We cam make these
drugs better by pharmacodelivery.

29
 25/03/2024
Trastuzumab

• FDA approved in 1998

• Brand name: Herceptin
• The drug was first discovered at Genetech, Inc.
• The first clinical trials were with few women, a phase I clinical trials (1992) in patients with HER2-positive
metastatic breast cancer succeded
• Two small scale, phase II clinical trials wee conducted simultaneously
• Breast cancer
o It is the leading type of cancer in women, 100 times more common than in men
o Survival rates in the developed world are high, with between 80% and 90%
o It can be classified on histological features
▪ ER or hormone-receptor positive
▪ Human epidermal growth factor receptor 2 overexpression (HER2+)
▪ Triple negative breast cancer (TNBC)

• HER receptors
o Human epidermal growth factor receptor (HER) family can be used as target
o In cells with HER2-overexpression, the result is a spontaneous dimerization
o This leads to the phosphorylation of the tyrosine kinase, which triggers signaling downstream
pathways such as MAPK, PI3K, phospholipase C and protein kinase C
o Overexpression of HER-2, occurs in 15 to 20% of invasive breast cancer
o The trigger HER-2 pathways promote cell growth and division beyond normal limits
o Trastuzumab, a recombinant humanized ani-HER2 monoclonal antibody targets the extracellular
domain of HER2 with high affinity; it was the first clinically

• HER-2 antagonists
o When Trastuzumab is combined with chemotherapy, it produces a higher overall response rate
and longer time to disease progression
o Novel anti-HER2 therapies are needed to overcome acquired trastuzumab resistance
o Trastuzumab is used in combination with

• Development of trastuzumab
o Production of an anti-p185HER2 mouse monoclonal antibody

• Potential mechanisms of resistance

o Some HER2+ breast cancer cell lines display either primary or acquired resistance to
Trastuzumab caused by
▪ Truncated HER2
▪ Epitope masking
▪ Cross-signaling to HER2
o Hyperactivating Downstream signaling
o Failure to activate ADCC

Pertuzumab

• Monoclonal antibody that binds the extracellular subdomain II of HER2

• Synergism
• Current consensus guidelines agree that
o Dual blockade with trastuzumab + pertuzumab in the preferred regimen for the first line treatment
o T-DM1 is the second line treatment of choice and addition of lapatinib may be considered in the
later lines

Ado-trastuzumab emtansine (T-DM1)

30
 • Ab-drug conjugate targeting HER2
• Trastuzumab + stable linker + cytotoxic agent emtansine
• Dosage
• Costs

Cetuximab

• Chimeric antibody
• No “zu” suffix because it’s not humanized; all the variable portion of the murine molecule remain
untouched

Adalimumab
• Rheumatoid arthritis

The avidity effect is tapped because of the two arms

If one arm is detached, I don’t bind the molecule anymore

Etanercept
• Fusion protein
• Artificial molecule that does not exist in nature

CLINICAL STUDIES AND TRIALS MUST BE PRESENT: p-value is the most important <0.05

Certolimumab Pregol

Belimumab

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 26/03/2024
Rituximab
• Non-Hodgkin’s lymphoma
• It was first produced by Genentech (Rituxan) which was acquired by Roche (MabThera)
• It is a chimeric antibody of 145 kDa
• The Fc fragment has glycosidic groups
• Acts against the CD20 antigen: only targets Pre-B to immature B cells
• CD20 has an outer structure that is composed of two loops, one of them being the target of rituximab
• CD20 is a strong tumoral marker because it is overexpressed, never internalized and does not shed

• Non-Hodgkin lymphoma
o The incidence of non-Hodgkin lymphoma varies by geographical region. The areas with the
highest incidence of NHL are North America, Europe and Australasia, as well as several countries
in Africa and South America
o About 10-12% of people worldwide are affected by this tumor
o Different organ settings → lymph nodes, stomach, gut and skin
• Intravenous administration
• Side effects
o Weakness
o Low blood pressure
o Pain
o Flu symptoms
o Nausea and vomit
• The patent has expired in 2013: other companies can sell this molecule

Ibritumomab

• Ibritumomab tiuxetan is normally called Zevalin

• It was approved by the FDA in 2002
• Chimeric antibody
• Mechanism of action is slightly different from rituximab
• Injection for intravenous use
• Combined with Rituximab

Reslizumab
• Asthma is a chronic and heterogenous airway disorder, characterized by recurrent respiratory symptoms,
due to the inflammation of the bronchial tubes with increased production of sticky secretions inside the
tubes
• This widespread airway disorder affects over 315 million people worldwide and it projected to reach
higher numbers in the future
• Asthma’s manifestation can range from mild to very severe
• It affects people of all ages and often starts in childhood, although it can also develop for the first time in
adults
• Genetic, environmental and occupation factors can be linked to developing asthma
• Eosinophilic asthma: 5-10% of asthmatic patients have severe asthma that is poorly controlled by drugs.
It is marked by high levels of white blood cells, eosinophils, that are involved in inflammation and swelling
in the respiratory system. Eosinophils are the inflammatory cells most frequently infiltrating the airways
of asthmatic patients
• Reslizumab is a human monoclonal antibody that sequesters the ligand to the IL-5 receptor (so IL-5)
• Pharmacokinetics
o ADME
▪ Adsorption → Peak serum concentrations of 80 µg/mL are often observed at the end of the
infusion; the serum concentration declines in a biphasic manner
▪ Distribution → Volume of distribution of 5 L, indicating a minimal distribution to the
extravascular tissue
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 ▪ Metabolism → it is metabolized by enzymatic proteolysis into small peptides and amino
acids
▪ Elimination → It is cleared at about 7 mL per hour and has a half-life of 24 days

Benralizumab
• Monoclonal antibody which was developed by MedImmune for the treatment of eosinophilic asthma. It
is directed against the alpha-chain of the interleukin 5 receptor
• Humanized monoclonal antibody against a subunit of IL-5
• It works with a different mechanism of action in respect to Reslizumab, because it bind to the receptor
of IL-5
• It works as an antagonist of IL-5R

33