Protein Therapeutics

Protein TherapeuticsPrior to recombinant DNA, protein pharmaceuticals

were very difficult and expensive to produce. They were also available in very limited quantities and their biological modes of action were not well understood.

Several thousand different potentially therapeutic proteins were cloned and expressed in both mammalian and bacterial host cells.

More than 250 of these “biotechnology drugs” have been approved for use in the U.S. or E.U.

Examples of “biotechnology drugs” have been approved for human use in the U.S. or E.U.

Antibodies can be used as theraputic agents to target specific cell to be destroyed.

PharmaceuticalsInterferons: a class of proteins produced

largely by virus infected cells that are secreted to the bloodstream and activate resistance pathways in other cells.

PharmaceuticalsPotential use as a drug in many disorders (MS,

hepatitis, etc.)Produced in three forms in humans (IFN-α, IFN-

β, and IFN-γ) with different biological activity.IFN-α and IFN-β, are produced as a result of

exposure to viruses, encoded by a family of 13 different genes and two genes, respectively.

IFN-γ is as a result of growth stimulating factors, encoded by a single gene.

Overview of protocol used to isolate IFN cDNA.

Isolated cDNAs are clones into expression vectors for production in E. coli.

Interferon gene shuffling.In human INFs, researchers found that the level of

anti-viral activity varied greatly within the subtype.IFNα-2 and IFNα-3 had common restrictions sites

allowing the production of hybrid genes to create proteins with novel IFN activities.

Enhanced production of oligoisoadenylate synthetase.

It produces 2’-5’ linked oligonucleotides that activate a latent endoribonuclease which attacks and degrades viral mRNAs.

Hybrid genes construction.

Others had antiproliferative activities against human cancers.

Longer-acting interferons.Fusing an IFN gene with the gene for a stable

protein, human serum albumin, produces a stable hybrid protein.

Human growth hormone: stimulates tissue and bone growth, increases protein synthesis and mineral retention, & decreases body fat storage.

Effective in treatment of dwarfism, infants and children who lack sufficient endogenous levels of human growth hormone, chronic renal insufficiency, and Turner’s syndrome.

Human growth hormone was one of the first therapeutic proteins approved for human use.

The first recombinant growth hormone was called somatrem.

The modification of the native protein was done to eliminate side effects.

It is desirable that growth hormone will bind only to growth hormone receptors but not prolactin receptors.

Site-directed mutagenesis was used to alter 3 amino acids which bind zinc ions (His-18, His-21, and Glu-174).

These modifications yielded human growth hormone derivatives that bound only to growth hormone receptors.

These derivatives are being tested for safety and efficacy in humans.

It is advantageous to have a long-lasting form.

The receptor fragment is fused to growth hormone resulting in dimerization which in turn stabilizing the growth hormone in vivo.

Another method to produce a long-lasting human growth hormone - Albutropin.

The N-terminous of human growth hormone is fused to the C-terminous of human serum albumin .

Tumor necrosis factor alpha (TNF-α): a potent antitumor agent, but not used widely due to severe toxicity.

If the protein could be delivered to the site (tumor), it could be used in lower doses and the unwanted side effects would be diminished .

Adding 6 amino acids targeting peptide to the N-terminus results in TNF-α with tumor specificity.

In mice, this fusion was 12-15X more effective, and much higher percentage of survival.

This work is preliminary until it can be tested in humans.

A 6-amino-acid targeting peptide (red) fused to its N terminous.

Survival of lymphoma-bearing mice following treatment with native and modified TNF.

Therapeutic enzymesDnase I: used in the treatment of cystic fibrosis

patients to relieve the severe symptoms by decreasing the viscosity and adhesivity of mucus in the lungs, made it easier to breathe.

Therapeutic enzymesActin binds very tightly to Dnase I and inhibits

its ability to cleave DNA.Changing one amino acid (either ala to arg or tyr

to arg) decreases actin binding and increases activity 10-50X.

Alginate lyase: also used in cystic fibrosis.Cystic fibrosis patients often have

Pseudomonas aeruginosa infected in the lungs which is impossible to treat with antibiotic treatment due to the biofilm.

It secretes an alginate-like polysaccharide that increases viscosity of mucus.

Alginate lyase can liquefy bacterial alginate, together with or prior to antibiotic treatment, significantly decreased the number of bacteria in biofilm.

This result suggests that, in addition to the Dnase I treatment, alginate lyase would help.

Isolated alginate lyase gene from Flavobacterium and cloned into E. coli for screening.

cDNA was cut to process a lower MW enzyme with higher specificity that easily hydrolyzes bacterial alginates.

DNA construct encoding the 40,000-Da alginate lyase is fused to the leader peptide from a B. subtilis α-amylase gene.

The construct is under the transcriptional control of a B. subtilis penicillinase gene expression system.

When these transformants were grown in liquid medium, the recombinant alginate lyase was secreted into culture broth.

Phenylalanine ammonia lyase (PAL)Phenylketonuria (PKU) results from a defect in

phenylalanine hydroxylase. This enzyme produces tyrosine from phenylalanine.

Results in mental retardation due to a build up of phenylalanine.

Treatment entails a controlled semisynthetic diet with low levels of phenylalanine through infancy and possibly for life.

The administration of the enzyme is an alternative treatment but it is not very stable and requires a cofactor for activity.

Treatment with PAL degrades phenylalanine to ammonia and trans-cinnamic acid.

The experiment in mice indicated the lower levels of plasma phenylalanine.

α1-Antitrypsin: Many pathogens use human proteases in processing their proteins.

A therapeutic agent that blocked its activity might act as a broad-spectrum antiviral and antibacterial agent.

When tested in culture, a variant of alpha-antitrypsin blocks the production of HIV type 1 glycoprotein gp160, measles virus protein F0, and human cytomegalovirus.

Clinical efficacy is as yet unknown.Could be very useful due to variety of viruses

inhibited.

The antitrypsin will block the proteolytic activity preventing the production of infectious pathogen.

Glycosidases: Possible use in preparing blood for transfusions.

ABO blood types correspond to specific carbohydrates on surface of red blood cell.

A = N-acetylgalactosamineB = galactoseO = neitherThere are specific glycosidases that cleave

these sugars from the surface and convert type A, B, and AB into type O.

Digestion of monosaccharides that determine blood groups A and B to obtain blood group O.

Lactic acid bacteria are widely used in the production and preservation of fermented foods.

Lactic acid bacteria-many used as a probiotic that claim to offer a health benefit by altering the indigenous microflora of the intestinal tract.

They can be used to treat several gastrointestinal disorders, including lactose intolerance.

Lactobacillus lactis has been developed as a host system for cloning and expressing proteins to be delivered to the human gut.

The treatment of ulcerative colitis and Crohn’s diseases is to lower the levels of cytokines, TNF-α.

Interleukin-10 secreting L. lactis show promise in alleviating bowel inflammation in victims of Crohn’s disease and ulcerative colitis.

To prevent any plasmid-borne antibiotic resistance marker genes spreading to other bacteria in the environment, the construct is inserted into L. lactis chromosomal DNA by homologous recombination.

The recombinant bacteria grew well in the laboratory and produce interleukin-10 when thymine or thymidine was added.

Leptin secreting L. lactis are a possibility for treating severe obesity. It has been shown to reduce food intake and body weight loss in obese leptin-deficient mice.

Leptin was produced in L. lactis without formation of inclusion body and was secreted.

Nisin is a 34-amino-acid-residue polycyclic peptide that has antibacterial activity.

HIV inhibitor: Cyanovirin N (isolated from Nostoc cyanobacteria) is an inhibitor of HIV infection.

It is possible that Cyanovirin N secreting Lactobacillus jensenii could be used as a topical microbicide to help prevent HIV infections in women.

Monoclonal antibodies as therapeutic agentsAntibodies were first used to treat disease about

100 years ago in diphtheria with a polyclonal horse serum raised against Cornyebacterium diphtheriae.

It generally was effective if used in the first few days of infection, but often sensitized individuals for a second treatment causing anaphylactic shock and death because patients often develop antibodies against the foreign proteins.

It is now possible to engineer antibodies with a greatly reduced level of immunogenicity in humans.

A continuous supply of pure monospecific antibody can be maintained.

Problems with cross-reactivity and anaphylaxis though have still recurred to a lesser extent.

Human monoclonal antibodies with both specific immunotherapeutic properties and lowered potential for immunogenicity have been produced.

See Table 10.3 and Boxes 10.2 & 10.3.

Structure & function of antibodies: Each antibody is made up of 2 identical light and heavy chains held together by both hydrogen and precisely localized disulfide linkages.

The N-terminal portion of the L and H chains together determine antigen recognition site.

The site consist of three complementarity determining regions (CDR’s) that lie within the variable regions at the N-terminous.

The Fab fragment retains the antigen-binding activity after digested with the proteolytic enzyme papain.

In fact, the N-terminal half of the Fab, the Fv, contains all the activity.

The Fab portions can be used in treatments since they maintain the same binding but do not elicit some of the same responses.

The Fc portion elicits several immunological responses after antigen-antibody binding.

The compliment cascade-breaks down membranes, activates phagocytes, and signals to the rest of the immune response.

Antibody-dependent cell-mediated cytotoxicity (ADCC)-as a result of binding Fc portion to receptors on ADCC effector cells. These release compounds that lyse foreign cells.

Fc portion binds to receptors on phagocytes causing them to engulf the antibody-antigen complex.

Preventing rejection of transplanted organs:The idea was to use a specific antibody that

bind to certain lymphocytes diminishing immune response against the transplanted organ.

Mouse monoclonal antibody OKT3 was approved as an immunosuppressive agent.

OKT3 binds the CD3 receptor on T-cells and a full immunological response is blocked.

Mouse monoclonal antibodies show some problems including poor coupling to drugs/proteins as well as sensitization.

Due to difficulties producing human monoclonal antibodies, hybrid mouse/human monoclonal antibodies have been produced.

These are produced by genetically engineering mouse myeloma cells to produce modified human antibodies.

The chimeric antibody with the antigen-binding specificity of the mouse monoclonal antibody diminishing immunogenicity and introducing human Fc effector capabilities.

The humanized antibody replacing the CDRs from the mouse in human antibody. The product has antigen-binding specificity of the mouse and all other properties of human antibody.

The primers complementary to the rodent CDR with 5’ ends that complementary to human antibody. Then, by PCR, the amplified rodent CDRs are spliced into human antibody.

Generation of a Xenomouse that can synthesize only human Ig. They are selected, immunized, and used to make hybridomas producing human antibodies.

IgG is the main antibody found in mammalian serum, and it is native form that is used in therapeutic antibodies.

A variety of IgG derivatives or fragments that may be used instead of whole antibody molecule.

In addition, a protein-coding sequence can be linked to a single-chain antibody sequence to create a dual-function molecule that can both bind to a specific target and deliver toxin or some other specific activity to a cell.

Most antibody-toxin combinations have been constructed using the gene for Pseudomonas endotoxin A.

It is 66-kDa protein with 3 domains; binding, translocation, and ADP-ribosylation.

An immunotoxin is made by replacing the N-terminal binding domain from the gene with the single-chain antibody sequence.

The molecules are very similar in size to the original toxin with the ability to bind, enter, and kill specific cell.

These hybrids bind specific cells and allow the toxin to penetrate easily.

Toxin molecules may also be directed to cancer cells by using a dispecific diabody that is engineered to bind to a surface-specific tumor-associated antigen and then to a toxin.

It is possible to create peptides that are smaller than scFvs and still retain the ability to bind to a specific antigen.

They are more likely to penetrate a tumor and stop the tumor growth.

Next, the peptide is coupled to a toxin molecule.

Production of antibodies in E. coli that acts as “bioreactors.”

Done by cloning H&L genes, combining them in phage vectors, screening, excising the exons as part of a plasmid, and transform E. coli.

Harvest antigen-binding Fv fragment from E. coli.

DNA constructs of an Fv combinatorial gene library cloned into bacteriophage λ DNA, thereby creating all possible combinations of L and H chain fragments.

Production of antibodies in the filamentous bacteriophage M13.

A combinatorial library of antibody fragments displayed on the surface can be screened by ALISA-like system.

This approach is easier than using plaque assay.

A combinatorial library of full-length antibodies were expressed in periplasm of E. coli with the antibody anchored to the inner membrane.

Screening is done by fluorescently labeled antigen.

Chemically-linked monoclonal antibodies: Many potentially useful drugs in vitro fail in vivo. This is often due to the inability to deliver the drug to the target cell in a sufficient concentration.

Increasing the dose of drug is not the answer, because high drug concentrations often have deleterious side effects.

Strategies have been developed to overcome this problem using monoclonal antibodies conjugated to the drug or to an enzyme which activates the drug.

A monoclonal antibody-based drug delivery system.

Dual-variable-domain immunoglobulin containing two tandem Fv regions, each with a different specificity.

Anticancer antibodies may also bind to some non-cancerous cell.

This approach presents researchers with only a limited number of targets for therapeutic antibodies.

Tumor cell are first treated with the chemotherapeutic agent irinotican, which induces the synthesis of a unique cell surface protein.

Then, a monoclonal antibody directed against the cell surface protein and conjugated to a toxin molecule is added.

The toxin is internalized after the binding, thereby killing the tumor cell.