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`PFIZER INC.
`
`PFIZER INC.
`Exhibit 1013
`
`Exhibit 101 3
`
`

`

`Downloaded from
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`jem.rupress.org
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` on September 9, 2014
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`PFIZER INC.
`Exhibit 1013-01
`
`COMPARISON OF THE EFFECTOR FUNCTIONS OF HUMAN
`IMMUNOGLOBULINS USING A MATCHED SET OF
`CHIMERIC ANTIBODIES
`
`BY MARIANNE BRLJGGEMANN,* GARETH T. WILLIAMS,4
`CAROL I. BINDON,* MICHAEL R. CLARK,* MATTHEW R. WALKER,*
`ROY JEFFERIS,t HERMAN WALDMANN,* AND MICHAEL S. NEUBERGER4
`
`From the *Division ofImmunology, Department ofPathology, Addenbrooke's Hospital,
`Cambridge CB2 2QQ ; the *Department ofImmunology, University ofBirmingham,
`Birmingham B15 2TJ; and the ¢Medical Research Council Laboratory ofMolecular Biology,
`Cambridge CB2 2QH, United Kingdom
`
`The five Ig classes have distinct biological roles. The IgG subclasses also show
`marked differences in their ability to mediate a variety of effector functions. A
`detailed comparison of the properties of the human Ig classes and subclasses is
`not only of interest for relating the functions of antibodies to their structures
`but is also of great importance for the implementation of therapy based upon
`immunological intervention . Indeed, this second aspect has become particularly
`significant as the development of techniques for the production of chimeric
`antibodies (1-3) should ensure that immunological intervention is now likely to
`make use of mAbs that have human effector functions; several cell lines have
`already been established that secrete chimeric antibodies directed against human
`cancer cells (4-6).
`Much of our knowledge of the properties of human Igs has been obtained
`from the study of myeloma proteins (reviewed in references 7-9). However,
`generally myeloma proteins do not bind identified antigens and, moreover,
`different myeloma proteins differ not only in their heavy chain class/subclass but
`also in their light chains and variable regions. As the initiation of antibody
`effector activity is usually a consequence of antigen binding and is indeed
`influenced by the quality of that binding, previous studies on myeloma proteins,
`although valuable, may not provide a sufficient picture of antibody effector
`function for therapeutic purposes. To carry out a detailed and controlled
`comparison of the effector functions of the different human C regions, we have
`established a panel of cell lines that secrete a matched set of human chimeric
`antibodies. These antibodies are directed against the hapten 4-hydroxy-3-nitro-
`phenacetyl (NP).' This specificity for a known hapten has allowed us to compare
`the effector functions of the IgG subclasses not only when interacting with
`soluble antigen but also when interacting with cell-bound antigen. This has
`enabled us to determine the efficacy with which different subclasses lyse their
`This work was supported by grants from the Medical Research Council and Wellcome Biotechnology.
`' Abbreviations used in this paper.
`ADCC, antibody-dependent cell-mediated cytotoxicity, NP,
`nitrophenacetyl; NIP, 5-iodo-4-hydroxy-3-nitrophenyl .
`J. Exp. MED. © The Rockefeller University Press - 0022-1007/87/11/1351/11 $2.00
`Volume 166 November 1987
`1351-1361
`
`1351
`
`
`(cid:9)(cid:9)
`(cid:9)
`
`Published November 1, 1987
`
`

`

`Downloaded from
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` on September 9, 2014
`
`PFIZER INC.
`Exhibit 1013-02
`
`1352
`
`EFFECTOR FUNCTIONS OF HUMAN CHIMERIC ANTIBODIES
`
`target cells by means of both cell-mediated and complement-dependent mecha-
`nisms.
`
`Materials and Methods
`Plasmid pSV-VNP (Fig. 1) contains a single Bam HI site for the insertion of
`Plasmids.
`restriction fragments containing C exons; this plasmid, as well as pSV-VNPHe, has been
`previously described (3). Derivatives of pSV-VNP with Xba I or Hind III sites in place of
`the Bam HI site were created by inserting octameric linkers into the Bam HI site that had
`been blunted using Escherichia coli DNA polymerase I Klenow fragment . Construction of
`the Hind III vector required prior destruction of two other Hind III sites in pSV-VNP by
`Klenow treatment. The origin and manipulation of the DNA inserts specifying the
`1 .
`different C genes is described in the legend to Fig.
`Cell Lines' and Transfection. J558L plasmacytoma cells (10) were obtained from Dr.
`S. L. Morrison (Dept. of Microbiology, College of Physicians and Surgeons, Columbia
`Univ., New York) and were grown in DME containing 10% FCS. Transfection was
`performed by spheroplast fusion (11) in the case of the IgG2-, IgG4-, and IgE-secreting
`cell lines, and by electroporation for the other constructs. For electroporation (12), 2 x
`10' cells that had been washed and resuspended in 0 .2 ml cold PBS were mixed with
`linearized plasmid DNA (15 gg in 20 Al H2O) and placed in a 1 cm x 0.4 cm x 4.5 cm
`plastic microcuvette that had been equipped with aluminum electrodes. The sample was
`given 15 2-kV pulses at 1-s intervals from an Apelex power supply, left on ice for 30 min,
`and then resuspended in 50 ml DME/10% FCS/gentamycin before transferring to 24-
`well plates. Selective medium containing mycophenolic acid was applied 24 h later (11).
`Anti-NP antibody in the culture medium was assayed by RIA (13) and cells were cloned
`by limiting dilution.
`For antibody purification, cells were allowed to grow
`Protein Purification and Analysis.
`to saturation in 2 liters ofDME/10% FCS. The culture medium was filtered, supplemented
`to 0.05% in NaN3, and passed over a 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP)-caproate
`Sepharose affinity column. This column was prepared according to a procedure provided
`by M. Cramer (University of Cologne, Cologne, Federal Republic of Germany). 80 ml of
`Sepharose CL-4B (Pharmacia Fine Chemicals, Uppsala, Sweden) that had been washed
`and resuspended in an equal volume of ice-cold 2 M Na2CO3, was activated by mixing
`with 4 ml of 0.5 g/ml CNBr in acetonitrile. After extensive washing, the activated
`Sepharose was mixed with 40 ml of bis(3-aminopropyl)amine that had been brought to
`pH 10 by the slow addition of 12 M HCl at VC.C. After gentle mixing overnight at VC,C,
`the derivatized Sepharose was washed in H2O, resuspended in cold 3% NaHC03 and
`mixed with 60 mg of NIP-caproate-O-succinimide (Cambridge Research Biochemicals,
`Harston, Cambridge) that had been dissolved in 1 ml dioxane. After mixing overnight at
`4°C, the NIPcap-Sepharose was washed with 0.1 M glycine/HCI, pH 3, followed by PBS
`and then stored in PBS/0.05% NaN3. After samples had been passed through the column
`and extensive washing, antibody was eluted from the sorbent with 0.5 mM NIP-caproate
`or NP-caproate in PBS and dialyzed extensively. Antibody samples were centrifuged
`extensively to remove aggregates.
`Protein samples were analyzed on SDS/PAGE gels (14). Samples were also purified on
`NIP-caproate Sepharose from cells labeled with [ 'Cl-L-lysine in the presence or absence
`of 10 pg/ml tunicamycin as previously described (15).
`Serological Assays.
`Serologic analysis was performed in an ELISA assay as follows: wells
`of flat-bottomed microtiter plates were coated overnight at 4°C with 100 ill purified anti-
`NP antibody diluted to 1 jig/ml in 0.2 M NaHC03, pH 9.6, or with purified human
`paraproteins of serologically defined class, subclass, and allotype . After washing with
`PBS/0 .05% Tween 20 (PBS/Tween), 100 pl of a 1 :100 dilution of mAb ascitic fluid in
`2 The cell lines will be deposited in the European Centre of Animal Cell Cultures, PHLS Centre
`for Applied Microbiology and Research, Porton Down, Salisbury, Wilts.SP4 OJG, United Kingdom,
`where they may be obtained by scientists affilitated with nonprofit research in pursuance of an
`academic research program .
`
`(cid:9)(cid:9)(cid:9)(cid:9)
`
`
`
`Published November 1, 1987
`
`

`

`(cid:9)(cid:9)(cid:9)
`
`
`Published November 1, 1987
`
`Downloaded from
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`jem.rupress.org
`
` on September 9, 2014
`
`PFIZER INC.
`Exhibit 1013-03
`
`1353
`BROGGEMANN ET AL .
`PBS/Tween was added to each well and incubated at 37°C for 2 h. After extensive
`washing with PBS/Tween, 100 A,1 of a 1 :4,000 dilution in PBS/Tween ofpolyclonal sheep
`anti-mouse Ig antibody conjugated to horseradish peroxidase (BDS Biologicals Ltd .,
`Birmingham, United Kingdom) was added and incubated for a further 2 h at 37'C before
`repeated washing in PBS/Tween. Bound sheep antibody was revealed by addition of the
`substrate (2 .2 mM o-phenylene diamine). The reaction was quenched after 15 min by the
`addition of 50 g.l 12.5% H2SO4 and the OD295 was measured using a Titertec Miniscan
`(Flow Laboratories, Irvine, Scotland). Positive reactions were scored where the OD295 was
`0.5 OD units above control values.
`Purified antibodies (100 tig/ml in PBS) were coated onto the wells
`Protein A Binding.
`ofmicrotiter plates (Cooke; Dynatech Laboratories, Inc., Alexandria, VA) before blocking
`with PBS/3% BSA. The wells were then incubated with ["'I]protein A (50,000 cpm per
`well; Amersham International, Amersham, United Kingdom), which had been diluted in
`citrate/phosphate buffer of the appropriate pH. The wells were washed at the same pH.
`Parallel incubations were also performed in which the plates were washed at the various
`pHs and the immobilized antibody developed with radio-iodinated monoclonal anti-A,
`antibody Ls136 (13) in PBS/1% BSA. This confirmed that the pH dependence observed
`in the protein A binding assays was indeed a result of the pH dependence of anti-
`body/protein A interactions rather than artefact due to washing the chimeric antibody
`off the microtiter plate.
`Human Clq that had been radioiodinated by the
`C1q Binding and Hemolysis Assays.
`lactoperoxidase method was a gift from Dr. N. HughesJones and B. Gorick (MRC Centre,
`Cambridge, United Kingdom). Human erythrocytes were coupled with NIP-kephalin (gift
`of Dr. U. Weltzien, Max Planck Institute For Immunology, Freiburg, Federal Republic
`of Germany) as previously described (16), and, if required, were labeled with sodium
`[5'Cr]chromate (Amersham International) as previously described (17). For C 1 q binding
`assays, washed NIP-red cells (20 t1 at 109 cells/ml) in PBS/1% BSA were coated with
`saturating amounts of antibody (10 tL1 at 200 ug/ml) and then supplemented with
`['251]Clq (10 Al at 10-60,ug/ml). After rotation at 37 ° C for 1 h, samples were centrifuged
`in microfuge tubes through 150 Al of oil of density 1 .028 (made by mixing four parts di-
`n-butyl phthalate with one part dinonyl phthalate). The cell pellets were separated by
`clipping off the bottom of the tube and the radioactivity in the bound and free fraction
`was determined. Controls were performed without added antibody; nonspecific binding
`was always <1 %.
`For hemolytic complement assays, NIP-human red cells were labeled with 15'Cr]-
`chromate, washed, and 50 ul of cells (109/ml) were mixed in microtiter wells with 50 tul
`of appropriate dilutions of the chimeric antibodies. After 10 min at room temperature,
`100 Al of diluted human complement was added to give a final concentration of 20%.
`After a 30-min incubation at 37°C, the cells and supernatant were separated by centri-
`fugation (100 g, 2 min). Samples incubated with no antibody were used to calculate the
`spontaneous "Cr release. The percentage specific "Cr release is calculated as: Percent
`release = 100 X [(Test release - spontaneous release)]/[(Total radioactivity) - (sponta-
`neous release)].
`This was performed essentially as de-
`Antibody-dependent Cell-mediated Cytotoxicity.
`scribed (18) but with modifications. Cells (2 X 106) of the human T cell line HPB-ALL
`were labeled in 100,ul of medium with 50 tiCi of sodium [5'Cr]chromate for 30 min at
`37 °C, and 2 Al of NIP-kephalin (100 ug/ml) was then added and the incubation was
`continued for another 15 min. These target cells were then washed with Hepes-buffered
`Iscove's modified DME containing 1 % BSA. The effector cells were obtained from healthy
`donors by venipuncture and, after defibrination with glass beads, mononuclear cells were
`isolated by centrifugation on Ficoll-Hypaque (19) and cultured overnight in Iscove's
`MDM/5% FCS. Antibody-dependent cell-mediated cytotoxicity (ADCC) was measured by
`mixing labeled target cells (50 Al at 4 X 105/ml) with dilutions (100 Al) of the chimeric
`antibodies and then supplementing with the effector cells (50 11 at 1 .2 X 10'/ml). The
`cells were pelleted (200 g, 10 min) and incubated at 37'C for 4 h. The radioactivity in
`the supernatant was then measured. Assays were performed in triplicate and controls
`
`

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`(cid:9)
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`(cid:9)
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`(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)
`(cid:9)
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`(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)(cid:9)
`
`
`(cid:9)(cid:9)(cid:9)
`Published November 1, 1987
`
`1354
`
`B
`
`EFFECTOR FUNCTIONS OF HUMAN CHIMERIC ANTIBODIES
`pSV - VNP
`
`0
`
`L VNp
`r,a
`
`gpt
`
`amp
`.-
`
`Bam or xba or Hind
`
`1
`
`2
`
`3
`
`4
`
`X
`
`'
`
`n 2 3
`
`'y 1
`
`_
`
`n 2 3
`1M=
`2 3
`
`n
`
`n z 3
`
`72 = M
`
`-y 3
`
`y4
`
`_ 0
`
`=_
`=
`
`E
`m
`
`E
`m
`
`tkb
`
`m
`
`3
`,
`E ,
`E
`2
`m~Lm
`0 ME=
`'
`
`2
`
`3
`
`e
`
`tx 2
`
`a
`
`= Nor-
`
`Downloaded from
`
`jem.rupress.org
`
` on September 9, 2014
`
`PFIZER INC.
`Exhibit 1013-04
`
`Structure of plasmids. (A) The
`FIGURE 1.
`pSV-VNP vector. The mouse IgH enhancer (E)
`is located upstream of the leader (L) and V
`exons of the VNP variable region. The family
`of vectors have a Bam HI, Hind III, or Xba I
`site located downstream ofVNP for the insertion
`of germline C regions. The transcriptional
`orientations of the L/VNP, gpt, and ampicillin
`resistance (amp) genes are marked with arrows .
`(B) The CH region inserts. The exons for the
`3' end of the mRNA of the membrane forms
`of the heavy chains are not depicted, although
`they are probably present in their entirety in
`the y but not in the W, e, or a constructs. Only
`restriction sites at the ends of the Ca gene
`inserts are depicted. The C inserts originate:
`A, Xba I fragment of phage XC75R (20); 71,
`Hind III fragment of phage Charon 4A
`HIgy10 (21); y2 and y4, Hind III-Bam HI
`fragments of cosIg8 (22), which were obtained
`as Bam HI-Bam HI fragments after cloning of
`the genomic Hind III fragments in pUC19; y3
`was obtained as a 7 .4-kb Hind III fragment
`from either cosIg1 (22) yielding an IgG.G3m(g)
`antibody or from phage XEZZy3 (23) yielding
`an IgG.Gam(b) antibody. The a2 insert origi-
`nated from cosIgI0 (22) and was obtained as a
`Hind III fragment from a plasmid subclone
`whose insert extended from the Pst I site 5' of
`C 1 to the Bgl II site 3' of C.3. Plasmid pSV-
`VNpHe has been described previously (3).
`
`performed without effector cells (no lysis was then observed). The specific 5'Cr release
`was calculated as described above.
`
`Results
`The basic plasmid that was
`Cell Lines Expressing Human Chimeric Antibodies.
`used for the construction of the chimeric heavy chain genes (pSV-VNP) is depicted
`in Fig.
`1 . The plasmid contains the V gene of a mouse anti-NP antibody.
`Upstream of the V,, promoter is the mouse IgH enhancer element; downstream
`of VH is a unique Bam HI restriction site. Different human C,, genes were inserted
`either into this Bam HI site or into derivatives of pSV-VNP in which the Bam HI
`site was converted to a Hind III or Xba I site by insertion of linkers. The exact
`C,, fragments inserted are described in Fig.
`1 . In the case of 73, two constructs
`were assembled with different y3 genes that originated from different sources,
`having been cloned from different individuals.
`The gpt marker present in plasmid pSV-VNP allows stably transfected cells to
`be selected by virtue of their resistance to the drug mycophenolic acid. The
`plasmids were introduced into the mouse plasmacytoma J558L as described in
`Materials and Methods. This plasmacytoma secretes a X, light chain but expresses
`no heavy chain of its own. The VA of the endogenous light chain complements
`the VNP of the transfected heavy chain to yield an NP-specific antibody; such
`antibodies display a slightly greater affinity for the iodinated derivative, NIP,
`than for NP itself.
`Antibodies were purified to homogeneity from the culture medium of cloned
`
`

`

`
`(cid:9)(cid:9)
`(cid:9)
`(cid:9)
`
`Published November 1, 1987
`
`BROGGEMANN ET AL.
`
`1355
`
`Downloaded from
`
`jem.rupress.org
`
` on September 9, 2014
`
`PFIZER INC.
`Exhibit 1013-05
`
`Analysis of chimeric antibodies by SDS-PAGE. (A) Antibodies from cells biosyn-
`FIGURE 2 .
`thetically labeled in the presence or absence of tunicamycin (Tm) were analyzed on 10% SDS-
`PAGE gels after reduction. (B) Antibodies purified from culture supernatants were analyzed
`without reduction on a 7% SDS-PAGE gel. The positions of molecular mass (kD) markers are
`indicated.
`
`transfectants ; yields were typically in the range 2 to 10 mg/liter although
`sometimes up to 30 mg/liter was achieved.
`Analysis ofAntibodies on SDS/PAGE.
`To characterize the chimeric antibodies,
`purified samples were reduced and analyzed by SDS-PAGE. The mobilities of
`the heavy chains were much as predicted on the basis of their DNA sequences
`except for u, e, and a2. We have previously shown (3) that the chimeric e heavy
`chains become heavily glycosylated . We therefore resorted to biosynthetic label-
`ing to compare the antibody normally secreted by the transfectants with that
`made in the presence of the glycosylation inhibitor tunicamycin. The results
`(Fig. 2A) demonstrate the IgM, IgE, and IgA2 secreted by the J558L transfec-
`tants are indeed heavily glycosylated on their heavy chains. A much smaller
`amount ofglycosylation is apparent on the 7 heavy chains. The sizes of the heavy
`chains of the unglycosylated antibodies are exactly as predicted from the DNA
`sequences (the y3 heavy chains standing out from the other 7 chains because of
`the long hinge) .
`To examine whether the secreted antibodies were correctly assembled, unre-
`duced samples were analyzed on 7% SDS-PAGE gels (Fig. 2B). The IgGs and
`IgE exhibit mobilities consistent with their having the expected H2L2 structures
`whereas the IgM is clearly of very large molecular weight and, as expected for
`the pentameric form, scarcely enters the gel . IgA2 gives the most complex
`pattern and appears to contain H, HL, H2L2, and (H2 L2)2 forms; this may reflect
`the secretion of some noncovalently linked molecules.
`Serological Characterization.
`For potential therapeutic applications, it is clearly
`important to establish whether chimeric human antibodies secreted by a mouse
`plasmacytoma are indeed homologous to human mAbs. While the analysis on
`SDS-PAGE indicated that the chimeric antibodies resembled their human coun-
`terparts as regards the molecular weights of both the native and the unglycosy-
`lated heavy chains, we decided to extend this characterization to include a wide
`
`

`

`(cid:9)
`
`
`(cid:9)(cid:9)(cid:9)
`Published November 1, 1987
`
`1356
`
`EFFECTOR FUNCTIONS OF HUMAN CHIMERIC ANTIBODIES
`
`Downloaded from
`
`jem.rupress.org
`
` on September 9, 2014
`
`PFIZER INC.
`Exhibit 1013-06
`
`Cy3
`
`8a4
`J1 0d
`x3.8
`1al
`3910
`49/2C3
`RJ4
`
`non-IgG4
`non-IgG4
`non-IgG4
`non-IgG4
`IgG4
`
`I HINGE
`
`SG4-IgG3
`
`
`
`I INTERDOMAIN I
`
`Cy2
`
`We10
`QC1l
`WC2
`QF1"
`VC9'
`F7c*
`
`JD312
`G7c
`JL512
`NL16
`GON1
`GON2
`GB7B
`HP6031
`A55
`F10F
`JD79
`HP6080
`
`IgG1
`IgG1
`IgG2
`IgG2
`IgG4
`non-19G2
`non-G3m(g)
`non-G3m(g)
`non-G3m(g)
`G3n(O)
`
`Cyl
`
`TN15
`HP6044
`HP6046
`TN10 - non-IgG1
`TH14
`Glm(f)
`IgG2
`HP6014
`
`anon-19G3
`Molecular localization of epitopes recognized by mAbs to human IgG. For the
`FIGURE 3.
`origin and specificity of these antibodies see references 24 and 25. The anti-IgA antibodies
`are described in reference 26.
`
`range of serological markers. The chimeric antibodies were therefore typed
`using 37 different monoclonal anti-human antibodies (Fig. 3). On the basis of
`this typing (Table I), it will be seen that the two IgG3s are allotypically distinct.
`The y3 gene of the XEZZy3 clone (23) yields an IgG3 of the nG3m(g) iso-
`allotype; in other words, it is not of the G3m(g) allotype. Indeed, the DNA
`library from which the XEZZ-y3 clone was isolated was made from a Tunisian
`individual known to be homozygous for the G3m(b) allotype (23). However, the
`y3 gene from the cosIgI clone (22) yields an IgG.G3m(g) antibody. The chimeric
`IgA2 types as nA2m(2), a result that is predicted from the sequence of the a2
`gene used here which shows it to be of the A2m(1) allotype (27). Thus, all the
`chimeric antibodies type exactly as expected and the chimeric antibodies are not
`therefore serologically distinguished in these assays from authentic human anti-
`bodies as regards the CH region determinants.
`The chimeric antibodies were further tested in their
`Binding to Protein A.
`binding of radio-iodinated Staphylococcal protein A over a pH range from 3 to
`10 . The results (Fig. 4) indicate that the chimeric IgG1, IgG2, and IgG4 bind
`well to protein A and show a very similar pH dependence, binding occurring
`down to pH 4.5 . This similarity of pH dependence could be due to titration of
`one of the conserved residues in the antibody CH2/CH3 domain binding site for
`protein A (see review in reference 9) or could be due to titration of one of the
`protein A side chains themselves. No protein A binding was detected with IgM,
`IgE, IgA2, or either of the IgG3s.
`The binding of C1q to the chimeric antibodies was
`Binding of Human CIq.
`
`

`

`
`
`(cid:9)(cid:9)(cid:9)
`
`(cid:9)(cid:9)
`Published November 1, 1987
`
`BROGGEMANN ET AL.
`
`135 7
`
`TABLE I
`Serological Typing of Chimeric Antibodies
`
`Downloaded from
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`PFIZER INC.
`Exhibit 1013-07
`
`Specificity
`
`Number
`used
`
`IgG2
`IgGI
`+
`+
`7
`IgG
`-
`+
`1
`IgGI
`-
`-
`1
`Glm(f)
`+
`-
`1
`IgG2
`+
`-
`I
`IgG2,3,4
`+
`+
`5
`IgG1,2,4
`-
`-
`1
`IgG3
`-
`-
`1
`G3m(u)
`+
`+
`4
`nG3m(g)
`-
`-
`1
`IgG4
`+
`+
`2
`IgG1,2,3
`ND
`IgA
`ND
`1
`ND
`ND
`3
`IgAI
`ND
`ND
`I
`IgA2
`ND
`ND
`1
`nA2m(2)
`IgM
`6
`ND
`ND
`* The y3 C region derives from clone coslgl (22).
`$ The y3 C region derives from phage XEZZy3 (23) .
`
`Chimeric antibody
`IgG3*
`IgG3$
`IgG4
`+
`+
`+
`-
`-
`-
`-
`-
`-
`-
`-
`-
`+
`+
`+
`-
`-
`+
`+
`+
`-
`+
`+
`-
`-
`+
`+
`-
`-
`+
`+
`+
`-
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`
`IgA2
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`+
`-
`+
`+
`ND
`
`IgM
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`ND
`+
`
`Ig6l
`
`IgG2
`
`IgG4
`
`zo,ooo
`
`5,000
`
`1,000-
`
`2001W
`
`'9'
`
`a v
`
`c0aa
`
`c a
`
`4
`
`5
`
`i3
`
`-
`
`7
`6
`7
`6 7 8
`pH
`pH
`pH
`Binding of protein A to chimeric antibodies. The amount of radio-iodinated
`FIGURE 4.
`protein A bound to immobilized antibodies is plotted as a function of pH. No binding was
`detected to the IgM, IgE, IgA2, or either of the IgG3s over the entire pH range tested .
`
`9
`
`10
`
`3
`
`4 5
`
`6
`
`8
`
`9 10
`
`3
`
`4
`
`5
`
`8
`
`9 10
`
`assayed using various concentrations of radiolabeled C1qand hapten-derivatized
`red cells that were coated with amounts of antibody that were shown to be
`saturating . The results (Fig. 5, left) show that Clq binding is detected with the
`IgM, IgGI, and both IgG3 antibodies but not with IgG2, IgG4, or IgE. The
`binding to IgM is weaker than to IgG 1 and IgG3 and, ofthese two IgG subclasses,
`the IgG3s bound more Clq than did the IgGI .
`Antibodies were tested over a wide concen-
`Complement-mediated Hemolysis .
`tration range for their ability to lyse hapten-coupled human red cells in the
`presence of human complement . The results (Fig. 5, right) show that IgG2,
`IgG4, and IgE did not mediate hemolysis, whereas IgM, IgG1, and both IgG3s
`were effective. Indeed, it is notable that the IgG 1 was considerably more effective
`in this hemolytic assay than the IgG3s. We have previously demonstrated (17) that
`
`

`

`1358
`
`EFFECTOR FUNCTIONS OF HUMAN CHIMERIC ANTIBODIES
`IgG.G3m(g)
`
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`
`jem.rupress.org
`
` on September 9, 2014
`
`PFIZER INC.
`Exhibit 1013-08
`
`((tg/ml)
`Concentration
`Antibody
`Input C1q Concentration (NWml)
`Binding of Clq and complement-dependent hemolysis by chimeric antibodies.
`FIGURE 5.
`(Right) Binding of various concentrations of radiolabeled Clq to NIP-human red cells that
`have been coated with saturating amounts of chimeric antibody. (Left) Lysis of [51Cr]NIP-
`human red cells by various concentrations of chimeric antibody in the presence of human
`complement . The allotypes of the two IgG3 antibodies [IgG.G3m(b) and IgG.G3m(g)] are
`indicated.
`
`" IgG1
`
`AL IgG .G3m(b)
`V IgG .Wrn(g)
`
`0 IgM
`I.G2
`+I :A
`" IgG4
`IgE
`
`1 -3 °f.2
`1.4
`5 I -5
`-1
`10
`10
`10
`10
`10
`10
`Concentration (mg/ml)
`Antibody
`
`FIGURE 6. ADCC by chimeric antibodies. The
`lysis of [51 Cr]NIP-conjugated human target cells
`(HPB-ALL T cell line) by human PBMC was meas-
`ured as a function of the concentration of anti-NP
`chimeric antibody .
`
`the efficacy of anti-NP antibodies in this hemolytic assay is critically dependent
`on their affinity for the hapten. However, this cannot explain the results obtained
`here. Binding-inhibition assays (not shown) indicate that the IgG 1 and IgG3
`antibodies have indistinguishable affinities for NIP-caproate. Furthermore, the
`IgGI and IgG3 samples gave very similar titers in hemagglutination assays using
`hapten-derivatized red cells. Thus, although IgG3 is the best subclass as regards
`C 1 q binding it is considerably less potent than IgG 1 in hemolytic assays. Inter-
`estingly, a similar but less marked reversal of hemolytic efficiency as compared
`with Clq binding is observed with the two IgG3 allotypes. Whereas the
`IgG.G3m(g) binds Clq slightly better, the IgG.G3m(b) is somewhat more effective
`in hemolysis.
`The efficacy of the antibodies in
`Antibody-dependent Cell-mediated Cytotoxicity .
`mediating ADCC was tested using a hapten-derivatized human T cell line (HPB-
`ALL) as target and mononuclear cells from human volunteers as effector cells.
`It was found (Fig. 6) that only IgGI and IgG3 were effective in mediating ADCC
`with the IgG 1 showing greater potency than either of the IgG3s.
`
`
`(cid:9)(cid:9)
`
`
`(cid:9)(cid:9)(cid:9)
`(cid:9)
`Published November 1, 1987
`
`w QVve0m
`
`

`

`(cid:9)
`Published November 1, 1987
`
`Downloaded from
`
`jem.rupress.org
`
` on September 9, 2014
`
`PFIZER INC.
`Exhibit 1013-09
`
`1359
`
`BROGGEMANN ET AL.
`Discussion
`The cell lines established during the course of this work provide a source of
`chimeric antibodies that can be easily purified to homogeneity in a one-step
`purification on hapten sorbents. To confirm that these chimeric human antibod-
`ies made in mouse plasmacytoma cells exhibit the features expected of antibodies
`possessing human C regions, we have characterized the antibodies as regards
`their serological properties, the mobilities in SDS-PAGE of both the native and
`the unglycosylated forms as well as their binding to protein A. In all these
`respects, the chimeric antibodies behave exactly as expected from their authentic
`human counterparts.
`The known antigen specificity of these chimeric antibodies has allowed us to
`assay their efficacy in complement-mediated lysis and in ADCC. This has given
`rise to some novel and unexpected findings. The hierarchy of the binding of the
`aggregated chimeric IgG subclasses to human Clq described here agrees well
`with the previous results on the hierarchy in C1 q binding of monomer IgG
`myeloma subclasses (28); however, aggregated IgG will bind with a higher avidity
`than monomeric IgG. Nevertheless, when we measured the efficacy of the
`antibodies in complement-mediated hemolysis, a very different result was ob-
`tained. It is evident that the IgGI is very much more effective than the IgG3 in
`this assay. At present it is not clear why there is this lack of correlation between
`Clq binding and hemolytic efficacy; it will clearly be of interest to compare the
`IgG1 and IgG3 chimeric antibodies in intermediate stages of the pathway such
`as C1 activation and C4 and C3 binding. It will also be of interest to determine
`how the comparative hemolytic efficacy of IgGI and IgG3 depends upon the
`density and nature of the antigen on the target cell.
`The results presented here also indicate that the IgGI and IgG3 are the only
`antibodies that are really effective in ADCC. The greater effectiveness of IgGI
`as compared with IgG3 is a novel and interesting finding. The relative inactivity
`of the IgG2 and IgG4 antibodies was, however, predictable. Although the exact
`nature of the effector cell responsible for mediating ADCC has not been unam-
`biguously identified, the Fc receptors found on the possible effector cell types
`(lymphocytes and monocytes) bind IgGI and IgG3 much better than IgG2 and
`IgG4 (reviewed in reference 9).
`Finally, it is worth noting that the results obtained in this work suggest that
`for many therapeutic purposes an IgGI antibody might be greatly preferred to
`the other IgG subclasses as it appears to be considerably more effective in
`mediating both complement-dependent lysis and ADCC.
`Summary
`Cell lines have been established that secrete a matched set of human chimeric
`IgM, IgG l, IgG2, IgG3, IgG4, IgE, and IgA2 antibodies that are directed against
`the hapten 4-hydroxy-3-nitrophenacetyl. These chimeric antibodies secreted
`from mouse plasmacytoma cells behave exactly like their authentic human
`counterparts in SDS-PAGE analysis, binding to protein A and in a wide range of
`serological assays. The antibodies have been compared in their ability to bind
`human Clq as well as in their efficacy in mediating lysis of human erythrocytes
`in the presence of human complement. A major conclusion to emerge is that
`
`

`

`Downloaded from
`
`jem.rupress.org
`
` on September 9, 2014
`
`PFIZER INC.
`Exhibit 1013-10
`
`1360
`
`EFFECTOR FUNCTIONS OF HUMAN CHIMERIC ANTIBODIES
`
`whereas IgG3 bound C1 q better than did IgG l , the chimeric 1gG1 was much
`more effective than all the other IgG subclasses in complement-dependent
`hemolysis. The IgG1 antibody was also the most effective in mediating antibody-
`dependent cell-mediated cytotoxicity using both human effector and human
`target cells. These results suggest that IgGI might be the favoured IgG subclass
`for therapeutic applications .
`We are indebted to Catherine Teale and Mark Frewin for invaluable technical assistance
`and to B . Gorick, T. Honjo, N. HughesJones, M.-P. Lefranc, C. P. Milstein, T. H.
`Rabbitts, and U. Weltzien for gifts of DNA clones or reagents.
`
`Receivedfor publication 27July 1987.
`
`References
`1 . Boulianne, G. L., N. Hozumi, and M. J. Shulman . 1984. Productio n of functional
`chimaeric mouse/human antibody . Nature (Lond.). 312 :643.
`2. Morrison, S. L., M. J. Johnson, L. A. Herzenberg, and V. T. Oi. 1984 . Chimeric
`human antibody molecules : mouse antigen-binding domains with human constant
`region domains . Proc. Natl. Acad . Sci. USA. 81 :6851 .
`3. Neuberger, M. S., G. T. Williams, E. B. Mitchell, S. S. Jouhal, J. G. Flanagan, and
`T. H. Rabbitts. 1985 . A hapten-specific chimaeric IgE antibody with human physio-
`logical effector function. Nature (Lond.). 314 :268 .
`4. Sahagan, B. G., H. Dorai, J. Saltzgaber-Muller, F. Toneguzzo, C. A. Guindon, S. P.
`Lilly, K. W. McDonald, D. V. Morrissey, B. A. Stone, G. L. Davis, P. K. McIntosh,
`and G. P. Moore. 1986 . A genetically-engineered murine/human chimeric antibody
`retains specificity for human tumor-associated antigen] Immunol . 137 :1066.
`5. Sun, L. K., P. Curtis, E. Rakowicz-Szulczynska, J. Ghrayeb, S. L. Morrison, N. Chang,
`and H. Koprowski . 1986 . Chimeric antibodies with 17-IA-derived variable regions
`and human constant regions . Hybridoma . 5:S17.
`6. Liu, A. Y., R. A. Robinson, K. E. Hellstr6m, E. D. Murray, C. P. Chang, and I .
`Hellstr6m. 1987 . Chimeric mouse-human IgGI antibody that can mediate lysis of
`cancer cells. Proc. Natl. Acad . Sci. USA. 84:3439.
`7. Spiegelberg, H. L. 1974 . Biological activities of immunoglobulins of different classes
`and subclasses. Adv. Immunol. 19:259.
`8. Winkelhake, J. L. 1978 . Immunoglobulin structure and effector functions. Immuno-
`chemistry. 15:695 .
`9. Burton, D. R. 1985 . Immunoglobulin G: functional sites. Mol. Immunol. 22:161 .
`10. Oi, V. T., S. L. Morrison, L. A. Herzenberg, and P. Berg . 1983 . Immunoglobulin
`gene expression in transformed lymphoid cells. Proc. Natl. Acad . Sci. USA. 80:825.
`11 . Neuberger, M . S., and G. T . Williams . 1986 . Protein engineering of antibody . In
`Protein Engineering: Applications in Science, Medicine and Industry. M. Inouye and
`R. Sarma, editors . Academic Press, New York. 311-317 .
`12 . Potter, H., L . Weir, and P. Leder. 1984 . Enhancer-dependent expression of human
`is immunoglobulin genes introduced into mouse pre-B lymphocytes by electropora-
`tion . Proc. Natl. Acad. Sci. USA. 81 :7161 .
`13. Reth, M., T. Imanishi-Kari, and K. Rajewsky . 1979 . Analysis of the repertoire of
`anti-NP antibodies in C57BL/6 mice by cell fusion. Eur. J. Immunol. 9:1004.
`14. Laemmli, U. K. 1970 . Cleavage of structural proteins during the assembly of the
`head of bacteriophage T4. Nature (Lond.). 2278:680.
`15. Galfre, G., and C. Milstein. 1981 . Preparation of monoclonal antibodies: strategies
`and procedures . Methods Enzymol . 73 :3.
`
`(cid:9)
`Published November 1, 1987
`
`

`

`(cid:9)
`Published November 1, 1987
`
`Downloaded from
`
`jem.rupress.org
`
` on September 9, 2014
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`PFIZER I