Immunology e Immunopathology!III year!!12/11/2015!!Antibodies: structure, functions, receptors and negative regulation. Monoclonal antibodies"!!!G. Palmieri!!!This material is for personal, educational, non-commercial use only!

Antibody structure !

membrane/secreted

Plasma cell!

The tertiary structure of an immunoglobulin domain!

CDRs show the maximal variability and are mostly responsible for epitope binding!

Antigen epitopes can be of different shapes!

Antibodies recognize different types of epitopes (antigenic determinants)!

Most antigens are multivalent!

Ag with different epitopes! Ag with repeated epitopes!

Affinity: The strength of the interaction between a single antigenic determinant and a single Ab combining site!

of the attractive and repulsive forces!

Antigen-antibody binding is a reversible reaction!

Ag + Ab Ag-Ab!

Keq = [Ag/Ab]

[Ag] x [Ab]!

....applying the Law of Mass Action:!

Antibody AFFINITY is expressed as: [antigen]-1 that saturates 50% of combinatory sites

Degeneracy of the antibody binding site !

The relative concentrations of multivalent antigen and antibody regulate the size (and biological properties!) of

immune complexes!

these are different!!

Three levels of antibody variability !

Antibodies can perform several effector functions!1!

2!

3!

4!

1. Antibodies can neutralize the infectious ability of pathogens

1. Neutralizing antibodies limit pathogen spreading

1. Antibodies can neutralize pathogen-derived toxins

The antibody couples the specific recognition of antigen to the activation of non-specific effector mechanisms!

binding! no binding!

Complement activation:! cytolysis! opsonization! inflammation!

ADCC! phagocytosis!

microbe 1! microbe 2!

2. Antibodies can enhance phagocytosis of pathogens (OPSONIZATION)

Antibody crosslinking is necessary for phagocyte activation

3. Antibodies can induce cytotoxicity by innate immunity cells (Antibody Dependent Cytotoxicity-ADCC)

Many innate cell populations are capable of ADCC!

Toxic proteins

4. Antibodies can induce the activation of COMPLEMENT

Antibodies can perform several effector functions!1!

2!

3!

4!

Five different Ig

classes!(ISOTYPES)!

The HINGE region!

IgM!

vPentameric (decavalent)!vMost abundant class in a primary response!vMost efficient activators of the complement cascade!

IgA!

v Dimeric (secretions), !!monomeric (serum)!

v Produced during memory !!responses (but also natural!)!

v The most abundant isotype in !body secretions (tears, saliva, !breast milk)!

A specific transport mechanism for secretory IgA !

| IgA and IgG antibody-secreting cells (ASCs) have distinctive trafficking patterns that depend on their site of induction. IgA ASCs mainly arise in mucosal lymphoid tissues in the gut or the upper aerodigestive tract and have trafficking patterns that are related to their site of induction (a). IgG ASCs mainly traffic to the bone marrow or inflammatory sites irrespective of their site of induction (b). Part of this specificity is due to the expression of tissue-specific integrin ligands on the endothelium in target tissues. Arrow thickness corresponds to the relative amount of IgA or IgG ASCs that traffic to each site. The colour of the organs corresponds to the expression of the alpha4beta7 integrin ligand mucosal addressin cell-adhesion molecule 1 (MADCAM1) (pink) or the alpha4beta1 (and alpha4beta7) integrin ligand vascular cell-adhesion molecule 1 (VCAM1) (red) that fundamentally subdivides intestinal and non-intestinal tissues (although the mammary gland and urogenital tract seem to express both MADCAM1 and VCAM1).!

Plasma cell (PC) recirculation!PC IgA! PC IgG!Upper aerodigestive

tract lymphoid tissue!

Upper aerodigestive tract lymphoid tissue!

Intestinal lymphoid tissues!

bone marrow!

Intestinal lymphoid tissues!

Secretory IgA in mucosal defense!

Production: >50 mg/kg body weight per 24h! Specific transport to mucosal secretions! Resistant to host proteases! Inhibit bacterial adhesion! Inhibit macromolecule absorption (including allergen binding

!to, or uptake by, mucosal target cells)! Inhibit inflammatory effects of other Ig isotypes! Neutralize viruses (both extracellularly and within epithelial

!cells) and bacterial toxins! Eliminate antigens in tissues through Poly Ig receptor- !

!mediated transport of immune complexes through epithelial !cells!

In breast milk, protect baby from pathogen attachment to gut !epithelium and infection!

Four different IgG subclasses!

Placental barrier crossing!Complement activation!Binding to Fc receptors!

+ +

++

- +/- +/-

+ ++ ++

+ - +

Figure 1 | FcRn mediates the perinatal transfer of igg. In rodents and humans, the neonatal Fc receptor for IgG (FcRn) binds to maternal IgG in an acidic environment, transcytoses it across a polarized epithelial-cell barrier and releases it at physiological pH. a | In rodents, FcRn is expressed on the cell-surface brush border of enterocytes. Shortly after birth, rodent pups ingest maternal milk containing IgG, which binds FcRn on the brush border in the acidic milieu of the duodenum. Upon binding, FcRn transcytoses IgG and releases it at neutral pH on the neonatal side. b | In contrast to rodents, the bulk of materno fetal IgG transfer in humans occurs antenatally across the syncytiotrophoblast of the placenta. Syncytiotrophoblasts are bathed in maternal blood and internalize serum containing maternal IgG. FcRn is expressed in the internal vesicles of the syncytiotrophoblast. On acidification in the endosome, FcRn binds to maternal IgG and transcytoses it to the fetal circulation where it is released a physiological pH.

FcRn mediates IgG transport at the materno-fetal barrier and into tissue interstitial space!

Figure 1 | FcRn mediates the perinatal transfer of igg. In rodents and humans, the neonatal Fc receptor for IgG (FcRn) binds to maternal IgG in an acidic environment, transcytoses it across a polarized epithelial-cell barrier and releases it at physiological pH. a | In rodents, FcRn is expressed on the cell-surface brush border of enterocytes. Shortly after birth, rodent pups ingest maternal milk containing IgG, which binds FcRn on the brush border in the acidic milieu of the duodenum. Upon binding, FcRn transcytoses IgG and releases it at neutral pH on the neonatal side. b | In contrast to rodents, the bulk of materno fetal IgG transfer in humans occurs antenatally across the syncytiotrophoblast of the placenta. Syncytiotrophoblasts are bathed in maternal blood and internalize serum containing maternal IgG. FcRn is expressed in the internal vesicles of the syncytiotrophoblast. On acidification in the endosome, FcRn binds to maternal IgG and transcytoses it to the fetal circulation where it is released a physiological pH.

FcRn-dependent recycling of IgG extends their serum half-life and mediates their transport in the tissue interstitial space

FcRn-mediated transport of IgG at the materno-fetal interface

IgE!

vActivate mastocyte degranulation (histamine release)!

vActivate eosinophil cytotoxicity!vCharacteristic of the response against

parasites!vResponsible for allergic diseases!

Avidity: The overall binding strength of the Antibody molecule for a multivalent Antigen!

Affinity = Avidity!

Distribution of antibody classes

Antibody titers in serum: IgG, IgA, and IgM levels slowly increase until young adulthood

and remain stable thereafter!

Protective effect of maternal antibodies in serum and milk!

(ADCC)!

The different Ig isotypes perform different effector functions!

The production of Ig isotypes is controlled by TH-derived cytokines!

Several cell receptors for Fc region of antibodies!

M!PMN!Eo!

Mast cells!Basophils!

High affinity (1x10-10M)!

A family of Fc receptors!

M, PMN, DC, Eo!Phagocytosis, ADCC!

NK,M, Eo!ADCC!

B, M, DC,!mast cells!

inhibition!

HIGH!Affinity!10-9M! LOW to medium affinity (10

-5-10-6M)!

PMN!

Two major mechanisms for the negative regulation of antibody responses!

blocking antibodies! inhibitory receptor!

mIg!

Ag!

mIg!

sIg!

sIgG!Ag!

FcRIIb!SHIP!

Phosphorylated ITIM sequences in the intracellular region of FcRIIb recruit and activate the lipid phosphatase SHIP1 (SH2-containing inositol-5-phosphatase), that degrades PIP3 (phosphatidyl-3,4,5 trisphosphate)!

FcRIIB inhibitory receptor controls the expansion of!

B cells and their differentiation into plasma

cells!

FcRIIb blocks BCR-initiated Ca++ influx!

PI3K!Btk!

PLC

syk

FcRIIB inhibitory receptor is important for the maintenance of B cell tolerance too!

Costimulatory and inhibitory receptors that regulate B cell activation and antibody production!

Schematic representation of an activation/inhibitory Fc receptor pair!

b FcRI,

Figure 1 Activating and inhibitory Fc receptors set a threshold for immune effector cell activation. (a) Immune complex (IC) binding to activating FcRs results in the ITAM phosphorylation of the receptor-associated c-chain, creating docking sites for Syk kinases. Syk activates several downstream kinases such as the phosphatidylinositol-3 kinase (PI3K), leading to the recruitment of Brutons tyrosine kinase (BTK) and phospholipase C (PLC) to the plasma membrane, and ultimately leading to calcium influx into the cytosol from intracellular and extracellular sources. (b) The inhibitory FcRIIB interferes with these activating signaling pathways by recruiting phosphatases such as SHIP (SH2-containing inositide phosphatase) that hydrolyze phosphatidylinositol signaling intermediates necessary for recruitment of BTK and PLC, thus limiting cell activation. [Reprinted from Springer Seminars in Immunopathology (2006), 28:30519, Copyright 2006 with kind permission of Springer Science and Business Media.]

Activating and inhibitory Fc receptors set a threshold for immune effector cell activation by immune complexes!

cell activation! inhibition of activation!

FcRI or FcRIII! Ca

2+!

FcRI or!FcRIII! FcRIIb!

M, DC, PMN!

Expression of activating and inhibitory Fc receptors! Functional polymorphisms of Fc receptors! Differential binding ability of IgG subclasses! Post-translational modifications of IgG!

Which factors modulate the pro-inflammatory vs anti-inflammatory function of IgG-containing immune complexes?!

Figure 2 | The functions of FcRIIB. a | Fc receptor IIB for IgG (FcRIIB) has an important role in controlling humoral immunity by regulating B cell activation, localization of B cells in the germinal centres, as well as plasma cell survival. FcRIIB regulates B cell activation by increasing the B cell receptor (BCR) activation threshold and suppressing B cell-mediated antigen presentation to T cells. Follicular dendritic cells (FDCs) express FcRIIB, which is thought to be important for trapping immune-complexed antigen for presentation to germinal centre B cells. The absence of FcRIIB on germinal centre FDCs results in impaired antibody and memory responses. Terminally differentiated plasma cells express little or no BCR but express high levels of FcRIIB, and cross-linking FcRIIB with immune complexes in vitro can induce apoptosis. b | FcRIIB influences antigen presentation by inhibiting FcR-dependent internalization of immune-complexed antigen by DCs, as well antigen presentation to both CD4+ and CD8+ T cells (cross-presentation). FcRIIB is also thought to provide a basal level of inhibition to DC maturation, as blockade of immune complex binding to FcRIIB results in DC maturation and type I interferon production. There is also a possibility that FcRIIB may deliver intact antigen to a non-degradative compartment, allowing its recycling to the cell surface where it could interact with the BCR and activate B cells. c | FcRIIB can also influence innate immunity: in macrophages, FcRIIB cross-linking inhibits FcR-mediated phagocytosis and cytokine release (including tumour necrosis factor, interleukin-6 (IL-6) and IL-1), as well as Toll-like receptor 4 (TLR4)-mediated activation. In neutrophils, cross-linking of activating FcRs results in phagocytosis, superoxide production and enhanced neutrophil adhesion, rolling and migration, all of which are probably inhibited by ligating FcRIIB. d | FcRIIB also inhibits IgE-induced mast cell and basophil degranulation, thus contributing to hypersensitivity responses. FcR, Fc recpetor for IgE; SCF, stem cell factor.

The functions of FcRIIB!

Figure 2. Biologic Activity of IgGFc Interacting Partners. IgGFc binds to a variety of proteins that can initiate both proinflammatory pathways (e.g., C1q and activating FcRs) and antiinflammatory pathways (e.g., inhibitory FcRIIB and SIGN-R1). These pathways, at least in part, require the presence of terminal sialic acid residues on Fc (-2,6-sialylated Fc). The neonatal Fc receptor (FcRn) interacts with a distal site on Fc, independent of the sugar side chain. SIGN-R1 denotes surface receptorspecific intercellular adhesion molecule 3grabbing nonintegrin-related 1.

Biologic activity of IgGFc interacting partners!

Figure 2. Multifunctional attributes of B cells. Selected examples of how B cells regulate immune homeostasis are shown; many of these functions are independent of Ig production. Illustration by A. Y. Chen.

B cells: more than antibody producers!!

Selected examples of how B cells regulate immune homeostasis;! many of these functions are independent of Ig production!

Monoclonal antibodies (Mab)!Antibodies produced by one single plasma cell, after immortalization by physical fusion with a myeloma cell (plasma cell tumor):!!!

HYBRIDOMA!!!

! ! ! ! !specificity!!Mab are HOMOGENEOUS !affinity!!

! ! ! ! !isotype!!Produced in unlimited amounts, at a relatively low cost!

Characteristics of the myeloma cell partner!

1. Immortal!

2. Does not produce Ig (mutant)!

3. Lacks HGPRT (hypoxanthine guanine phosphoribosyltransferase), thus:!

4. Cannot grow in HAT selective medium (Hypoxanthine, Aminopterin, Thymidine)!

Pathways for nucleotide biosynthesis!

The hybridoma technique for monoclonal antibody production (1)!

polyethylen glycol!

The hybridoma technique for monoclonal antibody production (2)!

Applications of monoclonal antibodies!In vitro!!Identification and quantitation of an enormous range of molecules in almost any biological sample!!Identification of cell subsets (lineage, differentiation step, activation state, functional capability) through the analysis of specific markers!

!!

IMMUNODIAGNOSIS!!In vivo!!Diagnosis, imaging!!Immunotherapy:!tumors, transplantation, autoimmune diseases, infectious diseases !

The trinity of therapeutic antibody development. The development of therapeutic antibodies is crucially dependent on three key factors: the target, the antibody, and the patient that during drug design and development should not only be considered individually, but also as an integrated whole. Each should be validated for suitability for the intended immunotherapeutic application. In general terms, the target should be drugable and play an important role in pathogenesis. The therapeutic antibody should have high specificity and affinity for the target and should be able to modulate target function. For the antibody Fc fragment there is the choice for an activating Fc-tail, which can recruit antibody-mediated immune effector function or, for other applications, a non-activating Fc-tail may be required. The ability to manufacture a well-behaved and consistent antibody product is essential and should be validated. It is essential to perform clinical trials in a suitable patient population for which there is an unmet medical need. The role of the target in disease pathogenesis should be validated. Genetic polymorphisms between patients and specific mutations in diseased tissues may impact therapy and should be investigated. Finally, immunogenicity that is dependent on antibody sequence but strongly modulated by patient, disease, and treatment factors may impact long-term therapy. An integrated science-based approach in which knowledge of target and antibody biology, as well as insights in disease pathology and patient heterogeneity are taken into account maximizes the chances for the successful development of novel antibody drugs.

The development of therapeutic antibodies is crucially dependent on three key factors:!

Antitumor mechanisms of action of therapeutic antibodies!

Potential targets for antibody therapy of cancer!

a) tumor-associated blood vessel

b) Vascular growth factors (i.e. VEGF)

c) Diffuse malignant cells

d) Tumor cells in a solid tumor

e) Tumor-associated stroma

Mechanisms of action of therapeutic antibodies!

Figure 2 | mechanisms of action of therapeutic antibodies. Five non-overlapping mechanisms of action are depicted. Examples of therapeutic antibodies are listed for each mechanism of action depicted. Ligand blockade with full length IgG therapeutic antibodies (for example, infliximab, adalimumab or golimumab), antibody fragments (for example, certolizumab pegol) or receptor immunoadhesins (for example, etanercept and those indicated with ) can prevent ligands from activating their cognate receptors. Binding of ligands (for example, interleukin-6 (IL-6)) to receptors (for example, IL-6R) can also be blocked by antibodies directed to their cognate receptors and inhibit receptor activation or function. Binding of cell surface receptors by antibodies can also result in their internalization and downregulation to limit cell surface receptors that can be activated by the ligand. Note that binding of cell surface receptors by antibodies (for example, L integrin by efalizumab) or binding of a ligand (for example, free serum IgE by omalizumab) can indirectly also result in downregulation of cell surface receptors available for cellular activation. Binding of cell surface receptors can result in depletion of antigen-bearing cells through complement-mediated lysis and opsonization, as well as Fc receptor for IgG (FcR)-mediated clearance. Therapeutic antibodies can also induce active signals that alter cellular fates. Binding of the T cell receptor (TCR)CD3 complex by teplizumab can induce TCR-mediated signals and alter T cell functions and differentiation. MAC, membrane attack complex; TNF, tumour necrosis factor; TNFRI; TNF receptor I. *Antibodies with several mechanisms of action.

Problems for the in vivo use of Mab!

1. !HAMA !human anti-mouse antibodies!!2. Murine mAb do not efficiently activate some effector

functions (human complement, binding to human Fc receptors)!

3. !Murine Ab have a short half-life (impaired binding to FcRn on endothelial cells, responsible for IgG recycling)!

Monoclonal antibodies can be genetically manipulated!!

Three types of monoclonal antibodies now in clinical use!

Muronomab!Anti-CD3!murine!

Infliximab!Anti-TNF!chimeric!

Rituximab!Anti-CD20!humanized!

Figure 1. Humanization of therapeutic antibodies illustrated with the anti-TNF agents used in clinical practice. Original mouse IgG1 monoclonal antibodies (MABS) contain mouse sequences only in every part of the variable (V) and constant (C) regions of both light (L) and heavy (H) chains. The 2 heavy chains are connected by the glycosylated hinge region, which separates the antibody binding Fab region from the effector, or complement binding, Fc portion. Mouse mAb are by convention called MOMABS, and induce anti-mouse antibodies in most if not all patients. Chimeric mAb (ximabs), such as infliximab, are only murine in the variable regions (VH VL) and the antidrug antibodies they induce are called human antichimeric antibodies (HACA). Humanized antibodies (ZUMABS) contain mouse sequences in only the complementarity-determining regions (CDR) and contain 95% human peptide sequences. Certolizumab-pegol is a humanized Fab fragment linked to 2 polyethylene glycol (PEG) molecules, which improve pharmacokinetics. Removal of all murine peptide sequences results in fully human antibodies (mumabs) such as adalimumab. They can induce human anti-human antibodies (HAHA), but these mAbs have the lowest potential for antigenicity. Etanercept is a fully human TNF receptor/Fc fragment fusion protein containing 2 receptor units and 1 Fc fragment.

Humanization of therapeutic antibodies illustrated with the anti-TNF agents used in clinical practice!

HAMA: human anti-mouse antibodies!HAHA: human anti-human antibodies!

Figure 2 | Antibody design to improve the pharmacological functions. A better knowledge of the structurefunction relationships of antibody molecules allows fine-tuning of their associated pharmacological properties. the variable domain, which is associated with antigen binding (Fab moiety), can be tailored to modulate binding affinity and specificity using well-described phage display techniques. Fab fragments can be used as a monovalent non-activating format with a long half-life (conjugated to polyethylene glycol (PeGylated)) or with a short half-life (naked). Depending on its origin, humanization or de-immunization (that is, the substitution of key amino acids predicted to abrogate binding to human MHc class II molecules in order to reduce a t cell immune response) techniques can greatly decrease the potential immunogenicity of an antibody. With regard to the antibody Fc portion, better knowledge of the Fc receptors present on immune cells allows the tailored engagement of associated effector functions (such as antibody-dependent cellular cytotoxicity (ADcc), complement-dependent cytotoxicity or phagocytosis) by modulation of the binding affinities to these Fc receptors through mutations and/or glyco-engineering. the antibody Fc domain is also the major binding region to develop immunoconjugates, by association with a radioactive label, cytotoxic drug or protein. cDr, complementarity-determining region; cH, heavy chain constant domain; cL, light chain constant domain; Fcr, Fc receptor for IgG; Fcrn, neonatal Fc receptor; vH, heavy chain variable domain; vL, light chain variable domain.

Antibody design to improve the pharmacological functions!

BISPECIFIC antibodies (BsAb)!Derive from the somatic fusion of two hybridomas!

QUADROMA!! Two different antigen specificities in a single molecule! BsAb re-directs effector cell function against

!therapeutical targets! BsAb-cytokine conjugate: functional enhancement of

!effector cells!!!T ! ! !tumor cell killing!NK! ! !tumor or infected cell killing!PMN ! !tumor or pathogen cell killing!Monocyte/ !tumor or pathogen cell killing,!

! ! !Ag presentation!Dendritic cell !Ag presentation!

E

T!

Figure 1. The BiTE antibody principle. Generation of a BiTE antibody from the variable domains of two distinct monoclonal antibodies is depicted on the left. The anti-CD3 specific single-chain antibody (green) is shared by all BiTE antibodies. The target antigen-specific single-chain antibody (red) is different for each BiTE antibody, and can recognize for instance CD19 (blinatumomab; MT103) or EpCAM (MT110). As shown on the right, a BiTE antibody can transiently connect a T cell and a cancer cell by simultaneously binding CD3 and a target antigen. This will trigger T-cell activation involving cytotoxic granule fusion, transient cytokine release, and proliferation. Redirected lysis of the attached cancer cell involves membrane perforation by perforin, and subsequent programmed cell death as induced by granzymes. Of note, BiTE-activated T cells recognize the same surface antigens as regular monoclonal antibodies.

The BiTE antibody principle!

Cloned transgenic farm animals produce a bispecific antibody for T cell-mediated tumor cell killing !

Cloned, transgenic cattle producing up to 0.1g/liter of a recombinant bispecific scFV antibody. The bispecific protein, designated r28M, is directed to the CD28 molecule on human T lymphocytes and to a melanoma-associated proteoglycan. Purified from serum the bi-scFV molecule is fully active in stimulation of human T cell proliferation and drives T cell-mediated killing of melanoma cells. PNAS, 4/5/2004!

Anti-CD28! Anti-melanoma!VH! VH!

VL!VL!scFv!

XENOMOUSE!!

It is possible to create antibodies with a desired specificity, affinity, etc.!!

in vitro generation of antibodies by the use of phage libraries!