Chap. 4 Monoclonal Antibodies in Therapeutic Applications

Paul Ehrlich: Idea of targeted therapy by using antibodiesHAMA response ---- recombinant chimeric and humanized, or human Ab for therapeutics

Mab for therapeutic applications

Cancer

[1] Cancer therapy with unmodified (naked) antibodiesHarness natural effector functions (tumor cell killing)

a) selection of suitable isotype i. mouse IgG2a --- human ADCCii. human IgG1 & IgG3 --- cell killing (IgG1: construction of humanized Ab)iii. CHO on CH2 --- important for eliciting the effector function

Ab as ligands eliciting anti-tumor effects by signal transduction--- arresting cell cycle or inducing programmed cell death

Ab to growth factor receptor or GF (EGF and IL-6)--- blocking GF-mediated tumor cell growth

Dimeric or polymeric Ab via X-linking--- complement activation and ADCC enhancement (homodimer – signalling activity for cell cycle arrest or apoptosis)

Not successful, but it’s promising. a) CAMPATH-1H (the first humanized CDR-grafted antibody)

--- complement activation against B-cell lymphomab) C2B8 against CD20 (B-cell surface phosphoprotein)

--- complement activation, ADCC, cell signalling mech. (B-cell lymphoma)

c) Ab against p185HER2 (tumor growth factor receptor, HER2/neu/c-erb-B2)i. growth inhibition by down-regulating the GF receptorii. signal transduction pathway leading to cell deathiii. ADCC

[2] Anti-idiotype antibodies

Vaccines to generate anti-idiotype responseAnti-idiotype antibody carries “internal image” of original Ag

[Ag – Ab1, Ab1– Ab2; Ab2(anti-idiotype antibody) – Ab3 (Ag recognition)]

Vaccination with Ab2 for poorly immunogenic target antigensa) Ab induction via breaking immunological toleranceb) induction of T-cell response (therapeutic significance)

i. 105AD7 (Ab2 mimicking a colorectal tumor-associated Ag)--- T-cell response on the turmor (delayed tumor growth

and increased survival period)ii. murine Mab-17-1A (epithelial memb. Ag on colorectal tumor cells)

--- anti-idiotype response in patients (high level of Ab3)

[3] Bispecific antibody-mediated effector cell targetingto recruit effector cells (Tc cells) to the tumor site

Mechanism of targeting effector cells to tumorsa) tumor recognizing antibody binding to Fc receptor on the effector cellb) bispecific antibody binding to markers on the effector and tumor cells

Activation of T-cell --- CD3/TcR complex in the presence of IL-2 & -7 plus co-stimulatorysignals of CD2 and CD28

cf) trispecific antibody made by crosslinking three Fab’ fragments(tumor Ag and two different T cell antigens)

Bispecific Ab via hybrid hybridomas, chemical crosslinking, recombinant DNA tech.(Bispecific diabody --- small size bringing the effector and tumor cells closer toge

ther)

[4] Other approaches to recruit the immune system using monoclonal antibodies

Superantigen (bacterial or viral proteins) --- [exotoxin from Staphylococcus aureus]a) most potent T-cell activator by recognizing both TcR and MHC-II on APCb) Fab’-superantigen fusion protein

i. tumor cell targeting with T-cell stimulation (replacing MHC-II binding tumor cell binding)

ii. T-cell infiltration and tumor attack increaseiii. residual MHC-II binding property (problem causing high toxicity)

mutations in the superantigen binding site for MHC-II

Antibody-cytokine fusion protein (chimeric 14.18 --- tumor Ab-IL2 fusion protein)a) cytokine alone --- severe systemic toxicityb) fusion protein --- targeting a high local conc. of cytokine to the tumor sitec) IL-2: T-cell proliferation and T-cell mediated killing TNF, TNF, GM-CSF, IL-5, IL-8, Interferon-

[5] Radioimmunotherapy

Tumor localization: 10% injected dose per Kg of tumorToxicity (especially to bone marrow)To reduce the toxicity

a) short-range radioisotopes (internalization for cytotoxicity)b) specific clearing mechanisms to remove circulating activityc) two-step targeting strategiesd) use of rapidly clearing antibody fragments

(1) Form of antibody

ideal molecule: (a) turmor localization in large amounts(b) delivery of high dose of radiation(c) rapid clearance from the circulation(d) minimizing non-specific toxicity

F(ab’)2 or chemically cross-linked F(ab’)2 fragments [tumor penetration, phamacokinetics,tumor accumulation]

cf) metallic isotopes --- intracellular accumulation or high levels in kidney

Chemically crosslinked tri-Fabs (TFM)high accumulation in tumor, rapid clearance from the blood (Fc-), low level in kidney

(2) Radioisotopes (Table 4.5)

isotopes with a high linear energy transfer (LET) [unlike the immunocytochemistry]a) Auger emitter – the highest dose of radiation, cellular internalization (short range)b) -emitter – high radiation dose over a short range (50 m in tissue)c) -emitter – less energy, but long range (1cm – favorable for large tumor)d) -emitter – no use for therapy, toxic to normal tissue

(3) Clearance mechanisms and two-step targeting

a) second Ab administration to remove hot Abb) extracorporeal immunoaffinity chromatography columnc) two step targeting protocols

i. bispecific antibody for both tumor and DOTA-90YFollowing removal of Ab, the isotope administration.

ii. streptavidin labeled Ab for tumor – cleared via biotin-protein conjugate -- labeling with biotin-DOTA-90Y

[6] Immunotoxins (Ab-toxin conjugates)

Protein toxins as potential cytotoxic agents for Ab targeted cancer therapy (protein synthesis inhibition via rRNA modification or EF-2 inactivation)

Requirements of immunotoxinsa) internalizationb) small numbers of toxin moleculesc) translocation (endosome or TGN)d) release of the toxin from Ab

following internalization

Subunits of toxin a) cell bindingb) internalizationc) toxic subunit

Conjugationvia chemical linkage or expression of recombinant fusions (toxin gene to Ab gene)a) must be intact in the circulation, but be able to release free toxin inside the cell

i. S-S bondii. hindered S-S bond with methyl or phenyl groupsiii. proteolytically sensitive peptide linker

Optimal form of antibodya) intact IgG --- efficient internalization, high level accumulation at the tumor,

but low tissue penetrationb) Ab fragments (scFv, dsFv) --- more effective tissue penetrationc) bispecific Ab --- tumor and toxin (saporin) [targeting, internalization, release]

reduced nonspecific binding

Clinical applicationsa) toxicity (hepatotoxicity, neurotoxicity, vascular leak syndrome)b) immunogenicity of protein toxins --- limits antitumor effectsto remove the immunogenicity (immunosuppressive agents or PEGylation)use human enzyme as a toxin (human ribonuclease) --- no immune reponse

poor cytotoxicity

[7] Drug conjugates

Chemotherapy (cytostatic or cytotoxic drugs) little selectivity for tumor cells --- normal tissue toxicity (severe side effects)

Target chemotherapeutic durgs via drug conjugates with antibodiesless immunogenic compared to immunotoxins (small size)reduced non-specific toxicity (well-characterized drugs)

Drug conjugation to Aba) loss of activities of both Ab and drugb) poorly active immunoconjugatesc) identify drugs of sufficient potencyd) suitable chemistry to generate linkage between Ab and drugs without loss of activity

(stable conjugates in circulation, release active drug during Ab degradation)e) many drug molecules per antibodyf) polymeric carriers for drugs such as HAS or dextran before a site-specific attachment

to antibodyg) immunoliposome to target a large amount of the drug to the tumor

PEG-modified phospholipid into the liposome --- reduced reticuloendothelial uptake

(stealth immunoliposome)h) development of more toxic cytotoxic agent

enediynes (calicheamicin), maytansinoids, tricothecenes, analogues of CC-1065

[8] Antibody-directed enzyme prodrug therapy (ADEPT)two step targeting method for cytotoxic drug therapy

Disadvantagesa) free drug may diffuse away from the tumor site --- affecting normal tissuesb) no human enzymes or no extracellular enzymes

to avoid activation of the prodrug in normal tissues (enzymes in Table 4.7)

Carboxypeptidase G2 (CPG2) a) converting Glu derivative prodrug into nitrogen mustard drug

b) three step systemantibody-enzyme conjugate --- clearance with galactosylated Ab --- Prodrug (anti-CEA F(ab’)2-CPG2)

c) use of small size conjugates such as scFv-enzyme fusionsd) immunogenicity of the enzyme in humans

i. humanized Abii. human enzymeiii. catalytic Ab (bispecific Ab with one for tumor Ag and

the other with catalytic activity)

[9] Vascular targeting

Shutting off tumor blood vessels --- tumor cell death by blocking effective blood supply

a) anti-vascular approach --- targeting existing blood vessels and killing tumor endothelial cells

i. Ab-toxin conjugateii. blood clot formation by targeting tissue factoriii. markers of tumor vasculature --- endosialin and endoglin

b) anti-angiogenic approach --- preventing the formation of the new blood vesselsi. antibody for bFGF and VEGF and their receptors

preventing tumor angiogenesisii. antibody for the integrin v3 (CD51/CD61)

cell adhesion control (crucial for angiogenesis)apoptosis of the proliferating cells

Infectious disease

[1] Antiviral antibodiesa) RSV (respiratory syncytial virus) --- peumonia and bronchitis in infants

neutralizing Mab against the surface Ab of F and G proteins of RSVi. long half-life of humanized IgGii. Anti-RSV Ab from phage display (overcome the dominance of non-neutralizing epitope)

b) HIVFab against gp120 (the CD4 binding site)

---- CDR walking (CDR mutations to improve the affinity to pM range)Anti-HIV antibody i. prevention of the gp120/CD4 binding ii. Fc mediated phagocytosis of the virus iii. anti-gp120 immunotoxin (anti-gp120-ricin A-chain conjugate)

[2] Bacterial Sepsisbacterial infection – endotoxin or LPS – inflammatory response – activation of hostdefense systems and release of inflammatory mediators (TNF and IL-1) –shock, organ failure, death

a) Mab to endotoxin of all Gram – bacteria (lipid A component of endotoxin) murine IgM,E5 and human IgM HA-1A

b) Mab to TNF --- inhibition of TNF (potential therapeutic for septic shock)Ab with human4 (inactive isotype)

Blood

Coagulation and Dissolution in dynamic equilibriumBlood coagulation following vascular injury --- Rapid and localized reaction (cascade)

Hemostasis (the cessation of bleeding)a) vasoconstrictionb) platelet aggregationc) fibrin clot formationd) fibrinolysis of the clot by plasmin

[Overview]

vascular injury platelet activation adhesion to the site of injury via von Willerbrand factor (collagen and gpIb/IX)

platelet aggregation via fibrinogen (platelet gpIIb/IIIa) and plug formation

fibrin clot formation through extrinsic pathway (negatively charged phospholipids)

growth and maintenance of the fibrin formation by intrinsic pathway until the tissue repair

Cardiovascular disease

[1] Inhibition of platelet aggregationMab (mouse/human chimeric Fab fragment 7E3) against gpIIb/IIIa receptor on platelets

blocking the interactions of fibrinogen and von Willerbrand factor --- no platelet aggregation

Chimeric 7E3 Fab (ReoPro) prevents complications following angioplasty.7E3 blocking --- reduced platelet crosslinking --- reduced platelet rich thrombus i. monovalent Fab, not bivalent IgG ii. chimeric Fab (CDR grafted humanized Fab) --- reduced immunogenicity iii. associated bleeding problem (heparin as anti-coagulant)

[2] Thrombosisthrombolytic therapy with plasminogen activator (acute myocardial infarction)

--- reocclusion and side-effects such as cerebral haemorrhagetargeting thrombolytic agents to blood clots

i. anti-Fibrin antibody and plasminogen activator (tPA or uPA) via chemical conjugation

ii. fusion proteins such as scFv-uPA toward fibrin (not fibrinogen)iii. bispecific Ab (fibrin and tPA) to target PA to the clotiv. bispecific conjugate (bispecific di-Fab)

anti-gpIIb/IIIa Fab’(7E3) and Fab’ recognizing tPA --- thrombolysis with anti-platelet effects

Disorders of the immune system and inflammatory diseases

[1] The inflammatory responseinflammation: the body’s protection reaction toward injury or the presence of foreign materials from infectioninflammatory response via pro-inflammatory cytokines and cell adhesion molecules

a) increased blood flow to the siteb) dilation of blood vesselsc) migration of lymphocytes across the vessel wall into the tissue

Autoimmune disease (chronic inflammation)--- inappropriate T-cell activation towards self-Ag

on APCAb to T-cell or APC – potential therapeutics

a) blocking T-cell activationb) cytokines (TNF)c) adhesion molecules (selectins and integrins)

the long lasting IgG with neutral isotypeblocking interactions in vasculature

without the effector functions

Parallel reagents: antibodies to the equivalent animal protein to generate information on potential efficacy

[2] Blocking inflammatory mediatorsa) Anti-TNF antibodyRheumatoid arthritis

i. increased TNF levelii. endothelial activation and adhesion molecule expressioniii. granulocyte activation (increased phagocytosis, degranulation, generation of

oxygen radicals and prostaglandin E2)iv. stimulation of fibroblast growthv. stimulation of cytokine productionvi. T-cell stimulation with IL-2

CA2 (mouse:human chimeric IgG1), CDP571 (engineered human IgG4)confirmation: reduction in pain, reduction in number of tender and swollen joints

IBDi. Crohn’s diseaseii. ulcerative colitis

b) Anti-C5 (activated component of the complement system as mediator of inflammation)Ab blocking C5i. no generation of the major chemotactic and pro-inflammatory factors

C5a & C5b-9 ii. amelioration of inflammatory diseases (systemic lupus erythematosus)

iii. anti-C5 scFv fragment : acute conditions (rapid penetration)myocardinal infarction/stroke/cardiopulmonary bypass

[3] Blocking adhesive interactionsbetween vascular endothelium and leukocytes

a) Ab to E-selectin on the endothelium without the Fc functionsanti-inflammatory effects leukocyte interaction with E-selectin for the adhesionIgG4 (neutral isotype)Leu235Ala in the CH2 domain – reduced Fc receptor binding

b) Ab to the leukocyte integrins and their receptors on the endothelium Ab to CD49d/CD29 (VLA-4)

i. effects on multiple sclerosis (autoimmune disease with demyelination of CNS)ii. activated T-cells crossing the blood-brain barrieriii. block binding to CD106 (VCAM-1) and inhibit leukocyte migration

[4] Antibodies which directly inhibit T-cell activation and proliferationa) T-cell receptor

i. –subunit of the CD3 complex: murine IgG2a (OKT3) potent immunosuppressive agentprevention of acute rejection

ii. T-cell activation by OKT3 via crosslinking between CD3 of T-cell and Fc-receptor bearing cells

iii. controlling immune response by OKT3humanized OKT3 / IgG4 (human) / Leu235Glu in CH2 domainreduced T-cell activation and human therapy

b) CD4 (TcR and MHC II interaction)i. anti-CD4 antibodies : down regulation of T-cell activity and

suppression of immune responsesii. non-depleting anti-CD4 antibodies : temporarily inhibit T-cell activation

with major inhibition of other immune functions ex) primatized Ab (monkey-human chimeric Ab)

c) IL-2 and its receptor interaction on Tc celli. Ab for -subunit (Tac receptor or CD25)

ADCC activationprolongs the survival of heart transplants

ii. Ab for -subunit (IL-15 receptor) with Ab for –subunit: synergistic effect leading to increased inhibition of

T-cell proliferationiii. Bispecific Ab for both and subunitsiv. Ab for (IL-4 and –7 receptor subunit)

d) Combining anti-CD3 and anti-IL-2 receptor chain (CD25)i. bispecific Ab for CD3 and CD25 : effective immunosuppressant

reduced toxicity by anti-CD3 Ab and inhibition of IL-2 interaction

e) Anti-CD25 antibodiesi. therapeutics for graft versus host disease (GvHD)

bone marrow transplantationmature T-cells from donor --- immune attack to the recipient’s tissuesT-cell depletion of the marrow before the transplantation

--- graft rejection 증가 , relapse of leukemia, delayed reconstitution of the immune system

anti-CD25(IL-2 receptor) Ab ---- loss of the IL-2 receptor on memory T-cells

f) CD5 (all T cells and a subset of B cells)i. anti-CD5 Ab : depleting T cells (RA and GvHD therapy)ii. anti-CD5 immunotoxins (ricin-A) : T-cell internalization and cell depletion in vivo

g) GvHDi. anti-CD25 antibodyii. anti-CD5 antibodyiii. anti-CD3 F(ab’)2-ricin A chain conjugate : effective T-cell depletioniv. recombinant immunotoxin : anti-CD3 scFv – truncated diptheria toxin

[5] Antibody treatment of allergyallergic reaction :

excessive immune response to a common normally harmless substanceatopic individual sensitization by 1st allergen exposure --- 2nd exposure --- hypersensitivity reaction

(high levels of circulating allergen specific IgE)

Allergen --- IgE production from B-cell ---

high affinity receptor binding on mast cells in tissueand bosophils in the circulation ---

second exposure --- allergen crosslinks IgE molecules

--- signal transduction and degranulation ---

inflammatory mediators such as histamine, cytokines

a) Mab to IgE i. IgE at Fc receptor binding siteii. block binding to basophils and mast cellsiii. down regulates Fc receptor on basophils

b) Ab for specific allergens i. allergen on mucosal surfaces (nasal linings or lower airways)ii. neutralization of IL-5

inhibiting eosinophil infiltration in the lung ---- prevent tissue damage and hyperreactivity

c) Ab to adhesion molecules (eosinophil migration into the lung)i. Ab to CD49d/CD29(VLA-4)

inhibiting eosinophil accumulation and asthmatic response