lec 12 - PK of monoclonal antibodies (glassman)

zauberkugel - the ‘magic bullet’

• concept proposed by paul ehrlich in 1960

• hypothesis = the ideal drug will selectively kill a microbe but will NOT harm healthy cells

  • salvarsam → anti-syphilis drug discovered by ehrlich that had minimal adverse effects (the first magic bullet)

• are monoclonal antibodies “magic bullets'“?

  • very high affinity and selectivity for pharmacologic targets

  • relatively low distribution into non-eliminating organs

antibodies as drugs - brief history

• 1890 → kitasato shibasaburo and emil von behring develop ‘antitoxins’ from the serum of immunized animals to treat diphtheria (bacterial infection)

  • the principle of convalescent plasma therapy

• 1944 → edwin cohn describes a method to isolate the immunoglobulin fraction of plasma (basis of IVIg therapy)

• 1975 → george kohler and cesar milstein describe the methodology to produce monoclonal antibodies

• 1986 → first mAb (orthoclone) approved by the FDA

  • prevention of kidney rejection in transplant

  • withdrawn from the market due to toxicities

• 2000 → first antibody-drug conjugate (mylotarg) approved

  • treatment of acute myeloid leukemia

  • withdrawn from the market in 2010 (reintroduced in 2017)

• 2002 → first fully human mAb (humira) approved

  • treatment of rheumatoid arthritis

• 2016 → first biosimilar mAb (inflectra) approved

• 2021 → 100th mAb product approved by FDA

antibodies are an important therapeutic class

• 3 of the top 10 selling drugs globally in 2022 were mAbs

• mAbs are projected to be 5 of the top 10 selling drug since 2023

antibody therapeutics are a growing drug class

• 12 antibody therapeutics gained first approval in the Us or EU from jan-nov 2022

  • 24 more were undergoing review as of nov 2022

  • 23 were expected to be submitted for approval in 2023

  • ~140 in late stage clinical trials

what are antibodies?

• types of antibodies

  • IgG = γ chain

  • IgA (dimer) = α chain

  • IgM (pentamer) = μ chain

  • IgD = δ chain

  • IgE = ε chain

• antibodies are released in response to antigens → part of the memory response

• each antibody monomer is a Y shaped molecule, composed of 2 identical heavy chains and 2 identical light chains

  • some antibodies function as multimers

• within a species, the Fc regions of antibodies within a given class are the same

• in mammals, antibodies can be distinguished into 5 classes by their heavy chains

key features of mammalian immunoglobulin

• IgG(monomer)

  • heavy chains = γ

  • # of antigen binding sites = 2

  • molecular weight = 150,000 Da

  • percentage of total antibody in serum = 80%

  • crosses placenta = yes

  • fixes complement = yes

  • Fc binds to = phagocytes

  • function = main blood antibody of secondary responses, neutralizes toxins, opsonization

• IgA (secretory dimer)

  • heavy chain = α

  • # of antigen binding sites = 4

  • molecular weight = 385,000 Da

  • percentage of total antibody in serum = 13%

  • crosses placenta = no

  • fixes complement = no

  • Fc binds to = nothing

  • function = secreted into mucus, tears, saliva, colostrum

• IgM (pentamer)

  • heavy chains = μ

  • # of antigen binding sites = 10

  • molecular weight = 900,000 Da

  • percentage of total antibody in serum = 6%

  • crosses placenta = no

  • fixes complement = yes

  • Fc binds to = nothing

  • function = main antibody of primary responses, best at fixing complement, the monomer form of IgM serves as B cell receptor

• IgD (monomer)

  • heavy chains = δ

  • # of antigen binding sites = 2

  • molecular weight = 180,000 Da

  • percentage of total antibody in serum = 1%

  • crosses placenta = no

  • fixes complement = no

  • Fc binds to = nothing

  • function = B cell receptor

• IgE (monomer)

  • heavy chains = ε

  • # of antigen binding sites = 2

  • molecular weight = 200,000 Da

  • percentage of total antibody in serum = 0.002%

  • crosses placenta = no

  • fixes complement = no

  • Fc binds to = mast cells and basophils

  • function = antibody of allergy and anti-parasitic activity
    

IgG - key structural features

IgG structural features

• molecular weight ~150 kDA

• consists of 2 heavy and 2 light chains held together by disulfide bonds

IgG key domains

• fragment antigen binding

  • contains CDRs → key site of antigen binding

• fragment crystallizable

  • key site for effector functions

  • confers long half-life on IgG

IgG - the primary serum Ig

• IgG isotypes = different functions in the body

• subtle changes in structure correspond to differences in function

development of antibodies as therapeutics

george kohler and cesar milstein won the 1984 noble prize in physiology or medicine for development of monoclonal antibodies

approaches for antibody development

• mouse hybridoma

  • immunize mouse with targets

  • harvest splenocytes, generate hybridomas

  • screen the hybridomas

  • end up with mouse mAb and can either:

    • chimerization → chimeric mAb

    • CDR graft → humanized mAb

• phage display

  • phage-displayed Ab libraries

  • biospanning with targets (3-5 cycles)

  • screening

  • construction of human IgG

  • human mAb

• transgenic mouse

  • immunize mouse with targets

  • harvest splenocytes

  • screening

  • human mAb

• single B cell

  • collect PBMC (which have B cells) from humans

  • sort B cells with labeled antigens

  • PCR construct V_H and V_L

hybridoma technology

1. animals (mouse, rat, rabbit) are repeatedly immunized with antigen of interest

2. splenocytes are fused with myeloma cells

3. apply pressure (HAT media) to select for fused cells

4. separate fused cells into individual colonies (single cell selection)

5. screen for binding to target of interest via methods such as ELISA or SPR

drawbacks for clinical development

• NOT amenable to development of human antibodies

• no direct path to engineer antibodies for optimal behavior

humanization of therapeutic antibodies

• molecular biology has allowed movement from fully mouse mAbs → fully human

• increasing “human” content correlates with decreased immunogenicity (reduction in the ability of a substance to stimulate an immune response; so the immune system wont recognize the antibody as something ‘bad’) and increased half life

recent developments in mAb

transgenic mice

• human immunoglobulin genes knocked in, rodent genes knocked out

• allows production of fully human antibodies in rodents

• ex. vectibix (anti-EGFR)

phage display

• in vitro selection of candidates and affinity maturation

• can produce antibodies derives from numerous species

• ex. humira (anti-TNF-alpha)

george smith and gregory winter won the 2018 nobel prize in chemistry for development of phage display

antibody disposition mechanisms

antibodies are NOT

• orally bioavailable

• extensively protein bound

• metabolized by CYP450s (or other DME)

• renally filtered

• substrates for ABC or SLC transporters

factors influencing mAb PK

Fcy receptor mediated clearance

• genotype

• ethnicity

• age

• disease

catabolism

• disease

• infection

FcRn recycling

• age

• disease

• genotype

• ethnicity

immunogenicity

• type of mAb & target

• disease

• protein modifications

• formulation

• dose site and regimen

• aggregation

• concomitant medication

• age

subcutaneous absorption

• disease

• age

• dose site

• exercise, heating, rubbing

linear vs non-linear PK

linear PK: all ADME processes can be characterized by processes that are dose/concentration-independent

• all rate processes can be described by first-order kinetics

• results in PK profiles that are dose-proportional

non-linear PK: saturable processes (non-linear rates) are requires to describe some or all ADME processes

• results in PK profiles that are NOT dose-proportional

how to identify non-linear PK

• systematic trends observed in dose-normalized PK profile and NCA parameters with non-linear PK

• greater than dose-proportional increases in AUC with dose suggests saturable clearance mechanism

linear antibody PK-

general expectations

• biexponential PK profile typically observed

• long terminal half life (2-3 weeks)

• low Vd

characteristics of mAbs with ‘typical’ PK

• NO target-mediated drug disposition

• typically bind to soluble targets

non-linear antibody PK

general expectations

• greater than proportional increases in AUC

• dose-dependent changes in CL, Vd, t_1/2

characteristics of mAbs with non-linear PK

• often due to target-mediated drug disposition

  • typical targets are either:

    • membrane bound - HER2, EGFR, CD20

    • soluble and form immune complexes - IgE

IgG elimination

• is concentration dependent

• inverse relationship between serum IgG concentrations and half life

  • shorter half life → greater IgG concentrations and vice versa

  • brambell proposed that this was due to the presence of salvage receptor for IgG

neonatal Gc receptor - FcRn

• identified as the protection receptor for IgG by 3 groups in 1996

• functions as a 65 kDA heterodimer

  • 15 kDa light chain - β-2-microglobulin

  • 50 kDa heavy chain - MHC1-like protein

• expressed throughout the body

• endogenous roles

  • IgG/albumin homeostasis

  • maternal transfer of IgG to fetus/neonate

how does FcRn protect IgG from catabolism?

1. proteins are internalized via fluid-phase pinocytosis (NO IgG-FcRn binding)

2. endosomal acidication causes protonation of key histidines in IgG and FcRn

3. protonation leads to favorable IgG-FcRn binding

4. IgG-FcRn complexes are recycled to the cell surface

   1. before this, non-receptor bound proteins are degraded in the lysosome

5. following exposures to extracellular pH, IgG-FcRn complexes dissociate

where does FcRn function?

main focus: protection of IgG from catabolism

endothelium

• NO FcRn-mediated recycling leads to:

  • reduced albumin and IgG serum levels

  • reduced albumin and IgG serum half-life

epithelium

• NO Fc-Rn-mediated epithelial transport leads to:

  • reduced amount of IgG present at mucosal surfaces

  • reduced transport of mucosal immune complexes to lamina propia

  • reduced transport of IgG from mother → fetus

  • reduced albumin serum levels

hematopoietic cells

• bone marrow cells

  • NO FcRn-dependent response to basal levels of IgG and IgC leads to:

    • reduced NK cell numbers and function

    • reduced tissue CD8T cell function

    • reduced TH1 polarization of tissue CD4*T cells

  • NO FcRn-mediated recycling

    • reduced albumin and IgG serum levels

    • reduced albumin and IgG serum half-life

• neutrophils (which come from bone marrow cells)

  • reduced FcRn-dependent phagocytosis of IgG leads to

    • reduced pathogen elimination

• APC (which comes from bone marrow cells)

  • NO FcRn-assisted antigen presentation leads to

    • reduced presentation of IgG associated antigens

    • reduced anti-tumor immune activation

    • reduced ability to acquire immunological tolerance during neonatal and fetal life

routes of admin - mAb therapeutics

• oral

  • bioavailability = negligible

  • t_max = N/A

  • barriers

    • low gastric pH

    • GI tract enzymes

    • Gi epithelium

• IV

  • bioavailability = 100%

  • t_max = immediately

  • barriers = N/A

• Subq and IM

  • bioavailability = 52-80%

  • t_max = 6-8 days

  • barriers

    • lymphatics

    • immune cells

• SC admin is very attractive for pharma development

  • absorption is largely via the lymphatics

  • resonable bioavailability

  • less pain compared to IM injections

  • more convenient to pts compared to IV

  • significant investment into absorption enhancing strategies

SC absorption of mAbs

for large molecule >20 kDa

• route to injection site = peripheral lymph → thoracic duct → subclavian vein

for small molecule <20 kDa

• route to injection site = systemic circulation to subclavian vein

PK following SC dosing

• relatively slow absorption → t_max ~1 week

• reasonable bioavailability (50-90%)

• often only see monoexponential PK

  • first phase may be masked by absorption

FcRn is relevant in SC absorption

• bioavailability of mAbs decrease at high dose levels

• genetic knockout of FcRn reduces bioavailability of mAbs to ~30%

IgG distribution

• pathway: diffusion

  • efficiency: negligible

  • barriers: cell membrane

• pathway: bulk fluid flow

  • efficiency: tissue-dependent

  • barriers

    • endothelial pores

    • interstitial pressure

• pathway: pinocytosis

  • efficiency: relatively low

  • barriers: endocytic rate

• pathway: receptor-mediated uptake

  • efficiency: target dependent

  • barriers

    • target expression

    • target accessibility

    • endocytic rate

    • binding affinity

• tissue concentrations « plasma concentrations

  • slow extravasation into tissues

  • relatively rapid drainage via lymphatics

• small Vss from NCA analysis

  • typically close to plasma volume

  • true Vss measurement requires tissue concentrations of mAbs

IgG elimination

• intracellular catabolism

  • following fluid-phase endocytosis (non-specific)

  • limited by interactions with FcRn

• target mediated elimination (specific)

  • cell surface receptor: internalization

  • soluble target: formation of large complexes - phagocytosis

• receptor-mediated elimination (Fcy receptors)

  • may trigger endocytosis and catabolism

• receptor-mediated protection: FcRn

  • level of plasma concentration of IgG

• immunogenicity

  • formation of anti-drug antibodies (ADA) resulting in accelerated clearance or loss of activity

renal clearance of IgG

• generally accepted molecular weight cutoff for glomeular filtration ~60 kDa

• in healthy individuals, <0.01% of IgG is expected to pass thru glomerulus

elimination following pinocytosis

• non-specific

• can occur in any cell

• results in catabolism → component amino acids

  • degradation products = non-toxic

• efficiency is blunted by FcRn recycling

  • requires extremely high dose of IgG to saturate

• at typical mAb doses, linear process

target-mediated elimination

• highly specific

• saturation level and rate depends on

  • target expression/accessibility

  • target turnover

• occurs following receptor binding/internalization

• typically in non-linear process

  • manifests as dose-dependent changes in CL/Vss

IgG clearance balances 2 saturable processes

• at low doses, any target mediated processes will dominate the profile

  • dose-dependent decreases in CL until saturation

    • at low doses = plenty of target receptors available so high CL → as dose increases, more IgG is in the system but but the # of target receptors is the same so CL goes down → once targets are saturated, CL does not change

    • target binding competes with FcRn → target binding prevents FcRn

  • target is usually not saturated

  • @ low doses, IgG binds to its target receptors → bound complexes are degraded → high clearance → as dose increases, target receptors become saturated, meaning not enough receptor to bind IgG so more of it remains in circulation so clearance decreases

    • target binding prevents FcRn

  • at linear PK the clearance is the same b/c target binding does NOT apply

    • target binding is saturated so NO degradation so only FcRn is active so its constant CL

• at extremely high doses, FcRn will be saturated

  • dose dependent increases in CL

  • greater clearance because all FcRn is saturated so it can’t protect IgG anymore so CL goes up

therapeutic mAbs - target types

• soluble

  • target is generally present in circulation

  • ex. vascular endothelial growth factor

• membrane, non-internalizable

  • target is expressed on cell surface but does NOT endocytose

  • ex. CD20

• membrane, internalizable

  • target is expressed on the cell surface but endocytoses (either naturally or stimulated by binding)

  • ex. epidermal growth factor receptor

• membrane, shaddable

  • target is expressed on the cell surface but may be released into extracellular space

  • ex. human epidermal growth factor recepetor 2

• target properties influence PK of mAbs

immunogenicity

• immunogenicity refers to the formation of antibodies against the therapeutic molecule

  • commonly referred as anti-drug antibodies (ADA)

  • recognition of the therapeutic as ‘foreign’ by immune system

• different types of ADA

  • clearing → adverse effects on PK

  • neutralization → inhibits binding to target/efficacy

  • non-clearing/non-neutralization → NO impact

factors associated with immunogenicity

• duration of therapy

  • incidence typically increases with treatment time

  • ex. infliximab

    • ADA rarely appears prior to 2 months

    • after 12 months, >90% incidence

• dose

  • ADA more frequently detected at lower doses of mAb

  • NB: may be due to assay interference

• route of admin

  • ADA often found more frequently with SC/IM dosing

• frequency/onset of ADA CANNOT be predicted

identification of non-linear PK

• key approach - dose-normalized PK

  • if profiles do NOT overlay = non-linear PK

  • in example, lower relative exposure at 1 mg/kg dose compared to 10 and 25 mg/kg

• other metrics - NCA derived parameters

  • check if CL and Vss have any trends with dose

  • in example, CL decreases and Vss increases with increasing dose

• for mAbs, non-linear PK is often attributable to target-mediated disposition

compartmental modeling of mAb PK

• 2 compartment model = most common base model structure for mAbs

  • clearance pathways are described using linear, non-linear or combo of terms

• with extravascular admin, model is sometimes compressed to 1-CM structrue

  • biphasic PK profile is NOT always apparent for SC dosing

target-mediated drug disposition (TMDD)

• mechanism: high affinity binding of a large fraction of the dose to its pharmacologic target will significantly impact PK

  • hypothesis made based on the increasing # of drugs in development with high affinity for targets

• general expectations

  • NCA-derived Vss and CL will decrease with increasing dose to a limiting value

  • greater than dose proportional changes in AUC at low doses

    • AUC = total drug exposure

    • @ low doses → most of the drug gets cleared b/c targets are NOT saturated (CL is high) but as dose increases the targets start to fill up and less of the drug is cleared via TMDD so more remains in circulation → hence, small increase in dose results in a larger than expected change in AUC

    • at higher doses this is not the case b/c targets are saturated so at that point nothing is getting cleared (CL rate stays the same) there is more of a difference at low doses b/c CL is a factor

mathematical modeling of TMDD

• accounts for:

  • kinetics of drug-receptor association and dissociation

  • receptor turnover

  • kinetics of drug-receptor complex elimination

• in many cases, the kinetics of binding occurs much more quickly than other PK processes

  • binding parameters might NOT be identifiable when fitting the model

  • published approximations that assume equilibrium binding

characteristics of TMDD for mAbs

• key features

  • non-linear PK

  • dose-dependent decreases in clearance

• typically observed for mAbs against cell surface targets

  • TMDD may occur with soluble targets, but less frequent observation

• TMDD is saturable in nature

  • dependent on target expression, accessibility, turnover

  • PK studies at a range of doses are required to characterize TMDD

application of TMDD models - TRX1

• TMDD model is able to well characterize

  • plasma TRX1 PK

  • free and total CD4 concentrations (PD)

• model-estimated affinity does NOT agree with in-vitro measurements

  • K_D (model) = 19.4 nM

  • K_D (measured) = 0.6 nM

• TMDD models are useful tools to characterize blood PK data

  • use caution with extrapolation of results beyond the range of data used for fitting

physiologically-based PK (PBPK)

• PBPK models allow:

  • integration of mechanistic determinants of mAb PK

  • description of both blood and tissue PK

  • relatively straightforward scaling between species

• PBPK challenges:

  • difficult model validation

  • complex system of equations

• gold standard for prediction of PK

  • generally more useful for prediction than fitting

PBPK as predictive tool

• PBPK model was used to make a priori predictions of the PK of an anti-CEA mAb

  • 2 tumor models (high/low target)

  • NO target expression

• WITHOUT fitting any parameters, the model was able to make relatively good predictions of both blood and tissue PK across a range of doses

• PBPK = excellent predictive tool

antibody-drug conjugates (ADC)

• ADCs combine the selectivity of mAbs with the potency of chemotherapeutic drugs

  • careful selection of target antigen is critical to avoid severe toxicities

• primary application: oncology

  • some investigational compounds in the area of infectious disease

• more than 10 FDA-approved ADCs

ADC PK

• ADC PK measurements require assays for several analytes

  • conjugated mAb (ADC)

  • free/total mAb

  • free/total drug

• drug to antibody ratio (DAR) affects PK/PD

  • increased DAR → faster clearance

    • more drug attached → more hydrophobic and unstable ADC may be which can increase uptake by liver or spleen

  • some degree of deconjugation can be expected with time

• understanding PK of all analytes = critical to proper characterization

bispecific antibodies (bsAb)

• bsAb have affinity for 2 distinct targets

  • several molecular formats can be produced

  • generally include fragments from 2 different mAbs

• examples of approved products

  • catumaxomab

    • maligant ascites in patients with epCAM-positive cancer

    • binds to epCAM and CD3

  • blinatumomab

    • philadelphia chromosome-negative relapsed ALL

    • binds to CD19 and CD3

  • emicizumab

    • hemophilia A

    • binds to factor IX and X

bsAb PK

• PK of bsAb is complicated by affinity for 2 targets

  • generally described using multiple TMDD models

  • target molecules are often moving throughout the body - have to consider target kinetics/cycling

  • few good examples of bsAb PK modeling

• bsAb = often eliminated very quickly

  • affinity for 2 targets

  • many are antibody fragments that CAN be renally filtered

  • blinatumomab = dosed as 28 day IV infusion to maintain efficacious concentrations

biosimilars

• FDA def: a biological product that is highly similar to and has no clinically meaningful differences from an existing FDA approved reference product

  • similarity is assessed by comparing purity, chemical identify and bioactivity

  • clinically meaningful differences are assessed by comparing PK, PD, immunogenicity

• biosimilars are interchangeable with reference products

  • may be substituted by pharmacist WITHOUT physician’s intervention

• biosimilar mAbs

  • more than 15 approved products

  • remicade - inflectra, renflexis, avsola

  • humira - amjevita, cyltezo, hyrimoz, hadlima, abiralada