Antibody therapy and engineering
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Transcript of Antibody therapy and engineering
ANTIBODIES IN THERAPYANTIBODY ENGINEERING
MONOCLONAL ANTIBODIES
ANTIBODIESSTRUCTURE AND FUNCTION
Antibodies Are Made Up Of:
– 2 Light Chains (identical) ~25 KDa
– 2 Heavy Chains (identical) ~50 KDa
• Each Light Chain Bound To Heavy Chain By Disulfide (H-L)
• Heavy Chain Bound to Heavy Chain (H-H)
• First 100 a/a Of Amino Terminal Vary of Both H and L Chain Are
Variable
• Referred To As VL , VH, CH And CL
• CDR (Complementarity Determining Regions) Are What Bind Ag
• Remaining Regions Are Very Similar Within Same Class
• Repeating Domains of ~110 a/a
– Intrachain disulfide bonds within each domain
• Heavy chains
– 1 VH and either 3 or 4 CH (CH1, CH2, CH3, CH4)
• Light chains
– 1 VL and 1 CL
• Hinge Region
– Rich in proline residues (flexible)
– Hinge found in IgG, IgA and IgD
– Proline residues are target for proteolytic digestion (papain and pepsin)
– Rich in cysteine residues (disulfide bonds)
– IgM and IgE lack hinge region
– They instead have extra CH4 Domain
• Digestion With Papain Yields
– 3 Fragments
– 2 identical Fab and 1 Fc
– Fab- Fragment That is Antigen Binding
– Fc - Found To Crystallize In Cold Storage
• Pepsin Digestion
– F(ab`)2
– No Fc Recovery, Digested Entirely
• Mercaptoethanol Reduction (Eliminates Disulfide Bonds) And
Alkylation Showed
ANTIBODIES ACT AS IMMUNOGENS
• Antigenic Determinants on Abs Fall in 3 Categories
– Isotypic
– Allotypic
– Idiotypic
• Isotypic
– Constant Region Of Ab
– If you inject Ab in a different species Anti-Isotype is
generated
– If within same species, No Anti-isotype
Allotype
– Even though same isotypes within one species small differences (1-4
a/a) arise in different individuals (form of polymorphism)
– If injected with such Ab you generate anti-allotype Ab
• Ex. During pregnancy
• Blood transfusion
Idiotype
– Unique VH AND VL binds antigen but can also behave as antigenic
determinant
• If you inject a monoclonal antibody into a genetically identical recipient
then anti-idiotypic antibodies are generated
• No anti-isotypic and no anti-allotypic Abs will be generated
Isotypic determinants are constant region determinants that
distinguish each Ig class and subclass within a species.
Allotypic determinants are subtle amino acid differences encoded by
different alleles of isotype genes. Allotypic differences can be
detected by comparing the same antibody class among different
inbred strains.
Idiotypic determinants are generated by the conformation of the
amino acid sequences of the heavy- and light-chain variable regions
specific for each antigen. Each individual determinant is called an
idiotope, and the sum of the individual idiotopes is the idiotype.
• IgG
– Most abundant immunoglobin 80% of serum Ig
– ~10mg/mL
– IgG1,2,3,4 (decreasing serum concentration)
– IgG1, IgG3 and IgG4 cross placenta
– IgG3 Most effective complement activator
– IgG1 and IgG3 High affinity for FcR on phagocytic cells, good for opsonization
ANTIBODY CLASSES AND BIOLOGICAL ACTIVITIES
• IgM– 5-10% of serum immunoglobulin– 1.5mg/mL– mIgM (also IgD) expressed on B-cells as BCR– Pentameric version is secreted– First Ig of primary immune response– High valence Ig (10 theoretical), 5 empirical– More efficient than IgG in complement activation
• IgA– 10-15% of serum IgG– Predominant Ig in secretions
• Milk, saliva, tears, mucus– 5-15 g of IgA released in secretions!!!!– Serum mainly monomeric, polymers possible not common though– Secretions, as dimer or tetramer+J-chain polyptetide+secretory
component (Poly IgR)
IgE
– Very low serum concentration, 0.3g/mL
– Participate in immediate hypersensitivities reations. Ex. Asthma,
anaphylaxis, hives
• Binds Mast Cells and Blood Basophils thru FcR
• Binding causes degranulation (Histamine Release)
IgD
– Expressed on B-cell Surface
• IgM and IgD, Expressed on B-cell Surface
• We Do Not Know Any Other Biological Effector Activity
• Low serum concentrations, ~30g/mL
• BCR Is An Antibody On
Surface Of Cell mIg
• Very Short Cytoplasmic
Tail, Cannot Transduce
Signal
• Heterodimeric Molecule
Ig-/Ig- Transduces
(long cytoplasmic tail)
B-Cell Receptor
• To Transport Abs Across Membranes
– Secretion of IgA Across Epithelium into lumen
– Transport of maternal Abs Across Placenta (IgG)
• Many Cell Types Use FcR
– Ex. Mast Cells, Macrophages, Neutrophils, B, T, NK
• Opsonization, ADCC
• Poly IgR
– Transport of IgA across epithelium
• FcRN
– Transport of maternal IgG to fetus
Fc Receptors (FcR) Functions
Fc Receptors (FcR)
ANTIBODY ENGINEERING
VDJ recombination system
• V- Variable segment
• D- Diversity segment
• J- Joining segment
• 2 variants in light chain: λ and κ chain
• Major enzyme is VDJ recombinase
• Different from Site Specific Recombination
VDJ recombination system
LIG
HT
CH
AIN
SY
NTH
ES
IS
HEAVY CHAINS
Joining of V,D and J regions
The joining of V, D and J segments. Joining is masterminded by the recombination activating genes RAG-1 and MG-2, the products of which cleave the DNA at the signal ends. RAG-1 and RAG-2 together produce several thousand times more efficient VDJ recombination than either alone.
The introns adjoining the V, D and J gene segments contain specialized recombination signal sequences (RSSs) which include conserved heptamers and monomers separated by spacers of either 12 or 23 base pairs.
The two joining segments, in this example V and J, are brought into proximity by interaction between their respective RSSs mediated by the DNA bending and looping high mobility group-l and -2 proteins (HMG-l and HMG-2).
RAG-1 and RAG-2 cleave the DNA to produce doublestrand breaks at the border of the RSS. The excised signal sequences are ligated to form the signal joint resulting in a piece of circular DNA containing the excised sequences.
This is probably maintained in the cell for a period of time before eventually being lost from the cell. The double strands of each coding segment form 'hairpin' ends
The enzyme Ku (a dimer of Ku70 and Ku86) binds to the DNA ends and stimulates DNA-dependent protein kinase (DNA-PK, mutation of which gives rise to mice with severe combined imunodeficiency (SCID)) which facilitates the opening of the hairpin.
Terminal deoxynucleotidyl transferase (TdT) adds nucleotides to the ends of the DNAstrands in order to generate N-region diversity.
Unlike the precision of the signal joint, the coding joint is variable because it can involve the addition of base pairs resulting from both the resolution of the hairpin loop (P-elements) and the TdT-mediated N-region diversity.
Nucleases remove any excess nucleotides and polymerases fill in any gaps before the DNAligase IV and XRCC4 enzymes carry out ligation of the two sequences. Since the coding elements are joined at random with respect to the reading frames, two out of three events have two coding elements out of frame.
Although apparently wasteful, this is evolutionarily tolerated because it confers so much benefit in the form of antigen receptor diversity. VDJ recombination products define the major antigen-binding domains.
Features of VDJ recombination
• Productive and non- productive Ig genes
• Allelic exclusion- similar to XIST and TSIX system
• Class switching or isotype switching
AN
TIB
OD
Y E
NG
INE
ER
ING
ANTIBODY ENGINEERING contd• Mouse antibody- can bind to Ag
effectively, but causes allergic reactions
• Human antibody- Technical problems in production
• Chimeric mouse-human Ab:-, Fc receptor binding, Can retain longer in blood circulation
• Grafted CDRs:- CDRs from mouse
• Chimeric immunotoxins:- Terminal Fc region is changed.
• Heteroconjugates:- Hybrid of two different antibodies
Most antigens offer multiple epitopes and therefore induce proliferation and
differentiation of a variety of B-cell clones, each derived from a B cell that
recognizes a particular epitope.
The resulting serum antibodies are heterogeneous, comprising a mixture of
antibodies, each specific for one epitope .Such a polyclonal antibody
response facilitates the localization, phagocytosis, and complement-
mediated lysis of antigen; it thus has clear advantages for the organism in
vivo.
Unfortunately, the antibody heterogeneity that increases immune protection
in vivo often reduces the efficacy of an antiserum for various in vitro uses.
For most research, diagnostic, and therapeutic purposes, monoclonal
antibodies, derived from a single clone and thus specific for a single
epitope, are preferable.
MONOCLONAL ANTIBODIES
(a) Toxins used to prepare immunotoxins include ricin, Shigella toxin, and diphtheria toxin. Each toxin contains an inhibitory toxin chain (red) and a binding component (yellow). To make an immunotoxin, the binding component of the toxin is replaced with a monoclonal antibody (blue).(b) Diphtheria toxin binds to a cell-membrane receptor (left) and a diphtheria immunotoxin binds to a tumor-associated antigen (right). In either case, the toxin is internalized in an endosome. The toxin chain is then released into the cytoplasm, where it inhibits protein synthesis by catalyzing the inactivation of elongation factor 2 (EF-2).
Preparing
MAb from Ig librarie
s
MONOCLONAL ANTIBODY APPLICATIONS
• Diagnostic Tests
– Abs are capable to detect tiny amouns (pg/mL) of molecules
– Ex. Pregnancy hormones
• Diagnostic Imaging
– mAbs that recognize tumor antigens are radiolabeled with iodine
I-131
• Immunotoxins
– mAbs conjugated with toxins
• mAbs To Clear Pathogens
MONOCLONAL ANTIBODY APPLICATIONS
• Used as invitro Monoclonal antibody diagnostic
reagents Includes
- products for detecting pregnancy
- Diagnosing numerous pathogenic microorgs.
- Measuring the blood levels of various drugs
- matching histocompatibility antigens
- detecting antigens shed by certain tumors.
Radiolabeled monolconal antibodies can be used- Invivo to detect or locate tumor antigens
- Permitting earlier diagnosis of some primary or metastatic tumors in
patients.
Recent products are Genetically engineered to
incorporate V regions (or) CDRs of non human
antibodies into C regions& framework of human
antibodies thus minimizing the possibility of rasing
an immune response to them.
Abzymes• Catalytic activity of Ab was highly specific.
• Hydrolyzed only esters whose transistion state structure closely
resembled Ts used a s a haptenin the immunizing conjugate.
• Catalytic antibodies called abzymes in ref to their dual role as antibody
& enzyme.
• Ag-Ab binding similar to enzyme-substrate binding
• When monoclonal Ab was developed against ester and when incubated
with ester can increase the rate of hydrolysis by 1000 fold
• Cleave blood clot, cleave viral glycoprotein
• But abzyme which act as peptidase is difficult to produce
Antibody therapy
Immunoglobulin for passive immunization is prepared from the pooled plasma of thousands of donors. In effect, recipients of these antibody preparations are receiving a sample of the antibodies produced by many people to a broad diversity of different pathogens.
In fact a gram of intravenous immune globulin (IVIG) contains about 1018 molecules of antibody (mostly IgG) and may incorporate more than 107 different antibody specificities.
During the course of therapy, patients receive significant amounts of IVIG, usually 200–400 mg per kilogram of body weight.
This means that an immunodeficient patient weighing 70 kilograms would receive 14 to 28 grams of IVIG every 3 to 4 weeks.
A product derived from the blood of such a large number of donors carries a risk of harboring pathogenic agents, particularly viruses.
The risk is minimized by the processes used to produce intravenous immune globulin. The manufacture of IVIG involves treatment with solvents, such as ethanol, and the use of detergents that are highly effective in inactivating viruses such as HIV and hepatitis.
ANTIBODIES IN PASSIVE IMMUNIZATION THERAPY
In addition to removing or inactivating infectious agents, the production process must also eliminate aggregated immunoglobulin, because antibody aggregates can trigger massive activation of the complement pathway, leading to severe, even fatal, anaphylaxis.
Passively administered antibody exerts its protective action in a number of ways. One of the most important is the recruitment of the complement pathway to the destruction or removal of a pathogen.
In bacterial infections, antibody binding to bacterial surfaces promotes opsonization, the phagocytosis and killing of the invader by macrophages and neutrophils.
Toxins and viruses can be bound and neutralized by antibody, even as the antibody marks the pathogen for removal from the body by phagocytes and by organs such as liver and kidneys. By the initiation of antibodydependent cell-mediated cytotoxicity (ADCC), antibodies can also mediate the killing of target cells by cytotoxic cell populations such as natural killer cells.
1. Infliximab and Adalimumab - anti rheumatoid-chimeral monoclonal antibody
which binds and neutralizes TNF-α
2. Omalizumab- Bronchial asthma- Neutralise IgE
3. Abciximab-Anti platelet-It is the Fab fragment of a chimeric monoclonal antibody
against GP lIb/IlIa
4. Muromonab CD3- Immunosuppressant-murine monoclonal antibody against
the CD3 glycoprotein
5. Antithymocyte globulin (ATG)-Immunosuppressant- It is a polyclonal
antibody purified from horse or rabbit immunized with human thymic
lymphocytes which binds to T lymphocytes and depletes them.
6. Anti-D immuneglobulin- Immuospressant- It binds the Rho antigens and
does not allow them to induce antibody formation in Rh negative individuals
7. Hepatitis B immuneglobulin-vaccine-It is 10-18% solution of human IG
ANTIBODIES IN TREATMENT
lnfliximab:It is a chimera! monoclonal antibody which binds and
neutralizes TNFu; 3-5 mg/kg is infused i.v. every 4-8 weeks. An acute
reaction comprising of fever, chills, urticaria, bronchospasm, rarely
anaphylaxis may follow the infusion. Susceptibility to respiratory
infections is increased and worsening of CHF has been noted. It is
usually combined with Mtx which improves the response and decreases
antibody formation against infliximab.
Adalimumab: This recombinant monoclonal anti-TNF antibody is
administered s.c. 40 mg every 2 weeks. Injection site reaction and
respiratory infections are the common adverse effects. Combination with
Mtx is advised to improve the response and decrease antibody formation
Omalizumab
It is a humanized monoclonal antibody against IgE. Administered i.v. or
s.c., it neutralizes free IgE in circulation without activating mast cells
and other inflammatory cells. On antigen challenge, little IgE is
available bound to the mast cell surface receptors (F,,R1) to trigger
mediator release and cause bronchoconstriction. In severe extrinsic
asthma, omalizumab has been found to reduce exacerbations and
steroid requirement. It is very expensive; use is reserved for resistant
asthma patients with positive skin tests or raised IgE levels who require
frequent hospitalization. It is not itself immunogenic; only injection site
local reactions have been noted so far. It is being tried in other allergic
diseases as well.
Abciximab
It is the Fab fragment of a chimeric monoclonal antibody against GP lIb/IlIa.
Given along with aspirin + heparin during PCI it has markedly reduced the
incidence of restenosis, subsequent MI and death. After a bolus dose
platelet aggregation remains inhibited for 12-24 hr, while the remaining
antibody is cleared from blood with a tllz of 10-30 min.
Dose: 0.25 mg/kg i.v. 10--60 min before PC!, followed by 10 ].1g/rnin for 12
hr. REOPRO 2 mg/ml inj.
Abciximab is nonantigenic. The main risk is haemorrhage, incidence of
which can be reduced by carefully managing the concomitant heparin
therapy. Thrombocytopenia is another complication. Constipation, ileus and
arrhythmias can occur. It is very expensive, but is being used in unstable
angina and as adjuvant to coronary thrombolysis /PCI with stent placement.
Muromonab CD3
It is a murine monoclonal antibody against the CD3 glycoprotein located near to the T cell
receptor on helper T cells . Binding of muromonab CD3 to the CD3 antigen obstructs the
binding of MHC II-antigen complex to the T cell receptor: antigen recognition is interfered,
so that participation of T cells in the immune response is prevented and T cells rapidly
disappear from circulation leading to an immune blocked state. The response to this
monoclonal antibody is less variable than to the polyclonal antithymocyte globulin. It is also
less likely to produce allergic reactions. Muromonab CD3 has been used as induction therapy
together with corticosteroids and azathioprine with delayed use of cyclosporine in 'sequential
regimen' for organ transplantation. This serves to postpone potential nephro- and
hepatotoxicity of cyclosporine. This sequential regimen has been found to be more effective
than the standard triple therapy in renal and hepatic, but not in cardiac transplant recipients. It
is also valuable for steroidresistant rejection reactions and has been used to deplete T cells
from the donor bone marrow before transplantation. The initial doses of muromonab CD3 are
associated with 'cytokine release' syndrome with flu like symptoms: chills, rigor and wheezing.
Occasionally aseptic meningitis, intragraft thrombosis, pulmonary edema, seizures and a
shock like state are produced. High dose corticosteroid pretreatment reduces the reaction.
Antithymocyte globulin (ATG)
It is a polyclonal antibody purified from horse or rabbit immunized with
human thymic lymphocytes which binds to T lymphocytes and depletes
them. It is a potent immunosuppressant and has been used primarily to
suppress acute allograft rejection episodes, especially in steroid-
resistant cases or is combined with them. It can also be used in induction
regimens, but responses are less consistent than with muromonab CD3,
and it has the potential to produce serum sickness or anaphylaxis, but is
less expensive than muromonab CD3.
LYMPHOGLOBULIN (equine) 100 mg/vial inj.; 10 mg/ kg/day i.v.;
THYMOGLOBULIN (rabbit) 25 mg/vial inj.; 1.5-2.5 mg/ kg/day. ATG 100
mg inj; 200 mg i.v./day.
Anti-D immuneglobulin
It is human IgG having a high titer of antibodies against Rh (D) antigen. It
binds the Rho antigens and does not allow them to induce antibody
formation in Rh negative individuals. It is used for prevention of
postpartum/post-abortion formation of antibodies in Rho-0 negative, DU
negative women who have delivered or aborted an Rho-0 positive, DU
positive baby /foetus. Administered within 72 hours of delivery I abortion,
such treatment prevents Rh haemolytic disease in future offspring. It has
also been given at 28th week of pregnancy.
Dose:250-350 )lg i.m. of freez dried preparation. RHIGGAL 100, 350 )lg
vial, RHESUMAN, RHOGAM 300 )Jg/ vial inj. Higher doses (1000-2000 ].lg)
are needed for Rh negative recipients of inadvertantly administered Rh
positive blood.lt should never be given to the infant or to Rho-0 positive, DU
positive individuals.
REFERENCES
Immunology -Janis Kuby
Roitt’s Essential immunology by lvan
M. Roitt, Peter J. Delves, 10th edition.
Essentials of Medical Pharmacology
by KD Tripathi