antibody-based therapy
`
`Immunotoxins: magic bullets
`or misguided missiles?
`
`Ellen S. Vitetta, Philip E. Thorpe and
`Jonathan W. Uhr
`
`Thirteen years have passed since specific in vitro and in vivo killing of
`tumor cells by immunotoxins was first described. Why, then, has it taken
`so long to determine whether these pharmaceuticals will have a major
`impact on the treatment of cancer, AIDS and autoimmune disease? The
`answer is that the transfer of basic discoveries to the clinic is a slow,
`multistep, interdisciplinary process. Thus, immunotoxin molecules must
`be designed and redesigned by the basic scientist depending on the efficacy
`and toxicity shown in vitro and in relevant experimental models. Next,
`each version must be evaluated by clinicians in humans through a lengthy
`process (1—3 years) in which the dose regimen is optimized and in which
`new problems and issues frequently emerge. These problems must again
`be modeled and studied in animals before additional clinical trials are
`initiated. In this article, Ellen Vitetta and colleagues discuss both basic
`and clinical aspects of the development of immunotoxin therapy.
`
`mAbs can be used as intact molecules or as frag-
`ments‘. While fragments are less immunogenic,
`they
`have a shorter half-life in vivo and are often partially
`inactivated by their coupling to toxins. These problems
`should be circumvented by generating fusion proteins
`containing portions of the constant
`regions of the
`heavy chain, which confer a long half—life
`in the
`circulation.
`Growth factors Other ligands for preparing immuno-
`toxins are growth factors”. Although these bind to
`normal cells, tumor cells frequently express elevated
`levels of growth factor receptors. Advantages of using
`growth factors as ligands include their relative lack of
`immunogenicity, high affinity for their receptors, and
`the availability of cloned genes for generating fusion
`proteins. Problems include rapid in vivo clearance,
`stimulation of target cells by small amounts of bound
`immunotoxin insufficient
`to kill
`the cells, and the
`presence of circulating ligands or soluble receptors that
`compete for the immunotoxin.
`
`Immunotoxins are chimeric molecules in which cell-
`binding ligands are coupled to toxins or their subunits.
`If the ligand moiety is tumor cell-specific, the immuno-
`toxin should kill tumor cells selectively, unlike conven-
`tional chemotherapy and radiotherapy, which kill
`rapidly dividing or metabolizing cells, whether malig—
`nant or normal.
`
`Components of an immunotoxin
`The toxins used for different types of immunotoxins
`are depicted in Table 1, and their components, ligand,
`toxin and crosslinker, are discussed below'.
`
`Ligand
`Monoclonal antibodies The ligand most frequently used
`is a cell-reactive monoclonal antibody ’mAb)‘. Although
`tumor—reactive mAbs often react with some normal tis-
`sue, crossreactivity does not necessarily prohibit their
`use. Thus, low antigen density, anatomical barriers or
`poor endocytcsis could prevent the killing of a cell that
`has a crossreacting antigen”. Conversely, some cross—
`reactions not detectable by conventional
`techniques
`can damage life-sustaining tissues]. Hence, a primate
`model in which the mAb reacts with the primate anti-
`gen is desirable to test the safety of an immunotoxin to
`be used in humans.
`Only a proportion of mAbs make potent immuno-
`toxins‘. Depending on their specificity, they may not
`be internalized or, if they are, they may not be routed
`to the appropriate intracellular compartment
`for
`translocation of their attached toxin into the cytosol.
`Hence, mAbs must also be screened for effectiveness as
`carriers of toxin.
`
`Toxin
`The toxins used for immunotoxins are derived from
`bacteria and plants and all inhibit protein synthesis (as
`described below; and see Table 1). Unlike chemothera-
`peutic agents, these toxins kill both resting and dividing
`cells. Hence, as immunotoxins, they have the potential
`to kill tumor cells that are not in cycle at the time of
`treatment (dormant
`tumor cells) and that may be
`spared by conventional chemotherapy. These toxins
`share common features7:
`1 They are all synthesized as single chain proteins and
`are processed either post translationally or in the
`0 I993. Elsevier Science Publishers Ltd, UK. 0l67-569v/9JISDG.00
`
`Immunology Today 252 Vol. 14 No. 6 1993
`
`Immunogen 2144, pg. 1
`Phigenix v. Immunogen
`|PR2014-00676
`
`Immunogen 2144, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`antibody-based therapy
`
`Table 1. Structure and function of toxins and RIPs used for immunotoxins
`
`
`
`LD,0 of immunotoxins
`(mice, mg kg"
`Structure of mature form
`Toxin
`A-chain action
`Toxin receptor
`total protein)
`
`Diphtheria toxin
`(DT)
`
`o—ss—o
`
`Truncated
`diphtheria toxin
`(DAB486)
`
`Pseudomonas
`exotoxin (PE)
`
`Truncated
`Pseudomonas
`
`exotoxin (PE40)
`
`Ricin/abrin
`
`Blocked
`ricin/abrin
`
`Ricin toxin
`A-chain (RTA)
`
`Ribosome
`inactivating
`protein (RIP)
`
`3;}.
`:3
`
`.
`‘3)
`‘
`
`
`
`3”
`
`heparin-binding
`epidermal growth
`factor-like precursor
`
`none
`
`ADP-ribosylation of
`elongation factor 2
`
`ADP-ribosylation of
`elongation factor 2
`
`uz—macroglobulin
`receptor-like molecule
`
`ADP-ribosylation of
`elongation factor 2
`
`none
`
`galactose
`
`none
`
`none
`
`ADP—ribosylation of
`elongation factor 2
`
`N—glycosidase for
`285 ribosomal RNA
`
`N—glycosidase for
`285 ribosomal RNA
`
`N-glycosidase for
`285 ribosomal RNA
`
`0.3
`
`>1.0
`
`0.1
`
`2.0
`
`0.1—0.2
`
`0.4-0.8
`
`20
`
`5—20
`
`N~glycosidase for
`285 ribosomal RNA
`
`
`none
`
`A, B: different polypeptide chains; I: hydrophobic region in the polypeptide; indentations: cell-binding sites; X: partial or complete
`blockade of Iectin activity at the binding site.
`
`target cell to which they are delivered into two-chain
`molecules with interchain disulfide bonds.
`2 The disulfide bond linking the two chains is critical
`for cytotoxicity.
`3 All
`toxins have subunits or domains devoted to
`binding to cells,
`translocation across membranes,
`and the destruction of protein synthesis in the cell.
`These domains can be separated or genetically
`manipulated to delete those that are unwanted.
`Plant toxins The most widely used plant toxins, ricin
`and abrin, consist of two disulfide-linked polypeptides,
`A and B (Ref. 8). The toxin binds via the B-chain
`to galactose—containing glycoproteins and glycolipids
`that are present on the surface of all cell types. The
`toxin is then endocytosed and routed to the trans-
`Golgi network which is believed to be the site where
`the A-chain translocates to the cytosol. The A-chain
`then kills the cell by enzymatically removing a crucial
`adenine
`residue
`from the 605 ribosomal
`subunit
`which is needed for the binding of elongation factor 2
`(EF-Z) during protein synthesis”. Ribosome inacti-
`vating proteins (RIPS) are single—chain proteins found
`in many plants”, and have the same enzymatic proper—
`ties as the A-chain of ricin”.
`Bacterial toxins The active form of diphtheria toxin
`
`(DT) is a disulfide-bonded two-chain molecule”. The
`toxin binds via the B-chain to an epidermal growth
`factor-like receptor that is present on most cell types
`in DT-sensitive species”. The toxin is then endocy-
`tosed and, within an acidic intracellular compartment,
`the B-chain undergoes a conformational change to
`expose hydrophobic regions, which are thought
`to
`be important in enabling the A-chain to translocate
`across the membrane to the cytosol“. The A-chain
`then kills the cell by catalysing a modification of
`EF-Z that prevents
`its
`participation
`in
`protein
`synthesis”.
`is produced by the
`Pseudomonas exotoxin (PE)
`bacterium as a single-chain protein“. It binds via its N-
`terminal
`region (domain I)
`to an az-macroglobulin
`receptor-like molecule present on the surface of most
`cell
`types”. The toxin is
`then endocytosed and
`becomes converted through the action of proteolytic
`enzymes into a disulfide-bonded two—chain form”.
`The C-terminus of domain Ill (the equivalent of the
`A-chain) has an endoplasmic reticulum retention se—
`quence, REDLK, which causes the toxin to concentrate
`in the endoplasmic reticulum — probably the site where
`domain III enters the cytosol. Once in the cytosol, the
`toxin kills the cell in the same manner as DT.
`
`Immunology Today
`
`253 Vol. 14 No.61993
`
`Immunogen 2144, pg. 2
`Phigenix v. Immunogen
`|PR2014-00676
`
`Immunogen 2144, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`antibody-based therapy
`
`.
`Crosslinker
`The crosslinker used to join the ligand and the toxm
`must remain stable extracellularly but be labile intra-
`cellularly so that
`the toxic fragment can enter the
`cytosol. The choice of crosslinker depends on whether
`intact toxins, A-chains or RIPs are used. A-chains and
`RIPS are coupled to ligands using linkers that intro-
`duce a disulfide bond between the ligand and the
`A-chain'l". Bonds that cannot be reduced render these
`immunotoxins much less toxic or nontoxic probably
`because the A—chain must be released from the ligand
`by reduction to be cytotoxic”. Such immunotoxins
`tend to be labile in UiI/O unless hindered crosslinkers
`are used. These crosslinkers place bulky groups around
`the disulfide bond to protect it from attack by thiols
`in the blood and tissues'. Intact
`toxins are usually
`linked to ligands using nonreducible (cg.
`thioether)
`linkages to prevent release of active free toxin in UIl/O.
`Recombinant
`immunotoxins have been prepared by
`splicing the genes
`encoding truncated DT (c.g.
`DAB486) or Pseudcmonas exotoxin (e.g. PE40) to the
`gene encoding the ligand and expressing the entire
`immunotoxin as a
`fusion protein”. Recombinant
`immunotoxins are highly stable in VII/O because they
`contain nonreducible peptide bonds.
`
`Preclinical evaluation
`Cytotoxic potency and specificity
`Immunotoxins prepared from holotoxins (i.e. com-
`plete toxins containing both A- and B-chains or anal—
`ogous domains) are usually more potent than those con—
`raining A-chains (or RIPS) because the toxin moiety
`can interact with toxin receptors in or on the target
`cellu'z‘. This enables the immunotoxin to enter and kill
`the cell by the highly efficient entry pathway used
`by the native toxin. Predictably, however, holotoxin—
`containing immunotoxins are highly toxic to animals
`because they can bind to toxin receptors that are pres-
`ent on non-target cells. The problem of nonspecific tox-
`icity can be reduced with ricin-containing immunotox-
`ins by reversibly blocking the galactose-binding sites of
`the toxin either Sterically by the ligand itself or with
`galactose-based affinity labels'iz‘. Such ‘blocked’ ricin
`immunotoxins appear to act by being degraded inside
`the cell to release unblocked ricin or ricin fragments.
`Immunotoxins prepared from ricin A-chain or
`RIPS are highly specific for their designated target cells
`but vary in potency depending on the affinity of the
`ligand, the cell surface molecule and epitope that it
`recognizes and the capacity of that molecule to enter
`an intracellular compartment
`that
`is
`favorable for
`toxin translocation'l‘”. Immunotoxins prepared from
`DAB486 or PE40, which lack cell-binding domains,
`are also highly specific in their cytotoxic action on
`target cellsu'”.
`It
`is unclear whether immunotoxins
`containing DAB486 or PE40 are more uniformly
`cytotoxic than their A-chain counterparts, as might be
`expected if the truncated toxins have, for example,
`hydrophobic regions that assist the ent: y of the enzy-
`matic fragment or subunit into the cytosol. Thus far,
`the evidence with PE40—containing immunotoxins is
`that they have variability in potency similar to that of
`A-chain immunotoxins.
`
`Immunotoxins prepared with A-chains can often be
`made more potent by lysosomotropic amines and
`carboxylic ionophores, which inhibit
`the fusion of
`endosomes with lysosomes (where the A-chains are
`destroyed) or retard the transit of the immunotoxins
`through
`a
`compartment
`favorable
`for A—chain
`translocationn'”.
`
`Toxicity
`Many immunotoxins can cause hepatotoxicity‘
`(Tables 1 and 2). In the case of ricin A-chain (RTA),
`mannose- and fucose-containing oligosaccharides bind
`to liver cells leading to rapid clearance and hepatic
`damage'. This problem has been successfully circum-
`vented either by deglycosylating RTA (chemically or
`enzymatically)‘ or using recombinant RTA (expressed
`in a non-glycosylating cell)". In the case of blocked
`ricin immunotoxins,
`the oligosaccharides on the A-
`and B-chains and the affinity labels used to block the
`B-chain’s lectin sites result in liver homing and liver
`damage“. Bacterial toxins and RIPs produce hepato-
`toxicity by binding to molecules other than carbohy-
`drate receptors on liver cells or by binding to serum
`proteins that have receptors in the liverl'”.
`RTA-based immunotoxins cause vascular leak in
`humans, which is manifested by extravasation of fluids
`and proteins from the vasculature into the periphery
`causing edema and weight gain, and, occasionally, life-
`threatening pulmonary edema“. The mechanisms
`underlying vascular
`leak are not known, although
`recent evidence suggests that they may be related to the
`binding of the RTA to vascular endothelial cells”. In
`addition, these immunotoxins cause myalgias (rarely,
`rhabdomyolysis) via unknown mechanisms.
`
`Pharmacokinetics
`An effective immunotoxin must have a serum half-
`life of sufficient duration for a cytotoxic quantity of it
`to access the target cells. When the target cells are
`intravascular (for example, circulating tumor cells or
`normal lymphocytes), access is not a problem and the
`immunotoxins are highly effective, but when the target
`cells reside in large solid tumor masses with a poor
`blood supply and high interstitial pressure“, the need
`for a long serum half-life becomes critical. The half-life
`of immunotoxins prepared with mAbs is longest when
`the mAbs are intact, the crosslinker is stable and the
`toxin moiety does not bind to normal
`tissues.
`In
`contrast, when the ligand is an antibody fragment or
`growth factor,
`the crosslinker is not stable, or the
`toxin displays some nonspecific binding, the half—life is
`short”. The problem of a rapid half-life can be partially
`solved by continuous intravenous infusion of
`the
`immunotoxin, although increasing the half-life may
`also increase the likelihood that these immunotoxins
`will gain access to other tissues and cause unwanted
`toxicities.
`
`Immunogenicity
`Individuals with a functional immune system make
`antitoxin antibodies even when humanized‘antibodies
`or human growth factors are used as carriers"-“.
`Strategies to decrease such immunogenicity, such as
`
`Immunology Today
`
`254 Vol.” No.61993
`
`Immunogen 2144, pg. 3
`Phigenix v. Immunogen
`|PR2014—00676
`
`Immunogen 2144, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`antibody-based therapy
`
`immunosuppressive
`concomitant administration of
`drugs, have not yet been successful
`in humans“. In
`contrast, multiple courses of immunotoxin can be
`given to highly immunosuppressed individuals, such as
`B-cell lymphoma patients, without a resultant immune
`response”. Even in these patients, when earlier disease
`is treated,
`immunogenicity will become a problem.
`Circulating antibodies can inhibit
`the efficacy of
`immunotoxins by increasing their rate of clearance,
`and/or by blocking the binding site on the antibody or
`the enzymatic site on the toxin. Despite these consider-
`ations, immunotoxins have been administered in the
`face of serum antibody and, in some cases, have been
`effective”. With immunotoxins of a very short half-life
`(e.g.
`IL-2-DAB486; Ref. 38),
`the binding of non-
`neutralizing antibody may,
`in fact, increase the half-
`life. Nevertheless,
`immunogenicity will
`remain a
`problem until the entire immunotoxin is humanized.
`This may be possible by using human ‘toxins’, such as
`ribonuclease, attached to human antibody“. However,
`even this strategy may not avoid the formation of new
`immunogenic epitopes created by linking autologous
`proteins.
`
`Immunotoxin-resistant mutants
`In several rodent tumor models, immunotoxins have
`produced excellent tumor regressions but have failed
`to cure the animals because immunotoxin-resistant
`tumor
`cells emerge”. These are usually antigen-
`deficient mutants whose outgrowth can be prevented
`by
`administering immunotoxin cocktails directed
`against
`alternative
`tumor—associated
`antigens‘3~”.
`However, mutants have also been observed that have
`defects in intracellular transport of the endocytosed
`immunotoxin".
`Importantly, mutants with toxin-
`resistant ribosomes have not been observed, suggesting
`that such mutations may be lethal.
`
`Difficulties in evaluating immunotoxins
`From experimental studies and theoretical consider-
`ations, the optimal efficacy of immunotoxin should be
`obtained by administration of a single short course in
`patients with minimal', dormant“, or premalignant‘”
`disease. The latter is a particularly attractive state for
`intervention since
`the development of
`full-blown
`malignancy appears to require an additional
`rare,
`stochastically determined genetic event. Hence, killing
`of 100—1000 premalignant cells would probably prevent
`development of malignancy.
`However, the design of clinical trials does not allow
`this strategy to be tested readily. The initial
`trials
`(Phase I) require treatment of patients with intractable
`disease. Dose escalations of the drug are performed in
`small cohorts of patients until the maximally tolerated
`dose (MTD) is established. Side-effects, pharmacokinetics,
`and immunogenicity are analysed. As in most Phase I
`clinical
`trials, clinical benefit
`is unlikely to occur
`because the patients have far—advanced, bulky tumors
`and organ damage from previous therapy. Alterations
`in the drug or the protocol are usually not acceptable
`until completion of the trial. Therefore, lack of efficacy
`in a Phase I trial should not preclude further clinical
`testing of the drug.
`
`then administered at a safe dose to
`The drug is
`patients with less advanced disease to determine effi-
`cacy (Phase II
`trial). Generally, a response rate of
`20—40% (partial or complete remissions) must be
`observed at Phase II, or drug development is halted.
`This may be too stringent a criterion for an agent that
`is likely to be most effective when used in the treat-
`ment of minimal disease and in combination with one
`or two other immunotoxins and chemotherapy. An
`additional problem is that an MTD established in
`patients with bulky tumor may be very different from
`that in patients with minimal disease.
`Phase III trials require several hundred patients to be
`treated in multiple clinical centers along with controls
`(who usually receive placebos or the current
`‘best’
`therapy) and, therefore, requires support by a pharma-
`ceutical company. The result of the above consider-
`ations is that few immunotoxins have proceeded beyond
`the stage of Phase I or II trials. Therefore it might be
`wiser to test immunotoxins by an alternative strategy,
`for example to establish MTD (Phase I)
`in patients
`with less bulky disease and then use a safe dose in
`combinatorial therapy (Phase II) before proceeding to
`randomized Phase III
`trials in which immunotoxins
`plus or minus additional therapies are compared for
`efficacy.
`
`Clinical trials
`
`The completed or ongoing clinical trials involving
`systemic therapy with immunotoxins are summarized
`in Table 2. The major findings to emerge are:
`1 The side-effects of immunotoxin therapy are differ-
`ent from those of conventional therapy, in that there
`is no damage to rapidly dividing normal
`tissues.
`Blocked immunotoxins consisting of ricin, DT and
`Pseudomonas exotoxin routinely cause hepatotox-
`icity. All
`the
`ricin-based immunotoxins
`cause
`reversible vascular leak and myalgias. The MTD
`appears inversely related to the half life and the
`stability of the immunotoxin are directly related to
`the size of the antigenic sink. Multiple courses of
`immunotoxin therapy have been well
`tolerated,
`indicating that toxicity is not cumulative.
`2 Severe neurotoxicity was observed in two trials and
`was due to cross-reactivities of the antibody portion
`of
`the immunotoxins with neural cells‘“. This
`emphasizes the importance of carefully screening
`antibodies for unexpected cross-reactivities with life-
`sustaining tissues and, when possible,
`selecting
`mAbs which cross-react with their homologs in non-
`human primates. Conversely, administration of an
`anti-CD19 immunotoxin that was known to cross-
`react with astrocytes“ did not cause CNS lesions,
`presumably because the astrocytes were inaccessible
`to the immunotoxin, or were insensitive to it.
`3 Optimal regimens for administration of the immuno-
`toxins have not yet been devised. The half-lifes in
`trials to date have generally been shorter than would
`be predicted to induce an optimal therapeutic index.
`4 A general problem is that techniques for isolating
`and immunophenotyping the malignant progenitor
`cells have not been developed for the majority of
`tumors. The assumption usually has to be made that
`
`Immunology Today
`
`255
`
`Vol. )4 No. 6 1993
`
`Immunogen 2144, pg. 4
`Phigenix v. Immunogen
`|PR2014—00676
`
`Immunogen 2144, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Trial
`Disease
`phase
`
`
`Metastatic
`melanoma
`
`Colorectal
`carcinoma
`Metastatic
`breast
`carcinoma
`
`Ovarian
`carcinoma
`Non-Hodgkin's
`lymphoma
`
`Hodgkin‘s
`disease;
`non-Hodgkin‘s
`lymphoma
`Hodgkin's
`disease
`B—cell chronic
`lymphocytic
`leukemia
`
`T-cell
`lymphoma
`B-cell acute
`lymphoblastic
`leukemia
`
`l
`
`ll
`
`II
`
`l
`
`l
`
`l
`
`I
`
`I/Il
`
`I
`
`l
`
`I
`
`I
`
`I
`
`l
`
`l
`
`l
`
`Xomazyme-Mel
`
`Xomazyme-Mel
`
`Xomazyme-Mel
`plus cyclophosphamide
`
`Anti-gp72—ricin
`toxin A—chain
`260F9—ricin
`toxin A-chain
`(bolus)
`260F9—ricin
`toxin A-chain
`(continuous infusion)
`Anti-OVB3-
`Pseudomonas exotoxin
`Anti-CD19-
`blocked ricin (bolus)
`
`Anti-CD19—
`blocked ricin
`(continuous infusion)
`Fab‘ anti-CDZZ-
`deglycosylated
`ricin A-chain
`
`IgG anti-CDZZ-
`deglycosylated
`ricin A-chain
`
`IL-Z-truncated
`diphtheria
`toxin (DAB 486)
`
`Anti-CD30—
`saporin
`Anti-CD5 (TI-OI)—
`ricin toxin
`A-chain
`
`Anti-CD5 (H65)—
`ricin toxin A—chain
`Anti—CD19
`(B43)—PAP
`
`>3
`
`n.d.
`
`n.d.
`
`>1
`
`0.05
`
`0.4
`
`n.d.
`
`0.25
`
`0.35
`
`1.8
`
`0.7
`
`1.5
`
`n.d.
`
`n.d.
`
`3.3
`
`Not yet
`reached
`
`1.3
`
`vascular leak syndrome, myalgia
`
`vascular leak syndrome
`
`vascular leak syndrome
`
`vascular leak syndrome, aphasia
`
`vascular leak syndrome, myalgia,
`paresthesia
`
`vascular leak syndrome, myalgia,
`neuropathies
`
`SGOT/SGPT elevations, abdominal
`pain, encephalopathy
`SGOT/SGPT elevations,
`thrombocytopenia
`
`SGOT/SGPT elevations,
`thrombocytopenia, edema
`
`Vascular leak syndrome, myalgia
`
`vascular leak syndrome, myalgia
`
`hepatic transaminase elevations,
`hypoalbuminemia, hypersensitivity,
`creatinine elevations, thrombocytopenia,
`renal insufficiency
`thrombocytopenia, SGOT/SGPT
`elevations, proteinuria
`fever
`
`vascular leak syndrome, dyspnea
`
`hypoalbuminemia
`
`vascular leak syndrome, myalgia,
`hematuria, tremors
`
`
`antibody—based therapy
`
`Table 2. Summary of clinical trials of immunotoxins
`
`Maximum
`tolerated
`dose (total
`Toxicity
`Immunotoxin
`mg kg")
`t_’____~___________—»———————~——_.____._.__.a
`
`Anti-CD5 (H65)—
`Steroid-resistant
`ricin toxin A-chain
`graft-versus-host
`isease
`
`
`ll
`
`"Human antibodies made by the patient against the two components of the immunotoxin. measured by radio- or enzyme~
`linked-immunoassay.
`ARE anti-ricin A-chain antibody; AM: anti-mouse lg antibody; ADT: anti-diphtheria toxin antibody; AIL-2: anti-lL-Z
`antibody; AS: anti-saporin antibody.
`
`Immunology Today 256 Vol, 14 No. 6 1993
`
`Immunogen 2144, pg. 5
`Phigenix v. Immunogen
`|PR2014—00676
`
`Immunogen 2144, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Antibody
`production
`in response to
`toxin‘
`
`17/21
`
`n.d.
`
`13/13
`
`15/16 AR
`16/17 AM
`4/4
`
`4/5 AR
`3/5 AM
`
`n.d.
`
`12/15 ARIAM
`
`26/43 ARIAM
`
`4/14 AR
`1114 AM
`
`8/24 AR
`7/24 AM
`
`45/109 ADT
`28/109
`AIL-2 (26%)
`
`4/4 AM
`4/4 AS
`1/4 AR
`0/4 AM
`
`n.d.
`
`n.d.
`
`n.d.
`
`n.d.
`
`8.3
`
`4—6
`
`n.d.
`
`n.d.
`
`n.d.
`
`1.4
`
`7.8
`
`0.1—0.33
`
`n.d.
`
`n.d.
`
`3/43 PR
`.
`1/43 mixed
`9/43 stabilization
`
`4/20 PR
`
`5/16 mixed tumor
`regressions
`1/4 resolution of
`lung nodule
`
`0/5
`
`0/23
`
`1/25 CR
`2/25 PR
`10/25 mixed or transient
`
`2/43 CR
`5/43 PR
`11/43 transient
`
`5/13 PR
`
`6/24 PR
`1/24 CR
`
`4/109 CR
`8/109 PR
`
`3/4 PR
`
`4/5 transient
`rapid fall in circulating
`leukemic cells
`
`antibody-based therapy
`
`
`
`tm (h)
`
`Clinical
`responsef
`
`
`Reference
`
`1/22 CR
`9/22 mixed or stabilized
`
`Spitler, LE. er al. (1987)
`Cancer Res. 47, 1717-1723
`
`Spitler, LE. et al. (1988)
`in Immtmotoxins (Frankel, A.E., ed.),
`pp. 493-515, Kluwer Academic Publishers
`
`Oratz, R. et al. (1990)
`]. Biol. Resp. Med. 9, 345—354
`
`Byers, V.S. er al. (1989)
`Cancer Res. 49, 6153—6160
`Weiner, LM. et al. (1989)
`Cancer Res. 49, 4062—4067
`
`Gould, 8.]. et al. (1989)
`1. Natl Cancer Inst. 81, 775—781
`
`Pai, LH. et a1. (1991)
`J. Clin. Oncol. 9, 2095—2103
`Grossbard, ML. et al. (1992)
`Blood 79, 576—585
`
`Grossbard, M.l.. et al. (1992)
`Blood 79, 576—585
`
`Vitetta, 12.5. et al. (1991)
`Cancer Res. 15, 4052-4058
`
`Amlot, P.L. et 11]., unpublished
`
`Uckun, EM. et al., unpublished
`
`Falini, B. et al. (1992)
`Lancet 339, 1195—1196
`Hertler, A.A. et al. (1989)
`Int. ]. Cancer 43, 215—219
`
`LeMaistre, C.F. er (ll. (1991)
`Blood 78, 1173~1 182
`
`Uckun, EM. et al., unpublished
`
`10/12 ARIAM
`
`1.2—2.9
`
`4/14 PR
`
`12/14 AM
`
`4.4—5.7
`
`80—99% decrease in
`circulating B cells in 4/5
`
`6/23 AM
`6/23 AR
`
`1.5-3.9
`Byers, V.S. et al. (1990)
`9/32 CR
`Blood 75, 1426—1432
`7/32 PR
`6/32 mixedM
`
`f Measured in terms of the change in ‘tumor burden’, which is the sum of the products of perpendicular diameters of all tumor
`nodules by C1" scans. CR: complete response = tumor burden l« 100%; PR: partial response = tumor burden l 50-99%.
`.
`n.d.: not determined; SGOT: serum glutamic—oxaloacetic transaminase; SGPT: serum glutamtc—pyruVm transammase;
`PAP: pokeweed anti-viral protein.
`
`Immunology Today 257 Vol. 14 No. 6 1993
`
`Immunogen 2144, pg. 6
`Phigenix v. Immunogen
`|PR2014—00676
`
`Immunogen 2144, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`antibody-based therapy
`
`the malignant progenitors bear the same antigens as
`their progeny. If, however, the major population of
`tumor cells derives from a progenitor cell lacking the
`target antigen, immunotoxin therapy will be palli-
`ative, not curative.
`5 Clinical responses in lymphomas have been excellent
`considering that most trials were Phase 1, patients
`had bulky disease, and a single immunotoxin was
`used.
`In eight separate trials,
`the percentage of
`patients achieving partial or complete remissions
`ranged from 12 to 75% (Table 2). To put this
`into perspective, of those drugs which are mar—
`keted today for the treatment of cancer, the great
`majority (>90%) produced fewer
`than a 5%
`response rate in Phase I trials. In addition, clinical
`responses have also been excellent (28% complete
`responses) when accessible circulating T cells have
`been targeted as a treatment for steroid-resistant
`graft-versus~host disease or autoimmune disease“.
`By contrast, responses of large tumor masses to
`immunotoxin therapy have rarely been seen in Phase
`I
`trials in patients with solid tumors, primarily
`because of inaccessibility of the cells to the immuno-
`toxins‘w’.
`
`Future prospects
`The ideal immunotoxin should be non-immunogenic
`and cause minimal toxicity to normal tissue, yet have
`access to, and the potency to kill, 10“—10lz tumor cells
`and their progenitors
`in solid and disseminated
`tumors. Immunogenicity might be eliminated by com-
`plete humanization of the immunotoxin molecule or
`by using a short course of a potent immunosuppressive
`agent (e.g. anti-CD4). Further analysis of the struc-
`ture-function relationships of immunotoxins reveal
`ways by which to increase the therapeutic index.
`Elucidation of the mechanisms underlying side-effects
`may allow their successful treatment with conventional
`pharmaceuticals.
`is
`The problem of treating large solid tumors
`presently being approached by targeting the immuno-
`toxin to the vascular endothelial cells responsible
`for supplying blood to the growing tumor mass
`(F. Burrows and P. Thorpe, unpublished). The advan-
`tages of this approach are that endothelial cells are
`accessible, that they are normal and,
`therefore, un~
`likely to mutate. Furthermore, a single immunotoxin
`should be useful for treating a variety cf solid tumors,
`since the destruction of a single blood vessel should
`result in the death of a large number of tumor cells of
`any type.
`Immunotoxins may also prove useful in eliminating
`other undesirable cells such as those infected with
`human immunodeficiency virus and those involved in
`autoimmune disease“.
`
`Conclusions
`
`Clinical research is now focusing on refining dose
`regimens for already—developed constructs with the
`long-term goal of using cocktails of immunotoxins
`together with chemotherapy to treat minimal disease.
`Basic research is aimed at eliminating immunogenicity,
`understanding the basis for side-effects and developing
`
`new second- and third—generation immunotoxin con—
`structs with improved therapeutic indices and ability to
`attack solid tumors. Considering that
`took several
`decades after the introduction of chemotherapy before
`regimens were developed to cure patients, progress
`with immunotoxins has been substantial by the stan-
`dards of conventional drug development. The early
`clinical
`trials demonstrate considerable biological
`activity. The continued refinement in design of these
`pharmaceuticals in basic science laboratories further
`suggests that improved immunotoxins may eventually
`be useful
`in the treatment of cancer, autoimmune
`diseases and viral infections.
`
`We thank Ms Cindy Patterson for secretarial assistance.
`
`E.S. Vitetta is at the Cancer Immunobiology Center,
`RE. Thorpe and ].W. Uhr are at the Departments of
`Microbiology and Pharmacology, University of Texas
`Southwestern Medical Center, Dallas, TX 75235,
`USA.
`
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