Biotech II Lecture 11 - Protein Therapeutics

what are therapeutic proteins

proteins based on or inspired by naturally occurring proteins in the human body

types of therapeutic proteins

antibodies, enzymes, cytokines and hormones and more

why are therapeutic proteins important

size and complexity gives them advantage over small molecules when targeting extracellular or membrane associated proteins, modulating immune responses or replacing dysfunctional/missing proteins

why do therapeutic proteins often provide better specificity, reduced off-target effects and more natural regulatory effects

due to their structural similarity to endogenous proteins

proteins which are engineered in the laboratory for clinical uses

therapeutic proteins

an approach in treating diseases using proteins and peptides

protein therapeutics

delivers protein to the body in specific amounts to treat diseases and is similar to gene therapy

protein therapy

first recombinant protein therapeutic

insulin

timeline of protein therapeutic development

1920 - isolation of insulin

1980 - FDA approval of insulin and antibody muromonab

1990 - FDA approval of pegylated drug, enzyme and Fc fusion

2000 - FDA approval of ADC and lipidated protein

2010 - FDA approval of gycoengineered bispecific albumin fusion proteins

2020 - FDA approval over 100 antibody drugs

most widely used type of biologic

monoclonal antibodies (mAbs)

what do mAbs bind and how do they bind them

bind to a single target, antigen, and with high specificity

how are mAbs created

cloning a single type of immune cell so that all the antibodies produced are identical

what does mAbs high specificity allow them to do

bind only to disease related targets such as cancer cell markers or inflammatory proteins

due to their precision, mAbs can treat…

a variety of conditions, including cancers, autoimmune disorders and infections

what is herceptin (trastuzamab) used for and what does it do

HER2-positive breast cancer and it targets cancel cells with the HER2 protein, sparing normal cells

why are therapeutic enzymes often used

to replace enzymes that are missing or defective in patients with genetic disorders

what do therapeutic enzymes do

catalyze specific biochemical reactions, to aid digestion in patients with pancreatic insufficiency, target tumor cells by converting prodrugs to active drugs

why can enzymes be used to carry out precise therapeutic functions

they are highly specific

long-term enzyme replacement therapy (ERT) in patients with Pompe disease.

myozyme® (alglucosidase alfa)

what is pompe disease

glycogen storage disease type II causes by acid alpha glucosidase (GAA) deficiency

GAA

enzyme in lysosome that degrades glycogen

Fc fusion proteins consist of what

biologically active protein domain fused to the Fc region of an antibody

how do Fc fusion proteins extend the half-life of antibodies in the bloodstream

Fc region binds to receptors that recycle antibodies rather than degrade them

different types of Fc fusion proteins

• monovalent hGh-Fc (just one hGH on top)

• monovalent Fc-hGh (just one hGH on bottom)

• dinvalent hGh-Fc (two on top)

• dinvalent Fc-hGh (two on bottom)

dont think its that deep but u rmr if hgH comes first it’s on the top

enbrel® (etanercept)

• a recombinant fusion protein, combining two soluble TNF receptor segments with the Fc portion of IgG1, used to treat rheumatoid arthritis

• blocks RA inflammation and lasts longer in the body

• acts as a soluble decoy receptor

TNF

tumor necrosis factor, a cytokine that promotes inflammation, joint destruction and stiffness

what are antibody-drug conjugates (ADCs) and how do they work

• combine the targeting ability of mAbs w the potency of cytotoxic drugs

• antibody part brings drug to specific cell type (cancer cell) and the drug/payload is released upon binding

• enables targets delivery of chemotherapeutics, minimizing damage to healthy tissues

ADCetris® (brentuximab vedotin)

for hodgkin lymphoma; ADC that delivers toxic drug to cancer cells for targeted killing

these are essential for stopping bleeding and forming blood clots

coagulation proteins: factor VIII and factor IX

how are patients with blood factor deficiencies treated

receive replacement therapies made from donated plasma or recombinant proteins

fusion proteins of these factors last longer in the bloodstream

eloctate ® (efmoroctocog alfa, recombinant factore VIII protein)

• treats hemophilia A

• PDD FVIII (plasma derived factor 8) linked to IgG1 fc region

• Fc interacts with FcRN, providing protection against lysosomal degradation

interferon vs interluekin

• both are cytokines

• interferons have antiviral activity and interleukins regulate immune cell communication and activation

what are therapeutic cytokines used for

conditions like multiple sclerosis, chronic viral infections and cancer

avonex (interferon beta- 1a)

modulations immune activity to slow nerve damage, used for multiple sclerosis

(Fear von bc he slows ur nerves)

what are subunit vaccines and which patients is it safer for

use purifies pieces of pathogen to stimulate an immune response without introducing live pathogens (hep b, hpv, covid)

safer for immunocompromised individuals

gardasil 9TM

protein subunit vaccine for HPV prevention, uses viral protein to train immune system without risk of infection

major class of chemical messengers in the body that can be lab made to restore normal function when out of balance

hormones

lantus

replaces natural insulin to regulate blood sugar in diabetes

well established protein modification strategies

site-specific mutagenesis, antibody-drug conjugates, PEGylation, Fc fusion, glycoenginerring, lipidation, albumin fusion or binding, computational

emerging chemical strategies on protein therapeutics

supercharging, attachment of non-PEG polymers, fusion to other partners, unnatural amino acids

desirable characteristics of protein therapeutics

stability, desired pk & pd, intracellular delivery

site specific mutagenesis

by changing amino acids in the protein sequence, make recombinant proteins have better pk profile, more stable, less immunogenic or better at binding to target

ex. betaseron® (interferon beta-1b), Lantus® (insulin glargine)

betaseron®

human interferon beta, cys 17 substitutes w serine, 165 residues, produced in e coli no carbs

albumin fusion/binding

therapeutic proteins can be directly fused to albumin or engineered to bind it non-covalenty

ex. albutrepenonacog alfa (idelvion®), caplacizumab

what does albumin fusion do to the drug

prolong the circulation time of drug, extend half life w reduced kidney filtration, protect from in vivo protease degradation, improve stability and solubility

what is PEG and what is pegylation

polyethlene glycol is a water soluble, biologically inert, non-immunogenic, synthetic polymer; pegylation is the chemical attachment of peg chains to proteins

what does pegylation do to the protein

• increases size, hides them from immune sys, slows renal circulation → extends circulation time

• improved pk and stability, reduced immunogenicity

adverse effects and disadvantage of pegylation

hypersensitivity reaction (anaphylaxis), anti-PEG antibodies, metabolite accumulation in the kidney impairing normal kidney function

not cost-effective

neulasta® (pegfilgrastin)

PEGylated form of the recombinant human granulocyte colony-stimulating factor (GCSF), stimulates production of wbcs/neutrophils to fight infection

used in cancer patients with chemother

antibody drug conjugate advantages

minimize systemic toxicity, targeted delivery

kadcyla® (ado-trastuzumabemtansine (T-DM1) with thioether linker)

antibody drug conjugate

adc moa

1. binding to cognate antigen

2. internalization of adc-antigen complex

3. lysosomal degradation of the antibody portion

4. release of payload/drug in cytoplasm

5. interaction with target (dna, microtubules, topoisomerase 1)

6. taken up by neighboring cells

Fc fusion protein

therapeutic protein can be fused to the Fc region of an antibody to extend its half-life and improve its stability, fc region interacts w recycking receptor and avoids degradation

Fc fusion protein advantages

• extended half life

• lower immunogenecity

• improved stability

• enhanced pk/pd

• some can recruit immune cells to target cancer or other pathogens

fc fusion protein examples

etanercept (enbrel®)

aflibercept (eylea®)

dulaglutide (trulicity®)

glycoengineering

changing sugar moieties on proteins to influence immune system’s response or how long the protein stays active

glycoengineering advantages

improve protein folding, enhance receptor binding, reduce immunogenicity, important for antibodies and glycosylated proteins

• improved pk and activity

• enhanced antibody function

• reduced side effects

• diverse production methods

erythropoietin vs darbepoetin alfa

darbepoetin alpha has a longer half- life than rhEPO, given once in one or two weeks bc it is hyperglycosylated

lipidation

adding lipid (fatty acid) to a protein to help it stick to albumin → longer blood circulation, delays clearance and helps maintain steady dug levels

commonly used in insulin modification and hormone therapies

lipidation advantages

reversible, extended release, intracellular delivery

insulin detemir (levemir®) modification

has an added fatty acid chain (lipidation)

emerging and advanced strategies

xtenylation, multi-specific proteins (bi-/tri- specific), conditional activation (prodrug proteins), self assembling protein nanostructures, glycan masking and steering, engineered scaffolds (non-antibody formats)

XTEN

unstructured, hydrophilic and biodegradable synthetic polypeptide, has excellent water solubility and high chemical stability

how is xten synthesized

using recombinant DNA technology (pegylation is chemical modification)

what are the 6 amino acids in xten that are used to extend half-life and improve stability/solubility of therapeutic peptides and proteins

GGPAST

glycine, glutamine, proline, alanine, serine, threonine,

which amino acids are avoided when designing protein therapeutics to extend half-life and why

• Hydrophobic amino acids: due to protein aggregation

• Amide-containing amino acids: unstable upon long-term storage

• Positively charged side chains: may cause binding to membranes

• Cysteine residues: may become oxidized or cross-linked

where is the encoding DNA of xten fused and where is the cellulose binding domain fused

dna is bound to 3’ end of target protein

cellulose binding domain is fused to therapeutic protein gene at 5’ end

how to remove the cellulose-binding domain from the target protein

digest it with a protease

how is xtenylation different from pegylation

its genetically encoded, uses long unstructured polypeptides, and is biodegrable (can be rapidly degraded upon internalization and reduces tissue accum/toxicity)

tunable, genetically encode its production and is more homogenous than products

xtenylation

how does xtenylation extend a proteins half-life

increases its size and reduces kidney clearance

xten proteins advantages

• Excellent water solubility

• Precise genetic encoding and expression of its sequence

• Unparalleled control over sequence fidelity and molecular weight

• Highly homogenous product

• XTEN fusion proteins are highly soluble,

properly folded in E. coli

• Non-immunogenic

• Biodegradable

Efanesoctocog alfa (Altuviiio®)

protein therapeutic advantages

• Highly specific

• Less adverse effects

• Protein replacement therapy

• Clinical approval time could be faster than small molecule drugs.

protein therapeutic disadvantages

• Solubility

• Distribution

• Stability

• Route of drug delivery (iv, sc, im)

• Physiologically active post translational modification

• Cost and storage