Lecture 13: Monoclonal Antibodies and Their Uses

Antibodies

• Made by plasma cells to recognise specific targets e.g. pathogen or infect cell - protect us against harmful infectious agents

• Also useful as a detection agent

• Very specific reagents - hypervariable regions

• Large proteins - easily “tagged” and stick stuff on to:

  • identify where they bind to an antigen

  • what antigen they bind to!

How To Make Antibodies

• Mimic what happens in an infection

• Inject small samples of antigens (mg, usually protein, but no necessarily)

• Immunised several times (over 10 weeks)

• Booster shots are given to amplify the immune response and maximise the production of antibodies in the serum.

  • Helps maximise class switching, avidity, and affinity for the antibody.

• The serum is enriched for specific antibodies against the injected antigen.

• Polyclonal response: Serum still contains antibodies to other antigens.

Why are Antibodies Polyclonal

• Antigens usually contain many different epitopes.

• The immune response is polyclonal because each antigen has multiple epitopes that stimulate different B-cells.

• Even though a more dominant antibody response may occur, antibodies targeting different epitopes will still be produced.

• Therefore, the antibodies in the serum are polyclonal, meaning they target more than one antigen.

Anti-Toxins

• Injecting animals with sub-lethal doses of toxins, which induced the production of antibodies in their serum.

  • Generated polyclonal antibodies to neutralise toxins.

• The first antitoxin against diphtheria was developed in 1890 by Emile Adolf von Behring.

• Diphtheria, a bacterial infection, was a major cause of death in children.

• Von Behring's discovery earned him the 1901 Nobel Prize.

Anti-Venom

• First successful antivenom was developed by Albert Calmette in 1895 using cobra venom.

  • He injected an animal with sub-lethal doses of venom and harvested its serum. This crude method is still used today.

• Anti-venoms are still used for venomous bites/stings (snakes, spiders, scorpions, frogs, etc.).

Polyclonal Antibodies in Disease Outbreaks

• Used to treat Ebola and COVID-19.

• William Poole used anti-serum during the Ebola outbreak.

• Serum from survivors was used to treat patients during the COVID-19 pandemic, providing passive immunity.

Convalescent Plasma

• Blood plasma from people recovering from an infection (esp. if severe), may contain high levels of polyclonal antibodies.

• It can confer passive immunity to recipients e.g. by neutralising viral particles.

• Administered either as convalescent plasma (mixed) or as purified polyclonal antibodies (hyperimmune globulin)

• Used during influenza pandemic of 1918, Ebola outbreak of 2014, and COVID-19 (2020/21 in the USA).

• Hyperimmune globulin is still used for post-exposure prophylaxis against viral infections like hepatitis B, chickenpox, and rabies.

Monoclonal Antibodies

• A breakthrough by Köhler and Milstein, providing antibody purity to one target.

• They developed a protocol to immortalise clones of cells capable of making antibodies.

• This is achieved by fusing a normal plasma B cell with a myeloma cell (a tumor cell) to generate a hybridoma.

• The hybridoma contains the antibody-producing genes from the B cell and the immortal properties from the myeloma cell, allowing the production of vast quantities of the same antibody.

In Vivo Generation of Monoclonal Antibodies

• Mice Immunisation: Mice are immunised with an antigen several times to enhance antibody class switching and increase affinity for the target.

• Cell Extraction: Spleen cells are removed from the mouse because they contain antigen-specific B cells, which are mortal (finite lifespan) and therefore classified as primary cells.

Making Plasma Cells Immortal

• Splenocytes (antigen-specific B cells, mortal) are fused in vitro with a myeloma cell line (immortal).

• The myeloma line:

  • Has a purine enzyme deficiency → cannot metabolise purine

  • Cannot secrete immunoglobulin (Ig).

• The fusion produces hybridomas:

  • Immortal from the myeloma cell.

  • Antibody-producing from the B cell.

• Culture media is used to select for cells that can both survive and metabolise purines.

• Only fused cells (hybridomas) are immortal, survive and make antibodies.

HAT Medium and Selection of Hybridomas

• Cells cultured in HAT medium:

  • Contains Hypoxanthine, Aminopterin, and Thymidine.

• Myeloma cells cannot survive in HAT (can't metabolise purines).

• Splenocytes (B cells) are not immortal and die out.

• Result:

  • Only fused hybridoma cells survive:

    • Can metabolise purine.

    • Are immortal.

    • Produce antibody.

• This process is time-consuming and includes single-cell cloning into wells, ensuring that each well ideally contains one hybridoma → for clonality.

• Some fusions will result in antibody production against the OG antigen and be the product on one B-cell

Screening Hybridomas for Monoclonal Antibodies

• Each well ideally contains one hybridoma cell, → ensures the antibodies are monoclonal.

• Screening is done to identify clones that produce antibodies against the original antigen/target.

• Successful hybridomas:

  • Are immortal.

  • Produce antibodies of defined specificity.

• These clones can be cultured to provide an unlimited supply of monoclonal antibodies.

  • Ideally generate immortal cell lines secreting monoclonal antibodies

• Process can be conducted on a large industrial scale

Advantages of Monoclonal Antibodies

• High specificity

• Low cross-reactivity – no other targets

• Standardised worldwide – Ab used in one lab will be the same as the Ab used in another lab across the globe

• Unlimited supply

• Used in immunoassays, diagnosis, tissue typing, separation of cell types, and clinical therapeutic agents

Issues Caused by Antibodies

• Used in laboratory and the clinic, BUT in vivo can trigger immune responses  - e.g. detected by Fc receptors or macrophages and act as an opsonin

• NOT access the sites needed – Abs are large proteins – difficult in delivery

• Causes off-target effects – often if there is a similar antigenic epitope located somewhere else in the body

  • Solution - use of bioengineering

Bioengineering Solutions for Antibody Production

• Insect systems used to produce antibodies with distinct sugar structures, making them less likely to cross-react with human immunity.

• Fab fragments (antigen-binding portions of antibodies) can be engineered for targeted function.

• Specialised delivery systems are developed to help antibodies reach specific target cells effectively.

• Plant-based systems:

  • Example: ZMapp, an Ebola treatment, was produced using nicotine plants.

Immunological Methods Using Monoclonal Antibodies

• Affinity chromatography to purify molecules

• Enzyme Linked ImmunoSorbant Assay (ELISA) and ELISpot

• Haemagglutination

• Immunodetection on tissue samples

• Western Blotting

• Flow cytometry

• Magnetic cell separation

Affinity Chromatography

• Often used to purify an antigen or protein, e.g. for preparing an inoculum/treatment

• Antibody target bound to beads in a column

• Addition of a mixture of molecules, e.g. culture medium serum, to allow for binding to occur  - only the antigen of interest will bind to the antibody

• Washing away will remove any unbound substance, and the beads will remain in the column

• Addition of elution mixture  to extract the purified compound

• Way to purify and enrich for specific targets, e.g.  used to purify and enrich convalescent plasma

Magnetic Separation of Cell Population: Immunoisolation

• Often used in the lab to obtain purified cell populations.

• May be used clinically, e.g. cellular immunotherapy

• Usually, Abs are attached to little metal beads and apply a magnetic force to retain anything bound to the metal beads – antigens of interest will be retained in the magnetic force until the magnets are removed

• Quick and easy process – may be used to purify DCs for a DC vaccine preparation - can target all the cells you don’t want – keep them attached to the magnet so that unbound cells will wash through

• Can have positive or negative selection

Agglutination

• Oldest method in immunology -  Identify if antibody is present for an antigen

• Visualise clumping- i.e. antigen/antibody complexes form

• Commonly used in blood typing

• Also to diagnose if a patient has HAD an infection

• e.g. Typhoid fever- mix serum with a culture of Salmonella typhi  - clumping indicates the patient either had or has an S. typhi infection.

• (N.B. antibodies persist beyond the infection)

Haemagglutination

• Blood type is dependent on the antigens on the RBC

• Mixing reactions with different combinations of those antibodies to see whether or not clumping occurs, to then determine blood type

• Visual process due to the presence of RBCs

Detecting Antibody-Antigen Interactions

• Antibodies are colourless, but can be tagged:

  • Fluorescent markers, e.g. FITC

  • Metal ions

  • Enzyme markers that change colour when a substrate is added, e.g. peroxidase

ELISA

• Often used clinically, e.g. viral diagnostics, levels of hormone, antibody, etc.

• MOST COMMONLY USED DIAGNOSTICALLY AS OPPOSED TO OTHER METHODS

• Used for serum, urine, supernatants etc., but can be used on cell lysates  - often uses liquids

Direct ELISA

• Simplest form

• The sample to be tested is added to the well and sticks to the plastic.

• Then add antibody, which is covalently linked to an antibody

• The antibody will bind to any target in the plastic wells

• Wash away anything unbound

• Add substrate to see a colour change – can measure the absorbance of this

• If a known standard is present can determine the absorbance for this standard curve and determine how much protein is present in the sample.

Increasing Sensitive to Antibody Detection

• Signal amplification is achieved using a secondary antibody (an anti-antibody).

• The secondary antibody is generated against and binds to the Fc region of the primary antibody.

• Multiple secondary antibodies can bind to a single primary antibody, amplifying the detection signal.

Antibody Detection - Fluorescence: Direct vs Indirect

• Direct fluorescence:

  • A single antibody is directly conjugated to a fluorescent molecule.

• Indirect fluorescence:

  • A primary antibody binds to the antigen.

  • Then multiple fluorescently-labelled secondary antibodies bind to the primary antibody.

  • = Signal is amplified — useful for detecting low-abundance targets

Indirect ELISA

• Well is coated with an antigen i.e. your sample

• The specific antibody to be measured is added

• Enzyme-conjugated 2^°Ab is added

• Substrate added and the amount of 2^°Ab (absorbance) measured by ELISA reader

ELISpot (Enzyme Linked Immunospot)

• highly sensitive assay used to measure the frequency of cytokine or antibody-secreting cells at the single-cell level.

• Widely used during COVID to study the immune response and identify major B-cell targets.

• Cells are cultured ± stimuli on a surface coated with a capture antibody.

• After incubation, the cells are washed away, but their secreted antibodies/cytokines remain bound to the surface.

• Detection is similar to ELISA, but instead of color change in solution, it uses a precipitating substrate to form visible spots.

• Each spot corresponds to a single responding cell (e.g. cytokine-secreting or antibody-producing).

• Results in wells with visible “blobs”—each representing an individual activated immune cell

Uses of ELISpot

• Used to detect any secreted protein.

• Commonly used to assess:

  • Frequency of specific T cell responses

  • Frequency of antibody-secreting cells during infection or in response to vaccination

• Helps identify common epitopes that stimulate B-cell responses.

• Useful in vaccine research, e.g. to study T cells producing IFN-γ, indicating an active immune response.

Immunohistochemistry and Immunofluroescence

• Shows localisation of antigen, can be quantitative – shows the location  of a responding cell

• Fresh, frozen, fixed tissues; cultured cells etc.

• Applications include:

  • DNA sequences on chromosomes.

  • Spatial-temporal patterns of gene expression within cells/tissues.

• Not readily used diagnostically

Immunohistochemistry

• Use of enzyme-substrate reaction to bind to specific targets

• Pancreatic tissue – Islets of Langerhans -stained with anti-glucagon antibodies (on the left)(α cells) and anti-insulin antibodies on the right (βcells). Antibodies were conjugated with peroxidase enzyme and detected with substrate

Immunofluroescence

• Counterstained with a fluorescent stain e.g. DAPI to highlight the nucleus – useful in allowing you to orientate yourself around the tissue and tells you if you have a positive cell

Western Blotting

• Qualitative and quantitative analysis of protein expression

  • e.g. from tissue homogenates or cultured cells. E.g. to see if a cell expressed CD3

• Can be used diagnostically

  • e.g. Identify if pathogen present e.g. Lyme disease, HIV

2 Principles Underlining Western Blotting

• Electrophoresis

  • A protein mixture is dissociated and run through a gel under an electric current.

  • Smaller proteins travel further than larger ones.

  • Migration also depends on protein charge.

  • Results in a distinct banding pattern showing protein separation.

• Antibody-Based Detection

  • Antibodies bind to specific target proteins on the gel.

  • This allows visualisation of the bands.

  • Detection methods include luminescence, counterstaining and other visualisation techniques.

Identifying Lymphocytes Using Flow Cytometry

• Different types of lymphocytes may look morphologically simillar e but express different cell-surface proteins.

  • Antibodies can be used to detect these unique markers.

• Flow cytometry is used to analyse these surface markers on cells.

• For example:

  • T cells express CD3

  • B cells do not express CD3

• This allows distinction between cell types using a relatively crude but effective method.

Flow Cytometry

• Uses lasers to detect fluorescent antibodies bound to cells.

• Antibody is added to a cell mixture, and different fluorochromes are added(fluorescent dyes).

• Cells flow through a system one at a time, and a laser hits each individual cell, providing information on cell size and granularity, indicating its function

• Light scattering:

  • Larger cells (e.g., mast cells) scatter light differently due to their size and granularity.

  • Smaller cells (e.g., lymphocytes) scatter light more efficiently and pass more light through.

• Depending on the fluorochrome, cells may be excited with a laser with different photomultiplier tubes to detect the light emitted when the laser hits the fluorochrome

• This generates scatter plots that help visualise cell populations based on size, granularity, and fluorescent marker expression.

Scatter Plots

• Generated from flow cytometry

• Cells isolated from the lung were labelled with fluorescent antibodies against:

  • CD3 (T cells)

  • CD4 (T helper cells)

  • CD8 (Cytotoxic T cells)

    • then analysed by flow cytometry

• Plot A: Granularity (side scatter) vs CD3

  • Helps identify general T cell populations.

• Plot B: CD4 vs CD8

  • Allows a clear distinction between T helper and cytotoxic T cells.

• The further along or up an axis = higher expression of that marker.

Uses of Flow Cytometry

• Used often → generates quantitative and qualitative information e.g. different cell types in a blood or tissue sample

• It is often used for

  • Levels of cell activation

  • Cell viability

  • Cytokines secreted

  • Rare cell types e.g. used to identify innate lymphoid cells

• Clinical uses common

  • e.g. Diagnosis/staging of patients with a haematological neoplasm

  • detecting rare tumour cells

  • monitoring cell numbers (e.g. CD4 cells in HIV)

Monoclonal Antibodies At Home - Pregnancy Tests

• Use ELISA-based technology to detect human chorionic gonadotropin (hCG), a hormone released from the placenta shortly after conception.

• The original concept was created and patented by Margaret Crane in 1967.

• Early pregnancy tests were crude, using hemagglutination reactions that looked for clumping of hCG in test tubes with mirrors to visualise the result.

• Modern tests are more specific, using antibodies and an indicator reaction based on ELISA principles.

Monoclonal Antibodies at Home - COVID-19 Tests

• Lateral flow tests use the same principles as pregnancy tests and ELISA

• They employ monoclonal antibodies and lateral flow immunoassay technology, derived from ELISA concepts.

• These tests are designed to detect viral proteins (antigens) using antibody-based capture and detection systems, providing a visible signal (e.g. a colored line) when the antigen is present.

Monoclonal Antibodies in the Clinic – TNF Therapy

• Marc Feldmann and Ravinder Maini created TNF therapy, using monoclonal antibodies therapeutically.

• Finola and her team hypothesised that active autoimmune diseases generate damaging cytokines, and that blocking one of these key cytokine may block all of the cytokines and the pro-inflammatory consequences

• A control test using antibodies against lymphotoxin (LT) had no effect on IL-1 levels.

• However, antibodies against TNF successfully blocked cytokine production, reducing inflammation and turning off part of the inflammatory cascade.

• This approach was game-changing and highly successful in the clinic.

Uses TNF-a Therapy

• Monoclonal antibodies to TNF-α now used for:

  • rheumatoid arthritis,

  • IBD,

  • Ankylosing spondylitis,

  • Psoriasis,

  • Psoriatic arthritis

Blockade of Immune Checkpoints to Enhance T-Cell Responses

• James Allison and Tasuku Honjo were awarded the Nobel Prize for developing checkpoint inhibitor therapy, a form of antibody therapy used in cancer treatment.

• Their research focused on understanding the immune response "off switches", particularly PD-1 and CTLA-4 (immune checkpoint molecules), which tumours exploit to suppress immune responses.

• They generated antibodies to block PD-1 and CTLA-4, preventing the immune system from being switched off.

• This allowed T-cells to remain ‘on’, boosting the immune response to help kill tumour cells.

  • Method of boosting the immune response to tumours

• Checkpoint blockade is now a successful and a used therapeutic approach in oncology.