Monoclonal Antibodies in Therapeautics

DR. SARJU R. ZILATE

Monoclonal Antibodies

Monoclonal antibodies (MAb) are antibodies that are

identical because they are produced by one type of immune

cell; all are clones of a single parent cell.

Monoclonal antibodies (MAbs) are an integral part of

targeted therapy approach for various diseases which

result in decrease in adverse effects and increase in

efficacy.

They target various receptors or various growth factors on

the cell surface and modulate their vital functions and

cause cell death by various mechanisms.

The Structure of an Antibody

Antibody FunctionAntibodies have two major functions:

•Recognize and bind antigen

•Induce immune responses after binding

The variable region mediates binding

•Affinity for a given antigen

•confers absolute specificity for an antigen

The constant region mediates immune response afterbinding

•Different classes of constant regions generatedifferent isotypes

•Different isotypes of antibody have differingproperties

Production of monoclonal antibodies

• Since 1975, when Keller and Milstein developed

hybridoma technology,

• technological strides towards the production of antibodies

have been made.

• The first success in generating human mAbs (hmAbs)

with predefined specificity was conducted in 1980

through

• the fusion of human spleen cells from patients with

human myelomas.

• Since then, several major methods have been

established to generate hmAbs, including

• 1) immortalization of antigen-specific human B cells;

• 2) acquisition of antigen-specific human B cells via

phage display technology;

• 3) the production of hmAbs from transgenic mice;

• 4) single human B cell cloning techniques to directly

clone and express immunoglobulin (Ig) genes in vitro

from antigen-specific B cells.

Conventional hybridoma technology

• Step 1: Immunization of mice

• Mice are immunized with an antigen (attached to

adjuvant).

• The antigen can be whole cells, membrane fragment, or

complex molecules.

• Mice serum’s are screened using various techniques such

as ELISA.

• When sufficient titre is reached the mice are euthanized

and spleen is removed as a source of cells for cell fusion.

• Step 2: Preparation of Myeloma Cells

• Myeloma cells are immortalized cells that are capable of

dividing indefinitely.

• These cells are treated with 8-azaguanine to ensure

sensitivity to HAT

• Step 3: Fusion of myeloma cells with Spleen cells:

• spleen cells harvested from mice are fused with myeloma

cells. ,through co-centrifuging in polyethylene glycol.

• Cells are plated in selection medium

• Hypoxanthine-aminipterin-thymidine (HAT) selection

medium –blocks nucleotide synthesis.

• Only fused cells with grow on HAT. Cells are distributed on

feeder cells (murine bone-marrow) to promote growth of

the hybridomal cells.

• Step 4: Cloning of Hybridoma cells .

• A mouse is inoculated with the cell and thereby becomes a

factory for producing the MAbs.

• Ascites fluid collected from the mouse

• Step 5: Antibody is screened and purified:

• Antibodies are screened using specific Ag binding.

Advantage of in vivo

• Step 6: Desired antibodies are cloned:

• This is done in vitro on culture bottle

Immortalization of antigen-specific humanB cell and hybridoma technology

• Hybridoma technology has contributed to virtually all

areas of biology and medicine

• and has been greatly refined since its introduction in 1975.

• human antibody-secreting cells can be immortalized by

fusion of antibody-producing human B lymphocytes with

either mouse or human myeloma or lymphoblastoid cells

by infection with the EBV.

Phage display(1985)

• successfully used

• to select antigen-specific variable region genes and

• to express functional antibody fragments with unique

specificity.

• the library of diverse human immunoglobulin-heavy

chain variable (VH) gene and light chain variable (VL)

gene segments are prepared by reverse transcription of

mRNA from B cells and PCR amplification.

• The gene encoding single chain variable fragment (scFv)

can be created by randomly combining VH and VL gene

segments using PCR.

• After bio-panning the phages bound to a range of antigens

or haptens, antigen-specific antibody fragments with good

affinities can be identified.

• One phage display-derived mAb has been approved by the

FDA and at least 35 human mAbs generated by phage

display technology have entered into clinical development.

Phage display(1985)

Transgenic mice

• Genetically engineered mice expressing humanantibody repertoires were first reported forgenerating hmAbs using the hybridoma technology in1994.

• disruption of endogenous mouse

Ig-heavy chain and

Igκ-light chain loci together

• with the introduction of

human Ig-heavy chain and

Igκ-light chain genes.

Generation of mAbs from single human B cells by Ig gene cloning and expression in vitro

This approach requires the

• isolation of individual B cells from mouse

spleen or human PBMCs using magnetic

microbeads conjugated with B-cell-selective

markers

•The light-chain and heavy-chain genes

• coding for variable fragments of antibody

• in each cell

•are separately amplified by RT-PCR

•and then combined with the sequences by anoverlapping PCR technique.

•For each cell,

• Ig heavy- and corresponding Ig light-chain gene

transcripts are amplified by nested RT-PCR and

• cloned into mammalian expression vectors to

produce hmAb with defined specificity in vitro.

Types of mAbs

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Purple denotes human component orange murine component

EVOLUTION OF MONOCLONAL ANTIBODY

1. TRANSGENIC

2. LIBRARIES

a.BACTERIOPHAGE

b. mRNA

c. Cell Surface

Ist

generation

mab

2nd generation mab

daclizumab

Types of monoclonal antibodies

1. Murine source MAbs: rodent MAbs with excellentaffinities and specificities generated usingconventional hybridoma technology.

2. Chimeric MAbs: chimers combine the humanconstant regions with the intact rodent variableregions. Affinity and specificity unchanged.

3. Humanized MAbs: contained only the CDRs of therodent variable region grafted onto human variableregion framework

4. Recombinant DNA engineered MAbs

Murine

Derived from mice

Patients treated with murine mAbs develop a human

antimouse antibody (HAMA) response

Rapid clearance of the mAb

Poor tumour penetration

Hypersensitivity reactions

90Y-ibritumomab

131I -Tositumomab

22

Chimeric Abs

Antigen binding parts (variable region) of mouse

Ab with effector parts (constant region) of

human

–Infliximab

–Abciximab

–Rituximab

23

Humanized

Human Ab with complimentary determining

region

(CDR) or hypervariable region from non human

source

–Daclizumab

–Trastuzumab

24

Human Abs

Recombinant DNA technology:

Genes for variable Fab portion of human Abs is

inserted in genome of bacteriophages & replicated

Mixed with Ag & complementary Ab producing

phages selected

e.g. Adalimumab

25

Nomenclature of Monoclonal Antibodies

Prefix Target Source Suffix

variable

-o(s)- bone -u- human

-mab

-vi(r)- viral -o- mouse

-ba(c)- bacterial -a- rat

-li(m)- immune -e- hamster

-le(s)- infectious lesions -i- primate

-ci(r)- cardiovascular -xi- chimeric

-mu(l)- musculoskeletal -zu- humanized

-ki(n)- interleukin -axo- rat/murine hybrid

-co(l)- colonic tumor

-me(l)- melanoma

-ma(r)- mammary tumor

-go(t)- testicular tumor

-go(v)- ovarian tumor

-pr(o)- prostate tumor

-tu(m)- miscellaneous tumor

-neu(r)- nervous system

-tox(a)- toxin as target

Nomenclature

Suffix

–Human: -umab

–Humanized: -zumab

–Murine: -momab

–Chimeric: -ximab

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Examples

•ab- + -ci- + -xi- + -mab: chimeric monoclonal antibody used on the cardiovascular system.

•tras- + -tu- + -zu- + -mab: humanized monoclonal antibody used against a tumor.

• Pali- + -vi- + -zu- + -mab

humanized mab used against a virus (RSV)

Pharmacokinetics: mAbs

• Routes of administration:

Subcutaneously (Rituximab, Trastuzumab, Adalimumab)

Intramuscularly (Palivizumab)

Intravenously

• IV route: preferred because of 100% bioavailability

• Route for elimination of antibodies

Via uptake & catabolism by reticuloendothelial system & target tissue.

29

P/K: mAbs

•Half-life

Chimeric : 4 –15 days

Humanized: 3 - 24 days

Recombinant human: 11– 24 days

•Human antimouse antibody (HAMA) response develops 7–10 days following exposure to murine antibody

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MOA

• 1.Make the cell more visible to the immune system:

• The immune system attacks foreign invaders in your body, but it doesn't always recognize cancer cells as enemies. A monoclonal antibody can be directed to attach to certain parts of a cancer cell. In this way, the antibody marks the cancer cell and makes it easier for the immune system to find.

• 2. Block growth signals- cetuximab

• 3.Deliver radiation to cells: By combining a radioactive particle with a monoclonal antibody, doctors can deliver radiation directly to the cancer cells.

Therapeutic uses

• Immunosuppression

•Autoimmune diseases

•Malignancies

•Antiplatelet therapy

• Infectious diseases

•Asthma

• Osteoporosis32

Monoclonal antibodies in therapeutic use:

• ANTI CD3:

• CD 3 is a co receptor which plays an important role inT cell receptor signaling.

• MUROMUMAB (OKT3) is a drug which blocks thisreceptor. It blocks killing by cytotoxic T cells and manyother T cell functions. It kills cells by blocking vitalfunctions of CD3, approved for treatment of renalallograft rejection crisis.

• TEPLIZUMAB is a newer anti CD3 drug. It isused for

• protecting remaining beta cells in newly diagnosedtype I DM. It is currently in phase III clinical trials.

• ANTI CD20:

• RITUXIMAB:

• Its mechanisms of action in killing cancer cells are-

ADCC ,CDC and direct induction of apoptosis with

proven efficacy against wide range of NHL B-cell

malignancies.

• Dose: 375mg/m2 IV infusion weekly for 4 weeks.

• Side effects are fever with rash, dyspnoea and late onset

neutropenia.

• OCRELIZUMAB is a newer CD20 drug .

• It targets mature B lymphocytes and thus is an

immunosuppressive drug.

• It is currently in Phase III.

• It is used in RA, SLE, MS and lymphomas.

• OFATUMUMAB

• is also a newer CD20 drug.

• It inhibits early B lymphocyte activation.

• It targets different epitope of that by rituximab.

• It was approved in February 2010 for refractory CLL.

Radiolabelled Anti CD20

• Radiolabelling the monoclonal antibodies increases

their efficacy.

• They can also be used for various imaging purposes.

• Yttrium 90-ibritumomab (Y90),

• Indium 111- ibritumomab (In111),

• Iodine 131- tositumomab (I131 sub) are the 3

commonly radiolabelled anti CD20 MAbs.

• They show increase in efficacy than their naked

counterparts.

• Effective in relapsed /refractory/advanced cases

of follicular B-cell lymphoma and NHL in the

doses of 0.4 ci/kg IV.

• Its side effects are myelodysplasia and

hematological toxicities

ANTI CD 22:

• CD22 is a co receptor important for B cell receptor

signaling.

• EPRATUZUMAB is a drug which blocks CD22

signaling.

• It is currently in phase III clinical trials.

• It is active against malignant B cells and used in SLE.

• It produces cell death by ADCC

ANTI CD52:

• CD52 is present on thymocytes, macrophages,lymphocytes and monocytes.

• ALEMTUZUMAB, is a drug which blocks the CD52

• signaling.

• It kills tumor cells by ADCC, CDC and apoptosis.

• It is Administered IV 30mg/day thrice weekly.

• Premedication with diphenhydramine andacetaminophen should precede this drug sincehypersensitivity reactions are common.

• It used in B cell and T cell lymphomas and MS.

• Its side effects are T cell depletion andimmunosuppression.

ANTI CD33:

• CD33 is a co receptor found on myeloid cell surface.

• GEMTUZUMAB OZOGAMICIN (MAb linked to a

toxin). Humanized MAb covalently linked to a

semisynthetic derivative of calicheamicin .

• It causes DS DNA breaks and cell death.

• Its dose is 2 doses of 9mg/m2 IV separated by 14 days.

• It is used in AML.

• Its side effects are hematopoietic suppression and vaso

occlusive disorders.

• LINTUZUMAB

• is a newer anti CD33 MAb.

• It is currently in phase III for AML.

ANTI CD 11a:

• CD 11a is a co receptor found on B cells and important

in cell to cell adhesion and co-stimulation.

• EFALIZUMAB is a drug that blocks CD11a signaling.

• It is approved for the treatment of adult patients with

severe psoriasis.

• It administered by SC injections.

ANTI HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR 2/ NEU:

• Human epidermal growth factor receptor is a factor which

plays an important role in growth of many breast cancers.

• TRASTUZUMAB

• is a drug which blocks this receptor.

• it causes cell death by inhibition of HER2 signaling with

G1 arrest and also by ADCC and apoptosis .

• Its dose is loading dose of 4mg/kg IV followed by 2 mg/kg

weekly.

• It is used in HER2-positive metastatic breast

cancers.

• Its side effects are cardiomyapathy and flu like

syndrome.

• PERTUZUMAB is a newer HER2 blocking MAb.

• It is currently in clinical trials

ANTI EPIDERMAL GROUTH FACTOR RECEPTOR (EGFR)

• Epidermal growth factor receptor plays a vital role in

growth of many cancers.

• CETUXIMAB is a drug which blocks EGFR signaling and

causes cell death by ADCC.

• Its dose is loading dose of 400mg/kg infusion followed by

250mg/kg weekly.

• It is used in metastatic colorectal and head and neck

cancers.

• ITS SIDE effects are infusion related toxicity and skin rash.

• PANITUMUMAB –

• It is an another EGFR receptor blocking MAb..

• Produces cell death by similar mechanism as

cetuximab.

• It was approved in September 2006.

• It is used in metastatic colorectal and head and neck

cancers.

• Its adverse effects are skin rash and fatigue.

• First MAb approved by FDA developed from transgenic

mice.

• MATUZUMAB and NIMOTUZUMAB

• are the other EGFR blocking MAbs

• which are in phases III of clinical trials.

ANTI VASCULAR ENDOTHELIAL GROUTH FACTOR (VEGF)

• Vascular endothelial growth factor plays an important role

in angiogenesis and neo vascularization of various tumors.

• BEVACIZUMAB is a drug which blocks VEGF.

• It inhibits angiogenesis and neovascularization

• in the dose of 5mg/kg IV every 14 days until disease

progression stops.

• It is used in colorectal and head and neck cancers.

• Its side effects are hypertension, pulmonary hemorrhages,

GI perorations and CCF.

• RANIBIZUMAB is a newer VEGF blocking drug.

• It is used in neovascular macular degeneration.

• It is injected directly into vitreous cavity.

Side effects of monoclonal antibody

• In general, the more common side effects caused by

monoclonal antibody drugs include:

• Allergic reactions, such as hives or itching

• Flu-like symptoms, including chills, fatigue, fever, and

muscle aches and pains

• Nausea

• Diarrhea

• Skin rashes

Rare, but more serious side effects of monoclonal antibody therapy may include:

• Infusion reactions. Severe allergy-like reactions can occur

and in very few cases lead to death.

• Dangerously low blood cell counts. Low levels of red blood

cells, white blood cells and platelets may lead to serious

complications.

• Skin problems

• Bleeding. Some of the monoclonal antibody drugs are

designed to stop cancer from forming new blood vessels.

There have been reports that these medications can cause

bleeding.

Agent Target(s) FDA-approved

indication(s)

Ado-trastuzumab emtansine

(Kadcyla)

HER2 (ERBB2/neu) Breast cancer

(HER2+)

HER2 (ERBB2/neu) exon 21

substitution

(L858R) mutations)

Aldesleukin (Proleukin) Renal cell carcinoma

Melanoma

Alemtuzumab (Campath) CD52 B-cell chronic

lymphocytic

leukemia

Brentuximab vedotin (Adcetris) CD30 Hodgkin lymphoma

Anaplastic large cell

lymphoma

Belimumab (Benlysta) BAFF Lupus

erythematosus

Belinostat (Beleodaq) HDAC Peripheral T-cell

lymphoma

Bevacizumab (Avastin) VEGF ligand Cervical cancer

Colorectal cancer

Fallopian tube

cancer

Glioblastoma

Non-small cell

lung cancer

Ovarian cancer

Peritoneal cancer

Renal cell

carcinoma

Canakinumab (Ilaris) IL-1β Juvenile idiopathic

arthritis Cryopyrin-

associated periodic

syndromes

Cetuximab (Erbitux) EGFR (HER1/ERBB1) Colorectal cancer

(KRAS wild type)

Squamous cell

cancer of the head

and neck

Denosumab (Xgeva) RANKL Giant cell tumor of

the boneIbritumomab tiuxetan (Zevalin) CD20 Non-Hodgkin's

lymphoma

Ipilimumab (Yervoy) CTLA-4 Melanoma

Nivolumab (Opdivo) PD-1 Melanoma

Obinutuzumab (Gazyva) CD20 Chronic lymphocytic

leukemia

Ofatumumab (Arzerra, HuMax-

CD20)

CD20 Chronic lymphocytic

leukemia

Panitumumab (Vectibix) EGFR (HER1/ERBB1) Colorectal cancer

(KRAS wild type)

Pembrolizumab (Keytruda) PD-1 Melanoma

Pertuzumab (Perjeta) HER2 (ERBB2/neu) Breast cancer (HER2+)

Ramucirumab (Cyramza) VEGFR2 Gastric cancer or

Gastroesophageal

junction (GEJ)

adenocarcinoma Non-

small cell lung cancer

Rituximab (Rituxan, Mabthera) CD20 Non-Hodgkin’s lymphoma

Chronic lymphocytic

leukemia Rheumatoid

arthritis Granulomatosis

with polyangiitis

Siltuximab (Sylvant) IL-6 Multicentric Castleman's

disease

Tocilizumab (Actemra) IL-6R Rheumatoid arthritis

Juvenile idiopathic

arthritis

Tositumomab (Bexxar) CD20 Non-Hodgkin's

lymphoma

Trastuzumab (Herceptin) HER2 (ERBB2/neu) Breast cancer

(HER2+) Gastric

cancer (HER2+)

TO SUMMARISE :

MAbs are highly specific Abs produced by a clone of

single hybrid cells formed by fusion of B cell with the

tumor cell.

The hybridoma formed yields higher amount of

MAbs.

MAbs can be produced in vitro and in vivo .

Animals are utilized to produce MAbs, but these

antibodies are associated with immunogenicity and

ethical problems.

Recombinant DNA technology, genetic

engineering and transgenic animals are used to

produce humanized MAbs or pure human MAbs,

with fewer ADRs

Used for treatment of cancer, autoimmune

disorders, graft rejections, infections, asthma etc.

•THANKS