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`
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`PHIGENIX
`PHIGENIX
`Exhibit 1021
`Exhibit 1 02 1
`
`

`

`IT:
`
`Molecular Immunology, Vol. 27. No. 3. pp. 273-282, I990
`Printed in Great Britain.
`
`0|6l-5890/90 $3.00 + 0.00
`Pergamon PreSs plc
`
`IMMUNOTOXINS OF PSEUDOMONAS EXOTOXIN A (PE):
`' EFFECT OF LINKAGE ON CONJUGATE YIELD,
`
`POTENCY, SELECTIVITY AND TOXICITY
`
`ALTON C. MORGAN JILL” GOWSALA SIVAM, PAUL BEAUMIER, ROBERT MCINTYRE, MIKE BJORN
`and PAUL G. ABRAMS
`
`NeoRx Corporation. 410 West Harrison Street, Seattle, Washington 98I I9, U.S.A.
`
`(First received 25 May I989; accepted in revised form 29 August I989)
`
`Abstract—Conjugates of monoclonal antibodies and Pseudomonas exotoxin A (PE) were formed with
`disulfide or thioether bonds. Thioether conjugates which formed with succinimidyl 4—(N-maleimidomethyl)—
`cyclohexane-l—carboxylate (SMCC) modified PE and reduced antibody formed with an 80% yield of
`equimolar conjugate within 30 min with an ofi‘ering of one to one (toxinzantibody). The efliciency and
`kinetics of thioether formation were much higher with SMCC than with other maleimidc reagents as well
`as more efficient than disulfide linkers. Thioether linkage resulted in immunotoxin consistently more
`potent and more selective in vitro than disulfide bonded conjugate. ,Thioether bonded conjugates also
`proved to have other favorable in viva properties compared to disulfide conjugates: (l) a longer half-life
`in serum; (2) increased tumor localization: and (3) reduced toxicity.
`Toxicity of thioether linked holotoxin conjugates was directed at the liver hepatocyte but was easily
`monitored by serum liver enzymes. The conjugates are currently undergoing clinical evaluation for
`treatment of ovarian cancer with intraperitoneal administration. Research is ongoing to further decrease
`residual toxicity without reducing the potency of the conjugate.
`
`INTRODUCTION
`
`Conjugates of monoclonal antibodies with toxins
`offer considerable potential for localizing therapy to
`tumor sites while sparing normal tissues of toxicity
`typically encountered with unconjugated cytotoxic
`agents. Molecules too toxic for use as free drugs can
`be delivered selectively to tumor cells with reduced
`toxicity as conjugates.
`Several strategies to create highly potent and selec-
`tive immunotoxins have been explored. The most
`common approach is to link the protein inhibitory
`portion of plant toxins, namely A-chains or hemitox-
`ins, or naturally occurring A-chain-lik‘e molecules
`called ribosomal
`inactivating proteins (RIPS),
`to
`monoclonal antibodies via disulfide linkages (Thorpe
`
`
`’To whom reprint requests should be addressed: Alton C.
`Morgan .Ir. NeoRx Corporation. 22021—20th Avenue
`S.E., Bothell, WA 9802l-4406, U.S.A.
`2Abbreviations used: RIP. ribosomal inactivating protein;
`PE, Pseudomonas exotoxin A; FPLC. fast protein liquid
`chromatography; SMCC, succinimidyl 4—(maleimido-
`methyl)cyclohexane-I-carboxylate; TAC. IL-2 receptor;
`PBS/BSA. phosphate buffered saline with 1% bovine
`serum albumin; DTT, dithiothreitol; PIP, paraiodo-
`phenyl: Tm.
`serum half~life; ADP.
`adenosine di‘
`phosphate: LDlm,
`lethal dose
`I00; LDH.
`lactate
`dehydrogenasc; SGOT. serum glutamic oxalate transam-
`inase; SGPT. serum glutamic pyruvic transaminase;
`DTNB.
`5.5’—dithio—bis-(2-nitrobenzoic
`acid); MBS,
`m-maleimidobenzoyl-N~hydroxysuceinimide;
`SMPB.
`succinimidyl 4-( p-maleimidophenyl)butyrate.
`"University of Washington. School of Medicine. Depart-
`ment of Radiology. Division of Nuclear Medicine.
`
`et al., I981, I982, I985; Hwang e! 01., I983; Rama-
`krishnan and Houston, I984). Although these con-
`jugates contain the enzymatic (toxic) portion of the
`protein, they have less potency than the holotoxin.
`This appears to be due to the lack of the B-chain
`which facilitates insertion and translocation of the
`
`I983;
`
`A-chain across membranes (McIntosh el al.,
`Youle and Neville, [982; Vittetta et (11., I984).
`An alternative strategy is to employ intact toxins
`(holotoxins) such as ricin or abrin or bacterial holo-
`toxins like Pseudomonas exotoxin (PE) conjugated to
`monoclonal antibody (Thorpe et al., I984; Fitzgerald
`er al.,
`I984). This results in agents of increased
`potency compared to the A—chain conjugates. This
`has been demonstrated in vitro by comparing conju-
`gates formed with the holotoxin PE or recombinant
`ricin A-chain and antibodies against both breast and
`ovarian cancer cell lines (Bjorn et (11., I985; Pirker et
`al.,
`I985). Similarly, comparisons have been made
`with whole ricin and ricin A-chain conjugated to the
`same antibody (Vallera e1 (11.. 1984).
`The clinical application of holotoxin conjugates is,
`however, compromised by toxicity, since the conju-
`gated holotoxin still retains some cell binding proper-
`ties. Several avenues have been explored to reduce
`, toxicity while retaining potency. Ricin B-chain bind-
`ing" can be inhibited by incubation with lactose or
`galactose or by covalent incorporation of these sac-
`charides into the B-chain lectin site (Quinones et a[.,
`I984; Houston, I983). Alternatively. holotoxins, with
`conjugation to antibody, can have reduced toxicity
`due to steric occlusion of cell binding (Thorpe er al.,
`
`273
`
`PHIGENIX
`
`Exhibit 1021-01
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`

`

`274
`
`ALTON C. MORGAN Ja er 11/.
`
`I984; Godal e1 (11., 1986). In our hands. even the last
`approach produces conjugates with some antibodies
`which still have a high level of non-specific toxicity
`(Godal et (11.. 1986: Morgan er a1., 1985). Non-conju—
`gated PE is less toxic to cells than abrin. ricin or
`diphtheria toxin.
`In addition, conjugation of PE
`further decreases its toxicity. The reduction in toxic-
`ity. together with high in vitro potency (len 10‘ ”’ to
`10"” M) and selectivity (3—4 logs) when linked to
`antibody.
`indicates the potential of this agent for
`clinical use. However, when conjugated via a disulfide
`linkage. PE conjugates still exhibit lethal toxicity at
`relatively low doses (300 rig/kg) when injected into
`primates (this publication).
`In this study. we examined holotoxin conjugates
`constructed with thioether linkages rather than con-
`ventional disulfide bonds. Contrary to prior reports.
`demonstrating reduced potency of toxin conjugates
`with stable linkages (Edwards er al.. 1983), thioether
`linked PE conjugates were equally potent on antigen
`positive cells and more selective than disulfide bonded
`conjugates. in addition. higher doses ofthe thioether
`conjugates could be safely administered to primates.
`Thus. potency was preserved and toxicity reduced
`simultaneously. The particular method of construct-
`ing the thioether immunoconjugate was also shown
`to provide a markedly improved yield,
`thereby im-
`proving the eventual efficiency and cost efiectiveness
`of therapy with these agents.
`
`-
`
`MATERIA LS AND METHODS
`
`Monoclonal antibodies and fragmentation
`
`Two murine lgG2a antibodies were used in these
`studies:
`anti-TAC (antibody
`recognizing
`IL—2
`receptor. kindly provided by Dr Tom Waldmann,
`National Cancer
`Institute. Bethesda, Maryland)
`(Uchiyama e! (11.. 1981a; Uchiyama (11121.. 1981b): and
`9.2.27. antibody recognizing a human melanoma-
`associated glycoprotein,’proteoglycan (Morgan 6! al..
`1981). Three murine lgGZb antibodies were also
`used: NR-LU-IO. directed to a pan-carcinoma
`antigen (Okabe e! 11]..
`I984). OVB—3 (Willingham
`er ul.. 1987) and NR-ML-US. directed to a different
`cpitope than 9.2.27 on the human melanoma associ-
`ated glycoprotein/proteoglycan (Woodhousc er (1]..
`1990).
`
`F(ab)§ fragments of 9.2.27 were prepared from
`whole antibody by digestion with immobilized
`pepsin. The digest was fractionated by ion exchange
`chromatography to remove peptides and undigested
`antibody and then concentrated by ultrafiltration.
`F(ab)§ fragments were homogeneous as assessed by
`both SDS—PAGE and FPLC gel filtration.
`
`Conjugation and ,f'rm‘rionulirm
`
`Disulfide bonded conjugates were produced ac-
`cording to previous published methodology (Pirker or
`a[., 1985) with some modification. Briefly. whole
`' antibody and PE were reacted with 2-iminothiolane
`
`(2-11) (molar ratio 5:1) at pH 9.5 in sodium bicar-
`bonate bufier
`(0.1 M. 0.15 M NaCl). Unreacted
`reagent was removed by gel filtration and derivatized
`antibody then reacted with DTNB and excess reagent
`removed. DTNB activated. derivatized antibody was
`then mixed with 2-IT treated PE at room temperature
`for up to 4hr. The conjugate mixture was then
`fractionated by FPLC gel filtration on a TSK 3000
`column in PBS pH 7.2 to remove unreacted PE.
`Conjugate corresponding in size to a l : 1 molar ratio
`of PE to antibody was pooled for subsequent testing.
`ThiOether linked conjugates were prepared by first
`reacting PE with
`succinimidyl
`4—(N-maleimido~
`mcthylkyclohexane-l-carboxylate (SMCC), succin-
`imidyl 4~(pvmaleimidophenyl)butyrate (SMPB). or
`m-maleimidobenzoyl-.N’—hydroxysuecinimide (M BS)
`at a molar ratio of 10: l in pH 9.0 sodium bicarbonate
`bufi'er and unreacted reagent removed by gel
`filtra—
`tion. Whole antibody was reacted with 25 mM dithio-
`threitol
`in 0.1 M phosphate buffered saline (PBS)
`pH 7.5 and excess reducing agent removed by gel
`filtration. The two conjugate components were then
`admixed and incubated at room temperature for up
`to two hours. Disulfide and thioether (SMCC) linked
`conjugates were also prepared with fragmented anti-
`body. For this.
`the above procedure was followed
`except
`that F(ab)§ was substituted for whole anti~
`body. Under the same reducing conditions as above.
`F(ab)§ was reduced to F(ab)‘ before conjugation.
`At
`least 2 batches of each type conjugate were
`produced with the batch size varying between I and
`5 mg. In the case. of OVB-3 conjugates the batch size
`was 800 mg as this was produced for a clinical trial
`(Morgan er (1].. submitted for publication).
`Crude conjugate mixtures were first separated by
`FPLC gel filtration. Fractions corresponding to a l :1
`molar ratio of conjugate and unconjugated antibody
`were pooled. Unconjugated antibody was removed
`by anion exchange chromatography on a Mono Q
`column (Pharmacia Fine Chemicals, Piseataway. NJ).
`Conjugate was eluted with a gradient of sodium
`phosphate starting with 5 inM sodium phosphate
`pH 7.6 and ending with 0.5 M sodium phosphate
`bufier pH 7.6 in 30 min (flow rate 0.5 mifmin).
`
`Biochemical aim/jails of conjugates
`
`Conjugate species separated by FPLC gel sieving
`or anion exchange chromatography were analyzed on
`SDS slab gels (10%). both reduced and non-reduced.
`Conjugates were also analyzed by analytical isolectric
`focusing using a ‘pH 3—10 gradient (Pharmacia. Pis»
`cataway, NJ). Unmodified 9.2.27 focused in multiple
`bands with isoelectric points of 7.4. 7.3. 7.1. 6.9, 6.8
`and 6.7 with an average pI of 7.2. Conjugate focused
`at an average pl of7.46. Anti-TAC focused in a series
`of more basic isoelectric points. but following conju-
`gation to PE focused at a pl similar to that of 9.2.27
`conjugate. Both NR-LU-IO and NR-ML~05 had
`slightly acidic pl (6.33-7.05) and showed little change
`after conjugation to derivatized PE.
`
`PHIGENIX
`
`Exhibit 1021 -02
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`

`

`Linkage of PE immunotoxins
`
`275
`
`Tumor cell lines and cytotoxicity testing
`
`In vitro cytotoxicity testing was performed as pre-
`viously described using Jl-l—leucine incorporation to
`measure surviving cells (Pavanasasivam et al., 1987;
`Morgan et al., 1987). 9.2.27 and NR-ML-OS (anti-
`melanoma) conjugates were tested on the human
`melanoma cell
`lines, A375 met mix (A375 M/M)
`which was antigen positive, =A375 primary which was
`antigen negative and the HT-29 colon carcinoma cell
`line which was also antigen negative. For anti-TAC
`(anti-IL-Z receptor) conjugates, HUT 102 cells were
`the antigen positive, and CEM the antigen negative
`targets,
`respectively (Fitzgerald et
`(11., 1984). For
`NR-LU-IO (anti-carcinoma) conjugates, PIT-29 was
`the relevant
`target and A375 M/M the irrelevant
`target. All cell
`lines were pretested for inherent
`sensitivity to unconjugated PE and showed similar
`sensitivity (IDSO = 100 ng/ml). 1D,.) values for conju-
`gates on cells. The IDSO determinations represent the
`average of at least three separate determinations.
`Conjugates were tested in two formats, short expo-
`sure and long exposure. For short exposure, conju-
`gate was incubated with cells for 1hr at 37°C, the
`‘cells gently washed and cultures continued for up to
`72 hr before addition of Jl-I-leucine. For long expo-
`sure, cells were exposed to conjugate for the entire
`72 hr of the culture period.
`
`lmmunoreactivity
`
`PE conjugates of 9.2.27 and NR-ML-OS were
`compared to unconjugated antibody for binding to
`target A375 M/M melanoma using flow cytometry as
`previously described (Pavanasasivam e! a[., 1987).
`Similarly, conjugates of TAC were compared to
`unconjugated TAC antibody on HUT 102 cells and
`NR-LU-IO conjugate to unconjugated NR-LU-10 on
`HT-29 cells. OVB-3 antibody and conjugates were
`analyzed on ALAB breast carcinoma cells. Briefly,
`target
`cells were
`suspended
`in PBS/BSA at
`l x 10’ cells/ml and incubated at 43C for 30 min with
`titrated doses of antibody or conjugate including
`saturating or subsaturating doses (1 to 0.01ug/ml).
`Cells were then washed twice with PBS/BSA, and
`incubated with 100 m1 (1 ug/ml) offluorescein labeled
`goat anti-mouse IgG for 30 min. Washed cells were
`then resuspended in PBS/BSA and analyzed for
`bound fluoresceinated secondary antibody using a
`cytofiuorograph
`(Coulter Diagnostics, Hialeah,
`Florida). Immunoreactivity was assessed by compari-
`son of
`the mean fluorescence index over
`1000
`
`channels for positive cells and then compared to a
`standard curve generated with fluoresceinated beads
`(Ortho Diagnostics) and expressed as fluorescein
`equivalents (FE). The percent of FE displayed by the
`conjugate was then expressed as a percent of the
`unmodified antibody at a subsaturation level. This
`type of assessment measures both changes in the
`percent of immunoreactive antibody molecules as
`well as alterations in affinity.
`
`MlMM 27:1—F
`
`ADP ribosylation
`PE as well as disulfide or thioether linked PE-
`
`conjugates were compared in a cell-free system as
`previously described (Vanness e! 01., 1980). ADP-
`ribosylation measures the ability of toxins such as
`Pseudomonas exotoxin A and diphtheria toxin to
`transfer labeled ADP-ribose to an intracellular accep-
`tor molecule which in the case of PE is elongation
`factor-2. Thus, the assay is a measure of enzymatic
`activity not dependent on cell binding or transloca-
`tion of the toxin as is required'for cell killing. Both
`disulfide and thioether conjugates were titrated in the
`assay after treatment with 8 M urea and 1 M DTT.
`
`Biodistribution, serum half-life and toxicology
`
`Thioether and disulfide linked conjugates of 9.2.27
`were compared in a nude mouse xenograft model of
`human melanoma
`for
`tumor
`localization and
`
`biodistribution (Hwang e! (11., 1985). PE was first
`radiolabeled with l2SI-para-iodophenyl (PIP) (Wilbur
`er al., 1986). By this method, the radiolabel is not
`subject
`to
`dehalogenation
`and
`thereby more
`accurately reflects the biodistribution of conjugates.
`The labeled PE was then incorporated into conju-
`gates and tested for retention of potency. Animals
`were administered 2—5 M g of
`labeled conjugate
`(2—5 uCi) intravenously and sacrificed at 20 hr post
`injection, organs blotted, weighed and counted as
`previously described (Wilbur et al., 1986), and per
`cent dose per gram calculated for each tissue. In
`addition, serum half-life of radiolabeled conjugate
`was estimated in non-tumor bearing Balb/c mice by
`retro—orbital sampling of whole blood (Wilbur et (11.,
`1986). Groups of four mice were used for both
`biodistribution and serum half-life determinations.
`
`Toxicity of disulfide and thioether conjugates was
`assessed both in mice and in cynomogolus monkeys.
`Both the 9.2.27 and anti—TAC PE conjugates
`were assessed in mice; only anti-TAC conjugates were
`assessed in monkeys.
`In both cases, comparisons
`were made to the corresponding disulfide conjugate.
`Groups of five mice were observed for death or
`survival as a final endpoint, whereas monkeys were
`monitored by liver enzyme levels and observation of
`other relevant symptoms including changes in behav-
`ior, appetite, nausea/vomiting and temperature. Lac»
`tate dehydrogenase (LDH) levels proved to be the
`most sensitive monitor of hepatic toxicity in mon-
`keys. LDH levels of treated monkeys were compared
`to averages obtained from normal monkeys tested at
`Microbiological Associates (Rockville, MD) over a
`period of years.
`
`RESULTS
`
`Production and analysis of conjugates
`
`Representative FPLC gel filtration profiles of cor-
`responding batches of disulfide and thioether linked
`conjugates of anti-TAC and PE are shown in Fig. 1.
`
`PHIGENIX
`
`Exhibit 1021 -O3
`
`

`

`276
`
`ALTON C. MORGAN JR et al.
`
`Q.
`
`(—m
`
`(.0!
`
`
`
`ODmtreLotiveunits)
`
`tonma:
`
`0
`
`600
`
`600
`Retention time (min)
`
`600
`
`60
`
`Fig. 1. Comparison of Pseudomonas exotoxirt immunoconjugates by FPLC gel filtration. Disulfide and
`thioether linked conjugates were analyzed on a Superose 12 column at
`lml,’min (Pharmacia). (A)
`Thioether reaction (SMCC). (B) Thioether reaction (SMPB). (C) Thioether reaction (M BS). (D) Disulfide
`reaction. Thioether reactions were analyzed 15 min post initiation. Disulfide reaction was analyzed after
`4 hr. 1. migration position of I:I molar ratio conjugate; 2, migration position of unconjugated antibody:
`3. migration position of unconjugated PE.
`
`Disulfide linkage employing iminothiolane (Panel D)
`produced conjugate species of varying sizes reflecting
`a range of molar ratios as reported previously.
`SDS—PAGE analysis of these fraction pools revealed
`size ranges corresponding to 2:1 and higher, and I :1
`molar ratios of PE:antibody. In contrast, thioether
`linkage (Panels A and B) primarily formed conjugate
`in a
`single size range which corresponded by
`SDS—PAGE analysis to a 1:] molar ratio of PE to
`antibody (peak I). Thioether conjugation efficiencies
`varied according to the reagent used for PE deriva-
`tization (Panels A through C). SMCC derivatized PE
`reacted with reduced antibody resulted in the best
`yields. e.g. 80% 1:] conjugate with an ofiering ratio
`of [:1 (PE to antibody). In comparison. disulfide
`conjugation typically gave a 30% yield of l : I conju-
`gate, with an offering of I0:I (PE to antibody). In
`addition, the best thioether conjugation required only
`a short incubation, with conjugation complete in 15
`to 30 min whereas the disulfide linkage required
`greater than 4 hours for optimal yields. Interestingly,
`the elution position of I : I conjugate differed between
`thioether and disulfide linked conjugate. One to one
`conjugate with disulfide linkage was well discrimi-
`nated from unconjugated antibody (compare peaks I
`and 2, Panel D) while I: l conjugate from thioether
`linkage migrated in the front edge of the antibody
`peak (compare peaks I and 2, Panel A).
`In fact,
`conjugation via thioether linkage was more easily
`monitored by disappearance of the unconjugated PE
`(peak 3) than formation of conjugate (compare Panel
`A, essentially complete conjugation.
`to Panel C,
`essentially
`no
`conjugate
`formation).
`Forming
`thioether linked conjugate with reduced antibody and
`SMCC—derivatized PE was the most efficient method
`for
`forming I:I conjugate. The combination of
`iminothiolane-derivatized
`antibody
`and SMCC-
`derivatized PE resulted in slower kinetics of forma-
`
`tion as well as the formation of higher molecular
`weight conjugate (not shown).
`Due to the difficulty in separating thioether linked
`conjugate from unconjugated antibody by gel filtra-
`tion, anion exchange chromotography was employed
`
`to further purify conjugate containing fractions (Fig.
`2). Conjugate but not antibody was bound to the
`Mono Q column under the conditions used and
`eluted with a hypertonic sodium phosphate buffer.
`Isolated lzl
`ratio thioether (SMCC) and disulfide
`conjugate as analyzed on SDS—PAGE under reducing
`conditions verified the nature of the bond (Fig. 3). As
`expected. reduction of disulfide conjugates gave three
`bands corresponding to PE and antibody heavy and
`light chains. Reduction of thioether conjugate gave
`bands corresponding to covalent adducts of PE with
`heavy and light chains with most of the adducts with
`the heavy chain as well as free heavy and light chain
`presumably
`from unconjugated
`antibody
`half
`molecules. Free PE was not apparent
`following
`reduction of thioether conjugate.
`
`lmmunoreacn'vity. potency and selectivity
`
`Immunoreactivity of antibody was consistently
`reduced by conjugation with PE (Table l). The loss
`of immunoreactivity was found regardless of which
`antibody and which linkage was used. This con—
`trasted to our previous experience with conjugation
`of A-chains or fibosomal inactivating proteins which
`did not alter immunoreactivity (Pavanasasivam e1 (11.,
`I987).
`Potency and selectivity of thioether and disuliide
`bonded conjugates were compared in vitro. Titration
`of ADP-ribosylating activity for disulfide
`and
`thioether conjugates of anti-TAC is shown in Fig. 4.
`In this cell-free system. PE requires unfolding by
`reducing and denaturing agents to expose the cofac-
`tor (NAO) binding site. Conjugates formed with the
`Mo types of linkages were equi-potent for enzymatic
`activity despite the demonstration by SDS~PAGE
`(above) that the thioether conjugate did not reduce to
`yield free PE.
`We also evaluated thioether and disulfide conju-
`gates of 9.2.27 in an in vitro cytotoxicity assay (Table
`2). 9.2.27 (whole antibody) thioether Conjugate was
`consistently more potent (and more selective) than its
`disulfide counterpart. Similar results were obtained in
`comparing thioether and disulfide linked TAC conju~
`
`PHIGENIX
`
`Exhibit 1021 -O4
`
`

`

`Linkage of PE immunotoxins
`
`277
`
`units)A(nm\lmtoo00234reLotive
`
`
`
`
`
`01
`
`
`Mot.wtx10's
`
`20
`
`30
`
`Retention time (min)
`
`Fig. 2. Separation of PE conjugated from unconjugated antibody by FPLC anion exchange chromatog-
`raphy. Top: chromatographic profile of an exchange column of thioether linked conjugate (Panel A,
`Fig. 1). Dark. broad line represents the gradient used for elution. Numbers 1, 2 and 3 refer to pools of
`material. Bottom: SDS~PAGE analysis (non-reduced) of pools from anion exchange chromatography.
`Lane I, unconjugated PE; Lane 2, starting material before anion exchange chromatography (after gel
`filtration); Lane 3. pool
`I from anion exchange column; Lane 4, pool 2 from anion exchange column;
`Lane 5, pool 3 from anion exchange column.
`
`gates (not shown). Thioether conjugates formed with
`F(ab)’ were slightly more potent than those made
`with whoie antibody but were considerably less selec—
`tive. It was also obvious that the use of a thioether
`
`linkage enhanced selectivity of both the intact and
`F(ab)’ forms of conjugates.
`Thioether-linked conjugates were also evaluated
`for selectivity using relevant and irrelevant conjugates
`reciprocally tested on antigen positive and negative
`cell
`lines (Fig. 5). Conjugates of both NR-ML-OS
`(anti-melanoma) and NR-LU-lO (anti-carcinoma)
`were highly potent when tested on antigen positive
`targets. approximately 10 to 100 fold more potent
`than unconjugated PE. In contrast, when tested on
`
`antigen negative targets, conjugates had approxi-
`mately l00 fold less toxicity than free PE. Both target
`cells were sensitive to the same extent
`to free PE.
`
`Thus, it appears that thioether linkage of PE to
`four different antibodies preserves the potency of the
`toxins in both cell-free as well as cytotoxicity assays.
`Moreover, thioether linkage seems to enhance selec-
`tivity, including those conjugates made with F(ab)’
`and to be highly efficient in yielding l:l molar ratio
`conjugate.
`
`Biodistribution and toxicology
`
`tumor localization, biodistribution and
`Toxicity,
`pharmacokinetics were compared for thioether and
`
`PHIGENIX
`
`Exhibit 1021 -O5
`
`

`

`278
`
`ALTON C. MORGAN In at u].
`
`
`
`Fig. 3. Comparison of Pseudomonas exotoxin immunoconjugates. SDS~~PAGE analysis. Pools corre-
`sponding to fractions from FPLC separation of disulfide and thioethcr conjugates were analyzed on
`reducing l0% SDS slab gels after radiolabeling (whole conjugate) with I35l—PlP. Left lanc = disulfide. right
`lane = thioether.
`
`disulfide linked conjugates. Mice were administered
`different doses of anti-TAC and 9.2.27 conjugates
`intraperitoneally. Disulfide
`conjugates
`exhibited
`an average LDloo of SOyg/kg difiering slightly de-
`pending on the batch. Thioether conjugates were
`reproducibly less toxic with an average LDM, of
`250 yg/kg.
`Prior data in monkey studies with disulfide conju—
`gates (Fitzgerald, Willingham, Pastan. unpublished
`observations) had suggested that toxicity was due to
`direct hepatocellular damage. Thus, we examined
`serial hepatic transaminase and lactic dchydrogcnasc
`
`lmmunoreactivity of thioether linked PE'
`Table l.
`conjugates (OVB—J)
`Antibody
`Dose
`Percent
`FE
`form”
`()1 gjml)
`positivc
`827
`OVB-3
`l
`94
`776
`0.1
`99
`457
`0.01
`98
`‘ 662
`l
`9‘)
`656
`0.l
`9K
`
`001 92‘ ll
`
`“Four other antibodies
`(NR-LU-IO.
`anti-TAC.
`’
`9.2.27 and NR-ML-OS)
`conjugated to PE
`showed similar reduction in immunorezlclivity.
`"Percent of control at subsaturation dose 2 20.
`
`OVB.3.’PE
`
`levels in monkeys administered PE conjugate (at-
`TAC). As shown in Table 3. LDH, the most sensitch
`indicator of hepatic toxicity;
`increased significantly
`after administration of only 300pg/kg of disulfide
`linked conjugate. In addition. hepatic transaminases
`like SGOT and SGPT were also elevated (not shown).
`Animals also exhibited behavioral evidence of toxic- >
`
`5
`
`pmixI000)
`
`c
`
`P)
`
`-1
`Conjugate Log ,19 AuDe)
`
`o
`
`t
`
`Fig. 4. Comparison of Pscudomonas exotoxin immuno-
`conjugates. ADP ribosylation. I disulfide: 1:) thioether.
`
`PHIGENIX
`
`Exhibit 1021 -O6
`
`

`

`Linkage of PE immunotoxins
`
`279
`
`Table 2. In uitra potency and selectivity of disulfide or thioether
`linked conjugate (9.2.27)
`Potency
`ODE)"
`
`Antibody form
`
`Selectivity”
`
`Type of linkage
`Disulfide—whole
`
`vs Antigen Positive
`vs Antigen Negative
`Thioether—whole’
`vs Antigen Positive
`vs Antigen Negative
`
`8 x l0'"
`4 x l0"°
`
`4 x 10'”
`2 x W”
`
`Disulfide——F(ab)‘
`
`3 x 10””
`vs Antigen Positive
`vs Antigen Negative 1.6 x 10' "
`
`LS
`
`3.5
`
`0.22
`
`'
`
`Thioether—F(ab)’
`3.5 x IO‘ '2
`vs Antigen Positive
`2.3
`l x 10“”
`vs Antigen Negative
`"Potency (IDso vs antigen positive cells as measured in the ’H-leucine
`incorporation assay).
`"Difl'erence. extrapolated from a graph, in IDso vs antigen positive
`and antigen negative cell (logs).
`rThioether conjugates using whole antibody were also evaluated for
`the four other antibodies: NR—LU-IO, OVBJ, TAC and NR-
`ML-OS. Potency against antigen positive cells varied from
`4 x l0"” to 4 x lO'”. Selectivity varied from 3 to 4.5 logs.
`
`ity at the same dose with loss of appetite, nausea and
`diarrhea.
`
`By contrast, up to 1 mg/kg of thioether linked
`conjugate produced only marginal elevation in LDH
`with little change in SGOT or SGPT. Animals exhib-
`
`l20
`
`100
`
`60
`
`4o
`
`20
`
`0
`
`l20
`
`Percentofcontrol
`
`too
`
`80
`
`20
`
`60
`
`40
`
`-2
`
`'-i
`
`2
`l
`o
`Canon (tog rig/ml )
`
`3
`
`4
`
`Fig. 5. Evaluation of PE conjugates for potency and selec-
`tivity. This was assessed by a long exposure format of a
`JH-leucine incorporation assay (Materials and Methods).
`Controls averaged 20,000 cpm/well. Top panel: HT-29
`colon carcinoma cells. NR-LU-lO/PE (relevant), E];
`unconjugated PE, I; NR-ML—OS/PE (irrelevant), 0.
`Bottom panel: A375 M/M melanoma cells. NILLU-lO/PE
`(irrelevant), C]; unconjugated PE, I; NR—ML-OS/PE
`(relevant). O.
`
`Table 3. Hepatic toxicity of PE immunotoxins (aTAC}—serum
`LDH values
`Dose post treatment
`
`'
`Dose
`5
`5
`4
`3
`2
`l
`(pg/kg)
`Bond
`ND
`7 l 5'
`ND
`4359‘
`2090‘
`566
`300"
`5-5
`ND
`ND
`ND ‘
`ND
`6000‘
`I725'
`600”
`5-5
`
`
`
`
`
`
`
`1500‘344333796‘389l000"S-C 386
`
`“Lack of appetite. nausea, diarrhea.
`"Lack of appetite. nausea, diarrhea, death.
`rNo symptoms, conjugate administered on day 0 and day 4 (l mg
`and 2mg, respectively).
`'
`'Values were 25D above the mean of normal. untreated monkeys
`(280), range 100—500.
`»
`ND = Not determined due to death or severe symptoms.
`Note: each set of values represents results from a single primate.
`
`ited no behavioral changes suggestive of toxicity. The
`thioether conjugates were thus less toxic than their
`disulfide counterpart as had been predicted by in vitro
`cytotoxicity and mouse toxicology studies.
`Conjugates of 9.2.27, using the two linkages, were '
`. also compared in nude mouse xenografts of human
`melanoma. The serum half-life of thioether linked
`
`increased to 3 hr as
`conjugates Tl,2 alpha was
`compared to 90 min for the disulfide linkage (Fig. 6).
`The beta phase was 10.4 hr for both conjugates. The
`resultant tumor localization and biodistribution at
`
`20 hr post administration is shown in Fig. 7. With the
`exception of kidney and intestine, higher levels of
`labeled PE were observed in tissues with thioether
`
`than disulfide linked conjugates, due to higher blood -
`levels. The tumor level of thioether linked conjugate ,
`was also increased, primarily. reflecting the higher
`blood levels; although with time this would be
`expected to result in improved tumor localization.
`
`DISCUSSION
`
`Holotoxin conjugates offer the potential for en-
`hanced potency when compared to hemitoxin or RIP
`conjugates (Quinones et al., 1984; Godal er al., 1986;
`Fitzgerald et al., 1984; McIntosh et al., 1983; Youle
`and Neville, I982; Vittetta et al., I984). As shown
`with ricin and diphtheria toxin, enhanced potency
`may be a reflection of enhanced internalization due to
`certain hydrophobic regions within B-chain (McIn-
`tosh er al., 1983; Youle and Neville, I982; Vitetta et
`
`IIO
`
`
`
`o 2466I0|2l4l5
`Timelhr)
`
`l820
`
`Fig, 6. Comparison of Pseudomonas exotoxin immunocon-
`jugates, serum half-life in Balb/c mice. 0, disulfide; A,
`thioether.
`
`PHIGENIX
`
`Exhibit 1021 -O7
`
`

`

`280
`
`ALTON C. MORGAN JR et a].
`
`4
`
`.
`E
`3'3
`3
`o
`‘7
`E
`
`E
`.
`.
`:3:
`i
`i
`M v
`.
`i
`i
`(I \fi
`i
`s
`\
`s.
`s
`if i! in ii tr #5 it
`it M u st 5! s! M
`LU
`LI
`sp sr
`TH
`KI
`[N
`
`t
`t
`i
`t
`§
`s
`n
`‘
`‘
`g
`i
`s
`N
`~
`t
`t
`w
`s
`V
`t
`V
`s
`V
`-w i.
`~
`'
`‘g‘
`~I
`5
`“iii“
`s
`o HMӤ5L!
`BL
`TA
`TU SK MU BO
`
`°
`
`l
`
`Fig. 7. Comparison of Pseudomonas exotoxin immunoconjugates, tumor localization and biodistribution.
`Animals were sacrificed and radiolabel in tissues assessed after 24 hr. I. thioether linkage; I, disulfide
`linkage. % inj. dose/gram = percent
`injected dose/gram. BL = blood, TA = tail = injection site.
`TU = tumor. SK = skin. MU = muscle. BO = bone, LU = lung. LI = liver. SP = spleen. ST = stomach,
`TH = thyroid. KI = kidney. IN = intestine.
`
`(11., 1984). Although no homologous hydrophobic
`region has been shown to exist
`in Pseudomonas
`exotoxin A. other as yet unidentified regions within
`PE may serve the same function (Allured e1 01., I986).
`Conjugates of PE are usually more potent
`than
`conjugates made with the same antibody and ricin
`A-chain (Bjorn et a[., 1985; Pirker et al., I985). This
`higher potency could translate in vivo to an ability to
`kill cells at lower antibody concentrations; i.e., condi-
`tions that may be most commonly achieved upon in
`viva administration. From studies with the 9.2.27
`
`it was shown that
`antibody in human melanoma,
`administration of 50 milligrams of antibody resulted
`in heterogenous antibody localization with higher
`levels in the most well vascularized regions and lower
`levels in sites more distant from blood or lymphatic
`vessels (Oldham et 0].. I984; Schroff et al., 1985). To
`achieve localization in more avascular sites doses of
`200—500 mg were required. Despite these dose limita-
`tions, antibody could be delivered to every tumor cell
`within a lesion with saturation of all antigen sites
`achieved in about half the patients. Thus it was
`extrapolated from these studies that for solid tumor
`therapy, conjugate would need to be administered at
`dose levels of 3—7 mg/kg in order to achieve complete
`or near saturation of antigenic sites. Thus,
`it
`is
`important
`to examine parameters which affect not
`only potency but also toxicity and delivery.
`One parameter which has a profound impact on
`these parameters is the type of bond between anti-
`body and the toxin. Ideally,
`it would be one that
`would produce the most potent conjugate with high
`selectivity, and high yields. By comparing different
`linkages with PE conjugates, we determined that
`thioether linkages yielded conjugates of at least equal
`potency to disulfide conjugates by in vitra evaluation
`in cell-free and cytotoxicity assays.
`In addition,
`
`thioether linkage produced highly potent conjugates
`with all four antibodies evaluated. Thioether bonds
`
`with A-chains or hemitoxins have usually resulted in
`conjugates with reduced potency (Lambert e! at,
`1985; Edwards et (11.. 1983). SMCC proved to be the
`optimum reagent
`for generating thioether bonds.
`resulting in easily controlled, rapid conjugate forma-
`tion. As far as we know, our results are the most
`efficient yet reported with regard to formation of l
`:
`l
`molar ratio conjugate with the lowest offering of
`toxin (1 : 1). This high efficiency is very important for
`large scale manufacturing necessary for clinical trials.
`In addition, we demonstrated that monovalent
`F(ab)’ conjuga