MONOCLONAL ANTIBODY :

ENGINEERING AND THERAPY

 Structure
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

 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.
ANTIBODY CLASSES AND BIOLOGICAL ACTIVITIES
• 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
• 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, 0.3g/mL

– Participate in immediate hypersensitivities reations. Ex. Asthma,

anaphylaxis, hives
• Binds Mast Cells and Blood Basophils thru FcR

• 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, ~30g/mL

ANTIBODY ENGINEERING
 ANTIBODY ENGINEERING
• 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
 MONOCLONAL 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 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.
Monoclonal Antibody
For Cancer Treatment
Cancer : An abnormal growth of cells which
proliferate in an uncontrolled way and
metastasize

Antibody : An immunoglobulin, a specialized

immune protein, produced because of the
introduction of an antigen into the body, and
which possesses the remarkable ability to
combine with the very antigen

Monoclonal Antibody : An antibody

produced by a simple clone of cells,
Monoclonal antibody is therefore a single
pure type of antibody
Metastasis is the spread of
cancer from one organ or
parts of the body to another
without being directly
connected .
 Benefits of using monoclonal antibody
Homogeneity : Single antibody molecule that binds to the antigen
with the same affinity and promote the same effectors function

Specificity : Highly specific towards the target antigen

Less side effects : Much lesser side effects with respect to

conventional chemotherapy

High efficacy : Efficacy has been found much higher in case of

• treating cancer over conventional approach

Tagging : Can be tagged with chemotherapeutic drugs, radioisotopes

and immunotoxins
NAKED & CONJUGATED MONOCLONAL ANTIBODIES
 Naked mAbs - Antibodies that work by themselves having no drug
or radioactive material attached to them. These are most common
in treating cancer.
Ex- Alemtuzumab, Trustuzumab, etc.

 Conjugated mAbs- Monoclonal antibodies joined to a

chemotherapeutic agent or to a radioactive particle are called
conjugated mAbs

1.Radiolabeled: ibitrumomab tiuxetan,

Tositumomab
2.Chemolabeled: Brentuximab vedotin,
trastuzumab emtansine
 Current cancer therapies provide only a 40% probability that a patients
diagnosed with cancer today will be alive in 5 years’ time. Furthermore
the relatively low therapeutic index of many anti-cancer drugs causes
severe toxic effect on normal tissues.

 As a consequence new cancer therapies are being investigated which

aim to specifically target tumour cells and sparing normal individuals
from exposure to the cytotoxic agent . One such approach is Monoclonal
Antibody directed cytotoxic therapy.
 Monoclonal antibodies (mAb) are antibodies that are made by identical
immune cells and are all clones of unique parent cell. Monoclonal
antibodies can have monovalent affinity, in that they bind to the same
epitope.

 Monoclonal antibody therapy is a form of immunotherapy that uses

monoclonal antibodies to bind monospecifically to certain cells. This may
then stimulate the patients immune system to attack those cells.

 Monoclonal antibodies directed cytotoxic agents are potent, specific and

relatively non toxic .
 Antibody-drug conjugates or ADCs are an important class of highly potent
biopharmaceutical drugs designed as a targeted therapy for the treatment of people
with cancer.
 It Contain a monoclonal antibody, a stable linker based on chemical motifs like
disulphide , hydrazones or peptides ( cleavable), and thioethers ( non-
cleavable),control the distribution and delivery of potent cytotoxic agent. The
availability of better and more stable linkers has changed the function of the
chemical bond. The type of linker, cleavable or no cleavable, lends specific properties
to the cytotoxic (anti-cancer) drug. For example, a non-cleavable linker keeps the
drug within the cell. As a result, the entire antibody, linker and cytotoxic (anti-cancer)
agent enter the targeted cancer cell where the antibody is degraded to the level of
an amino acid. The resulting complex – amino acid, linker and cytotoxic agent – now
becomes the active drug. In contrast, cleavable linkers are catalyzed by enzymes in
the cancer cell where it release the active agent.
 TUMOUR ASSOCIATED ANTIGENS
Antigen category Antigens Tumor types expressing antigen
Cluster of CD20 Non Hodgkin lymphoma
differentiation (CD)
antigen CD30 Hodgkin lymphoma
CD52 Chronic lymphocytic leukemia
EpCAM Epithelial tumors (breast, colon, lung)
gpA33 Colorectal carcinoma
Glycoproteins
Mucins Epithelial tumors (breast,colon,lung,ovarian)
CA-IX Renal cell carcinoma
PSMA Prostate carcinoma
Glycolipids Gangliosides Neuroectodermal tumors
VEGF Tumor vascularate
Vascular targets VEGFR Epithelium derived solid tumors
ErbB1/EGFR Glioma,lung,breast,colon,head,neck tumors
ErbB2/HER2 Breast,colon,lung,ovarian,prostate tumors
Growth factor
TRAIL R1,R2 Solid tumors & hematological malignancies
RANKL Prostate cancer & metastases
Pharmacologic mechanism of action

Adapted from Patrick GM et al., Cancer Biol Med, 2014

 FDA APPROVED MONOCLONAL ANTIBODIES IN TREATMENT
CANCER
TR

Recently approved drugs are Nivolumab (2015) for NSCL carcinoma, Olaratumab (2016) for Sarcomas, Avelumab (2017) for
metastatic merkel cell carinoma, Atezolizumab (2016) for Metastatic NSCL carcinoma, Durvalumab (2017) for Metastatic urothelial
carcinoma
MECHANISM OF ACTION OF Rituximab on non b cell
Hodgkin’s lymphoma

Adapted from Seyfizadeh N, et al., Crit Rev Oncol Hemtol, 2015

Case study-1

Random controlled trials done using conventional drug regimen and

Rituximab conjugated with conventional drug
Adapted From the clinical data of research work from Sehn LH., et al and
Fisher RL., et al.
MECHANISM OF ACTION OF CONJUGATED MONOCLONAL
ANTIBODY (TRaSTUZUMAB EMTANSINE)

Adapted from LoRusso PM., et al. Clin Cancer Res, 2011

Monoclonal antibodies CURRENTLY under clinical trials
 Limitations
 In murine xenograft models, mAbs directed against tumour specific antigens largely
remain in the blood and no more than 20% of the administered dose typically
interacts with the tumor

 Although great strides have been made in antibody engineering and cancer therapy,
production cost is estimated at twice that required for conventional drugs

 Among those mAbs approved for cancer therapy, only two (trastuzumab, cetuximab)
are mainly targeting solid tumours, whereas over 85% of human cancers are solid
tumours, clearly reflecting the current limitation of mAb treatment

 Although most therapeutic antibodies can trigger complement- dependent

cytotoxicity in vitro, this has not been demonstrated in vivo so far
 Limitations

 More than 90% patients on rituximab therapy experience infusion

related reaction such as cytokine release syndrome and tumor lysis
syndrome

 Intravenous administration of alemtuzumab is associated with

lymphopenia and concomitant immunosuppression

 Conjugated agents such as brentuximab vedotin precipitate cumulative

peripheral neuropathy and myelosuppression is the main toxicity of 131I-
tositumomab and 90Y-ibritumomab tiuxetan
 References
1. Scott AM, Allison JP, Wolhock JD. Monoclonal antibody in cancer therapy. Cancer immunity.
2012. 1-5.

2. Coulson A, Levy A, Williams MG. Monoclonal antibodies in cancer therapy: mechanism,

successes, limitations. West Indian medical journal. 2014. 650-652.

3. Barok M, Joensuu M, Isola J. Trastuzumab emtansine: mechanism of action and drug

resistance. Breast cancer research. 2014. 1-9.

4. Syfizadeh N, Syfizadeh N, Hasenkamp J, Yepeze SH. A molecular perspective on Rituximab: A

monoclonal antibody for B cell non Hodgkin lymphoma and other affections. Critical reviews
in oncology/hematology. 2015. 1-11 .

5. Pamela A Trail, Albert b Bianchi. Monoclonal antibody conjugates in treatment of

cancer.1999;11:584-588.