Antibody Based Approaches to cancer therapy 1

what is a biologic?

any medicinal product manufactured or extracted from biological sources

give some examples of biologics

• antibody therapies

• gene therapies (CRISPR-Cas9)

• recombinant therapeutic proteins

what is the name of the database used to check licensed antibody therapies?

therapeutic antibody database (TABS)

what are the strategies for the use of monoclonal antibodies as anticancer agents?

• single agents/naked antibodies (i.e. mono-specific)

• bispecific antibodies

• conjugated to cytotoxic agents (ADC)

• conjugated to radiopharmaceuticals

• conjugated to nanoparticles (experimental)

• ADEPT (experimental)

describe the nature of the actual protein that makes up antibodies?

• they are heavy globular plasma proteins with a molecular weight of ~150 kDa

• have sugar chains added to some of their amino acid residues :. glycoproteins

what is the basic functional unit of each antibody?

immunoglobulin monomer (containing only 1 Ig unit)

what are the different configurations of the Ig monomer when antibodies are secreted? what are the names for these configurations

• monomeric with 1 Ig unit → IgD, IgE, IgG

• dimeric with 2 Ig units → IgA

• pentameric with 5 Ig units → IgM

describe the anatomy of an antibody

• Fab region (AKA Fv) → variable region

• Fc region → constant region

• 2 x heavy chains

• 2 x light chains

how are the heavy and light chains in an antibody held together?

intramolecular interactions e.g. cysteine disulphide bridges

how was hybridoma technology significant in antibody production?

• antibodies are very hard to produce

• hybridoma technology is the discovery that prompted its production

how does hybridoma technology work?

• take single B cells from the spleen of a mouse :. each cell produces 1 antibody

• select myeloma cancerous B cells which are deficient in antibody production :. cancerous but cannot make antibodies

• fuse the myeloma B cells and mouse spleen B cells together to make a hybridoma cell

• hybridoma cells :. retain immortality of myeloma cancerous B cells & ability to generate specific antibody from spleen cells

• culture hybridoma cells in petri dish

• individual hybridoma cells are cloned and then tested for desired antibody

• monoclonal antibodies are purified

what were the initial problems associated with antibody therapy?

• immunogenicity → using mouse antibodies in humans generates immune response because antibody has mouse sequences

• short half life

• limited penetration to tumour sites

• poor ability. toreost effector functions

• hard to manufacture

what was hybridoma technologies later replaced with?

recombinant DNA technology

what are the 4 types of monoclonal antibodies developed for therapies?

• murine

• chimeric

• humanised

• human

what are murine monoclonal antibodies?

• initially obtained by hybridoma technology

• differences between the mouse and human immune systems led to clinical failure → different sequences

• major problems were: immunogenicity, short half life and reduced cytotoxicity

what are chimeric monoclonal antibodies?

• engineered antibodies

• sequence the genes in hydridoma cells

• remove the variable region of the mouse sequence and draft it into a human IgG sequence for the constant region

• 35% mouse, 65% human :. reduces immunogenicity and increases serum half life

what are humanised monoclonal antibodies?

• similar to chimeric but in chimeric you use the whole variable region → in humanised, you use the hyper variable region = smaller section of variable region that still retains binding site

• :. draft smaller hyper variable region into human constant region IgG :. 95% human, 5% mouse

what are human monoclonal antibodies?

• using stem cell engineering, you replace the mouse Ig gene with human Ig gene :. get transgenic mouse which expresses human IgG instead of mouse IgG

what are the 3 mechanisms of action that a single-agent (naked) antibody can use?

• interfere with cell signalling

• interfere with vasculature

• checkpoint inhibitors

give an example of a single agent with uses the cell signalling interference mechanism of action. how does it work?

trastuzumab

• targets human epidermal growth factor receptor 2 (HER2) = overexpressed

• inhibits HER2 signalling pathway once bound → growth factor inhibited :. tumour will not grow

• HER2 needs to be in dimer configuration to be active → trastuzumab inhibits dimerisation :. as a monomer, HER2 cannot signal

• used in breast cancer

how do antibodies enter cancer cells?

• too big to diffuse through membrane

• have to be actively transported

what is antibody dependent cell mediated cytotoxicity?

• when a single agent antibody is detected by immune system → T-cells and natural killer cells have Fc receptors on their surface

• interaction between Fc region on antibody and Fc receptor on effector cell causes release of chemical mediators which lead to apoptosis of cancer cell

how do single agent monoclonal antibodies interfere with vasculature? give an example of an agent utilising this mechanism

• targets VEGF growth factor :. prevents vascularisation :. prevents supply of nutrients :. prevents growth

• e.g. bevacizumab

what are checkpoint inhibitors? what is the effect of these in cancer cells?

• immune checkpoints = receptors in immune cells that prevent the superactivation of the immune system by preventing T cell activation :. response is proportionate to disease

• these checkpoint receptors are overexpressed in cancer cells :. weakens immune response and conceals cancer cells :. not recognised as foreign

give 2 examples of checkpoint inhibitor classes of drugs

• CTLA-4 inhibitors

• PD-1 or PD-L1 inhibitors

describe how PD-L1 or PD-1 inhibitors work

• PD-1 = checkpoint

• PD-L1 = antigen on APC (i.e. cancer cell)

• PD-L1 binds to PD-1 causing activation → PD-L1 can be overexpressed in cancer cells :. overactivation :. prevents T cell response

• inhibitors allow T cell activation

give an example of a PD-1 inhibitor

nivolumab

how does the number of mutations in a cancer cell impact the ability of checkpoint inhibitor drugs?

more mutations = works better because there are lots of mutant antigens for T cells to recognise and act upon once their response is re-initiated

what is one of the biggest problems currently associated with Mabs? how can this be overcome?

• price → very expensive

• biosimilars (generics off patent) can reduce costs

what are bi-specific antibodies?

• antibodies made of a heavy and light chain of 2 different monoclonal antibodies

• each arm is directed at a different antigen :. 2 Fab regions can bind to different antigen presenting cells

• allows antibody to recognise antigens on tumour cell and antigens on an effector cell (e.g. cytotoxic T-lymphocyte) :. activates immune cells against tumour cells

give an example of a bispecific monoclonal antibody drug

blinatumomab

what are antibody drug conjugates?

antibody conjugated to cytotoxic agent

• antibody provides specificity and cytotoxic agent (= payload) is responsible for killing cancer cell

• joined by a linker

give an example of an antibody drug conjugate

trastuzumab

describe the cleavage of the linker which joins the antibody to the drug in an antibody drug conjugate

• linker can be designed to cleave specifically at the tumour site :. releasing the cytotoxic agent

• important that payload is not released prematurely in the plasma otherwise toxicities and full cytotoxic effect won’t be achieved → should enter cancer cell and be released there

• bystander effect = when some release occurs externally to the cancer cell :. drug is free to diffuse to neighbouring cells → lose specificity but can be favourable if neighbouring cells have been affected by tumour

• bystander effect can also occur is drug diffuses out of cell or is pumped out by p-glycoprotein

what should be taken into consideration when the payload is being loaded onto the antibody?

Drug-Antibody Ratio (DAR)

• for maximum efficacy, a certain number of linker drug units needs to be attacked to an antibody = DAR

• DAR must be high enough to give good efficacy but not too high to affect antigen recognition and PK

• loading consistency is very important for regulatory and licensing reasons

what is the preferred linker-payload connection point? how can this pose a problem?

disulphide bridges at the hinge

• typical antibody has 8 inter-chain cysteine residues :. 4 disulphide bridges

• problem = can’t control how many payload molecules will conjugate per antibody molecule → problem with regulatory agencies as some antibodies will have e.g. 4 payload molecules and some will have 6 :. lack of consistency

how can payload attachment be controlled using advanced conjugated technologies?

ThioBridge

• attaches payloads at accessible disulphides with good control over DAR

• generates accessible sulphides using addition-elimination reactions :. can restrict number of payload molecules added by only exposing a finite number. of cysteines

what are protease sensitive linkers?

cleaves by specific proteins that are more abundant in tumour cells

what are pH sensitive linkers?

tumour microenvironment tends to be more acidic due to higher metabolism and secretion of lactic acid :. acid sensitive linkers