Ch. 10. Therapeutic Agents
I. Human proteins II. Enzymes III. Monoclonal antibodies IV. HIV therapeutic agents
I. Human proteins
II. Enzymes
III. Monoclonal antibodies
IV. HIV therapeutic agents
Therapeutic Proteins Produced by Recombinant Microorganisms that Express Human Proteins
See Table. 10.1 Treatments for: Immune system disorders Blood disorders: anemia, hemophilia, blood clots Wounds Hormonal deficiency: diabetes, development and growth Cancer Viral infections
See Table. 10.1
Treatments for:
Immune system disorders Blood disorders: anemia, hemophilia, blood clots Wounds Hormonal deficiency: diabetes, development and growth Cancer Viral infections
Immune system disorders
Blood disorders: anemia, hemophilia, blood clots
Wounds
Hormonal deficiency: diabetes, development and growth
Cancer
Viral infections
Cloning and Expression of Genes for Human Proteins
Human proteins may be present in very low concentrations in the body Ex. .Interferons, 10-10 to 10-15 M -Difficult to study function -Insufficient amounts for clinical testing - Not enough for therapeutic use
Human proteins may be present in very low concentrations in the body
Ex. .Interferons, 10-10 to 10-15 M -Difficult to study function -Insufficient amounts for clinical testing - Not enough for therapeutic use
Ex. .Interferons, 10-10 to 10-15 M -Difficult to study function -Insufficient amounts for clinical testing
- Not enough for therapeutic use
Genes that encode human proteins can be cloned and overexpressed to fullfill need for greater amounts
1. Isolate mRNA from cell line or tissues that express the protein => high levels of mRNA encoding the protein are present E.g. 70% of mRNA of islets cells in pancreas encodes insulin 2. Make cDNA from mRNA, insert into vector and transform host (cDNA library) 3. Screen library -DNA probe for target sequence or -Immunoassay for expressed protein product 4. Express cDNA gene in appropriate host for production of large amounts of protein with correct posttranslational modifications
1. Isolate mRNA from cell line or tissues that express the protein => high levels of mRNA encoding the protein are present
E.g. 70% of mRNA of islets cells in pancreas encodes insulin
2. Make cDNA from mRNA, insert into vector and transform host (cDNA library)
3. Screen library
-DNA probe for target sequence or -Immunoassay for expressed protein product
-DNA probe for target sequence or
-Immunoassay for expressed protein product
4. Express cDNA gene in appropriate host for production of large amounts of protein with correct posttranslational modifications
Small glycoproteins produced mainly by immune system cells Cellular functions Antiviral Inhibition of intracellular parasites Regulation of immune system Three types: IFNa 13 genes IFNb 2 genes IFNg 1 gene Therapeutic uses Treatment of: Viral infections Cancers Multipe sclerosis Different IFNs have different potencies in different cell types Genetic engineering of interferon genes may create interferons with new or improved threpeutic properties
Small glycoproteins produced mainly by immune system cells
Cellular functions
Antiviral Inhibition of intracellular parasites Regulation of immune system
Antiviral
Inhibition of intracellular parasites
Regulation of immune system
Three types:
IFNa 13 genes IFNb 2 genes IFNg 1 gene
Therapeutic uses
Treatment of: Viral infections Cancers Multipe sclerosis
Treatment of:
Viral infections Cancers Multipe sclerosis
Cancers
Multipe sclerosis
Different IFNs have different potencies in different cell types
Genetic engineering of interferon genes may create interferons with new or improved threpeutic properties
Genetically Engineered Human Interferons
See Fig. 10.2
To increase antiviral activity or create interferons with activitiy against different viruses and cancers
The genes are related and have similar nucleotide sequences with some common restriction enzyme sites.
Ex. Gene IFNa2 IFNa3
Ex.
Gene
IFNa2
IFNa3
Creation of hybrid interferons via gene shuffling
Cut cloned cDNA with restriction enzymes, mix and ligate fragments Insert hybrid cDNA into expression vector and test gene product for altered activity
Cut cloned cDNA with restriction enzymes, mix and ligate fragments
Insert hybrid cDNA into expression vector and test gene product for altered activity
Creation of a consensus interferon a (Infergen: Amgen)
1.) Amino acid sequences of several IFN-a proteins were compared 2.) Consensus sequence was determined Most commonly occurring amino acid at each position of the protein was identified 3.) Artificial gene having the encoding the consensus amino acid sequence was synthesized and expressed Encodes a 166 amino acid consensus protein Doesn't exist naturally 88% sequence similarity to IFN-a2a and IFN-a2b Antiviral activity was increased Approved by FDA to treat hepatitis C virus infection HVC infects 4 million in the U.S. Causes cirrhosis and cancer of liver
1.) Amino acid sequences of several IFN-a proteins were compared
2.) Consensus sequence was determined
Most commonly occurring amino acid at each position of the protein was identified
3.) Artificial gene having the encoding the consensus amino acid sequence was synthesized and expressed
Encodes a 166 amino acid consensus protein Doesn't exist naturally 88% sequence similarity to IFN-a2a and IFN-a2b Antiviral activity was increased Approved by FDA to treat hepatitis C virus infection HVC infects 4 million in the U.S. Causes cirrhosis and cancer of liver
Encodes a 166 amino acid consensus protein
Doesn't exist naturally
88% sequence similarity to IFN-a2a and IFN-a2b
Antiviral activity was increased
Approved by FDA to treat hepatitis C virus infection
HVC infects 4 million in the U.S. Causes cirrhosis and cancer of liver
HVC infects 4 million in the U.S.
Causes cirrhosis and cancer of liver
Therapeutic Enzymes for Treatment of Cystic Fibrosis (CF)
Inherited genetic defect of the CF gene Pseudomonas aeruginosa lung infections are common Produces alginate, a viscous polysaccharide polymer Lysis of bacteria in lungs releases DNA, another viscous polymer Mucus accumulates in lungs, breathing is impaired
Inherited genetic defect of the CF gene
Pseudomonas aeruginosa lung infections are common Produces alginate, a viscous polysaccharide polymer
Pseudomonas aeruginosa lung infections are common
Produces alginate, a viscous polysaccharide polymer
Lysis of bacteria in lungs releases DNA, another viscous polymer
Mucus accumulates in lungs, breathing is impaired
Therapeutic enzymes
1. DNase I - an enzyme that degrades DNA
Human gene was cloned Expressed in mammalian cell line (Chinese Hamster Ovary cells) Inhaled enzyme degrades bacterial DNA CF patients breath more easily Approved for use by the FDA Marketed as Pulmozyme by Genentech
Human gene was cloned
Expressed in mammalian cell line (Chinese Hamster Ovary cells)
Inhaled enzyme degrades bacterial DNA
CF patients breath more easily
Approved for use by the FDA
Marketed as Pulmozyme by Genentech
2. Alginate lyase - a bacterial enzyme that degrades alginate
May be used therapeutically as for DNase I Gene from Flavobacterium sp., was cloned and expressed in Bacillus subtilus as a fusion with the a-amylase leader peptide Secretion of the enzyme simplifies dowstream processing
May be used therapeutically as for DNase I
Gene from Flavobacterium sp., was cloned and expressed in Bacillus subtilus as a fusion with the a-amylase leader peptide
Secretion of the enzyme simplifies dowstream processing
Therapeutic Antibodies
I. Before recombinant DNA technology
Polyclonal antibodies obtained from serum of animal or human Ex. -Horses injected with an antigen (toxin) -Persons that recovered from an infection
Polyclonal antibodies obtained from serum of animal or human
-Horses injected with an antigen (toxin) -Persons that recovered from an infection
-Horses injected with an antigen (toxin)
-Persons that recovered from an infection
Antibodies were injected as therapy for patient with toxin or infection to: -Neutralize the toxin or -Target pathogen for destruction by immune system
Antibodies were injected as therapy for patient with toxin or infection to:
-Neutralize the toxin or -Target pathogen for destruction by immune system
-Neutralize the toxin or
-Target pathogen for destruction by immune system
Provides immediate protection, but: -May cause allergy or shock; -Some other infectious agent present in the antiserum of the donors
Provides immediate protection, but:
-May cause allergy or shock; -Some other infectious agent present in the antiserum of the donors
-May cause allergy or shock;
-Some other infectious agent present in the antiserum of the donors
II. Recent applications for therapeutic antibodies
See Fig. 10.14 Specificity of antibody binding to antigen can be used to target treatment to specific sites in the body. Drugs or enzymes may be coupled to a monoclonal antibody (Mab) Mab that binds to an antigen only present on diseased cells Lower drug dosages can be used, reduces side effects
See Fig. 10.14
Specificity of antibody binding to antigen can be used to target treatment to specific sites in the body.
Drugs or enzymes may be coupled to a monoclonal antibody (Mab)
Mab that binds to an antigen only present on diseased cells
Lower drug dosages can be used, reduces side effects
Problems
Mouse Mabs are recognized as foreign by human immune system May result in allergic reaction or shock
Mouse Mabs are recognized as foreign by human immune system
May result in allergic reaction or shock
Human monoclonal antibodies have not been developed -A human myeloma cell line is not available -Ethically, can't inject antigens into humans anyway
Human monoclonal antibodies have not been developed
-A human myeloma cell line is not available -Ethically, can't inject antigens into humans anyway
-A human myeloma cell line is not available
-Ethically, can't inject antigens into humans anyway
Genetic Engineering of Monoclonal Antibodies
See Fig. 10.17
I. Humanized monoclonal antibody (~5% mouse and 95% human)
1. Construct hybrid antibody light and heavy chain genes that encode: Antigen binding regions (CDRs) of a mouse monoclonal antibody that binds the target antigen Clone cDNAs encoding light and heavy chains from hybridoma cell line PCR amplify the sequence encoding CDRs Constant domains of light and heavy chains of human antibody Clone cDNA encoding light and heavy chains from human B cells Replace human CDRs with mouse CDRs 2. Insert hybrid genes into mammalian expression vector 3. Transfect and express genes in mammalian cell line
1. Construct hybrid antibody light and heavy chain genes that encode:
Clone cDNAs encoding light and heavy chains from hybridoma cell line PCR amplify the sequence encoding CDRs
Clone cDNAs encoding light and heavy chains from hybridoma cell line
PCR amplify the sequence encoding CDRs
Clone cDNA encoding light and heavy chains from human B cells Replace human CDRs with mouse CDRs
Clone cDNA encoding light and heavy chains from human B cells
Replace human CDRs with mouse CDRs
2. Insert hybrid genes into mammalian expression vector
3. Transfect and express genes in mammalian cell line
II. Production of fully human monoclonal antibodies in mice
Steps:
1. Destroy a mouse's immune system with radiation
Mouse can no longer produce mouse antibodies
2. Inject with bone marrow from an immune-deficient mouse (i.e. a SCID mouse with severe combined immunodeficiency)
Establishes a new blood-building system needed for survival of mouse
3. Inject human immune system cells
Establishes a human immune system in the mouse
4. Inject mouse with an target antigen to stimulate an immune response
B cells produce human antibodies specific for the antigen
5. Immortalize B cells and select hybridoma that produces a human monoclonal antibody that binds to target antigen
Therapeutic Agents for Treatment of HIV Infection
Acquired immunodeficiency syndrome(AIDS) is caused by the human immunodeficiency virus (HIV).
Infects helper T cells by binding to a viral glycoprotein (gp120) to the CD4 receptor on the surface of the cell membrane Infected cells display the viral glycoprotein on the cell surface
Infects helper T cells by binding to a viral glycoprotein (gp120) to the CD4 receptor on the surface of the cell membrane
Infected cells display the viral glycoprotein on the cell surface
A therapeutic protein might be produced as a fusion of CD4 and a toxin protein
Infected cells would be targeted and killed selectively
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