11Q)Am:S,~"1ff,Q ~'lf'lllliES~ ~ ll!ES !E~~ S BMID,~lU IEt:j
`UNITED STATES DEPARTMENT OF COMMERCE
`United States Patent and Trademark Office
`
`March 18, 2022
`
`TIDS IS TO CERTIFY THAT A~"NEXED HERETO IS A TRUE COPY FROM
`THE RECORDS OF THIS OFFICE OF:
`
`PATENT NUMBER: ./.,84./.,893
`ISSUE DATE: July .J, 1989
`
`By Authority of the
`Under Secretary of Commerce for Intellectual Property
`and Director of the Cnitecl States Patent and Trademark Office
`
`Certifying Officer
`
`Miltenyi Ex. 1005 Page 1
`
`

`

`United States Patent [19J
`H onsik et al.
`
`[111 Patent Number:
`[45) Date of P atent:
`
`4,844,893
`Jul. 4, 1989
`
`[54) EX VIVO EFFECTOR CELL ACTIVATION
`FOR 'f AR GET CELL .KILLING
`
`{75]
`
`[nventors: Cyril J. Honsik; Ralph A. Reisfeld,
`both of La Jolla, Calif.
`
`[73] Assignee: Scripps Clinic and Research
`Foundation, La Jolla, Calif.
`
`[211 Appl. No.: 916,173
`[22} Filed:
`Oct. 7, 1986
`Int. Cl.4 •••••.••••••••••••.•••••.••••••.••• ••••••••• A61K 39/395
`[51]
`[52] U.S. Cl. ..................................... 424/85.8; 424/93;
`435/240.2; 530/387; 530/828; 530/388;
`530/808
`[58) F ie.Id of Search ..................... 435/2, 4, 7, 29, 948;
`935/107, 108; 424/85, 93; 436/512, 519
`
`[56]
`
`Referenu s Cited
`U.S. PA TENT DOCUMENTS
`4,444,878 4/1984 Paulus .
`4,507,391 3/ 1985 Pukel er al ..
`4,675,287 6/1987 Reisfeld ................................. 435/ 68
`
`OTHER PUBLICATIONS
`Honsik, C. J. et al., Proc. Natl. Acad, Sci., U.S.A.,
`83:7893- 7897, (10- 1986).
`Cheresh, D. A. et al., Proc. Natl. Acad. Sci., U.S.A.,
`82:5155-5159, (8-1985).
`Park, M. M. et al., Cellular Immunology, 84:94-103,
`(1984).
`Mule, J. J. et al., Science, 225:1487-1489, (9-1984).
`
`Rosenberg, S. et al., New Engl. J. Medicin.e,
`313(23):1485-1492, (12-1985).
`Perez, P. et al., J. Exper. Med., 163:166-178, ( l-1986).
`Jung, G. er al., Proc. Natl. Acad. Sci., U.S.A.,
`83:4479-4483, (6-1986).
`Staerz, U. D. et al., Nature, 314:628-631, (4-1985).
`Cancer Research, (1984), 44:806-809, Dippold et al.
`Natural Immunity and Biological Response, (1985),
`4:253, Honsik et al.
`Eur. J. Immunol., (1986), 16:263-270, Staerz et al.
`Proc. Nat'l Acad. Sci. U.S.A., (1986), 83:1453-1457,
`Staerz et al.
`Proc. Nat'l Acad. Sci. U.S.A., (1985), 82:1242- 1246,
`Houghton et at.
`Primary Examiner- Margaret Moskowitz
`Assistant Examiner-Jeff. P. Kushan
`Attorney, Agent, or Firm- Dressler, Goldsmith, Shore,
`Sutker & Milnamow, Ltd.
`ABSTRACT
`[57]
`A method and composition for killing target cells is
`disclosed. The method utilizes ex vivo IL-2 activation
`of leucocyte effector cells and arming the activated
`leucocyte effectors with monoclonal antibodies whose
`Fe portions bind to the IL-2-activated effectors and
`whose paratopic portions immunoreact with an epitope
`expressed on the surfaces of the target cells. The com(cid:173)
`position contains a cytolytic amount of the armed, IL-2-
`activated effector cells clispersed in an aqueous physio(cid:173)
`logically tolerable diluent medium.
`
`20 Claims, 4 Drawing Sheets
`
`Miltenyi Ex. 1005 Page 2
`
`

`

`U.S. Patent
`
`Jul. 4, 1989
`
`Sheet 1 of 4
`
`4,844,893
`
`TARGET
`
`EFFECTOR
`
`COVA LENT BINDING
`
`FIG. I A
`
`T-Fc RECEPTOR BINDING
`
`FIG. I 8
`
`0
`
`'
`
`I/
`
`\\=v
`(C)'\
`---
`
`_ LYSIS_,,
`/
`
`E-Fc RECEPTOR BINDING
`
`FIG. ID
`
`--..: ___ __
`
`/
`'-..._ LYSIS _,,..,,
`
`Miltenyi Ex. 1005 Page 3
`
`

`

`U.S. Patent
`
`Jul. 4, 1989
`
`Sheet 2 of 4
`
`4,844,893
`
`FIG. 2
`
`u
`~
`u
`a.,
`C2. 40
`"' ~ 0
`
`20
`
`0 - - - - - - - - . - - - - - - - - - - -
`lDO
`25
`50
`Effector to Target Ratio
`
`Miltenyi Ex. 1005 Page 4
`
`

`

`U.S. Patent
`
`Jul. 4, 1989
`
`Sheet 3 of 4
`
`4,844,893
`
`FIG. 3
`
`100
`
`BO
`
`60
`
`V,
`
`·;:;;
`>-
`_,J
`
`L.l -u
`
`(U
`0..
`
`en 40
`~
`
`20
`
`D
`
`Normal #1
`
`Normal #2
`
`Patient #1
`
`Patient #2
`
`Patient #3
`
`Patient#4
`
`Miltenyi Ex. 1005 Page 5
`
`

`

`U.S. Patent
`
`Jul. 4, 1989
`
`Sheet 4 of 4
`
`4,844,893
`
`FIG. 4
`
`rll·2 AUGMENTATION OF AOC C
`
`100
`
`90
`
`80
`
`70
`
`·:;; 60
`.....
`>(cid:173)
`~ - 50
`u ..,
`~ 40
`
`C.
`
`30
`
`20
`
`10
`
`100:l
`
`50:1
`Effector: Target
`
`25: 1
`
`Miltenyi Ex. 1005 Page 6
`
`

`

`1
`
`4,844,893
`
`EX VIVO EFFECTOR CELL ACITV ATION FOR
`TARGET CELL KILLING
`
`The present invention was made with support of the 5
`Government of the United States, and the Government
`of the United States has certain rights in the invention.
`
`DESCRIPTION
`TECHNICAL FIELD
`The present invention relates to a cytotoxicity
`method, and particularly to a method for killing target
`cells such as tumor cells with antibody-directed, ex
`vivo-activated effector leucocytes.
`
`BACKGROUND OF THE INVENTION
`Neuroectodermal tumors are highly malignant, and
`include neuroblastomas, small cell cancinoma of the
`lung, gliomas, neuroblastomas and melanomas. Of the
`neuroectodermal tumors, neuroblastomas occur during 20
`infancy and early childhood. Except for Wilms' tumor,
`they are the most common retroperitoneal tumors in
`children. Neuroblastomas arise most commonly in the
`adrenal medulla, but they may also develop in other
`sympathetic ganglia within the thorax or abdomen. 25
`These tumors metastasize early with widespread in(cid:173)
`volvement of lymph nodes, liver, bone, lung and mar(cid:173)
`row. The prognosis is often good when the tumor is
`diagnosed prior to obvious metastasis, but with metasta(cid:173)
`sis, prognosis is poor despite the extensive use of radical 30
`surgery, deep X-ray therapy, and chemotherapeutic
`agents.
`Several antigenic determinants have recently been
`detected on neuroblastoma cells with monoclonal anti(cid:173)
`bodies (Mabs). See Seeger, Ann. Intern. Med., 91, 873 35
`(1982); Wikstrand et al., Cancer Res., 42, 267 (1982);
`Wikstrand et al., J. N euroimmunlogy, 3, 43 (I 982); Ei(cid:173)
`senbarth et al., Proc. Natl. Acad. Sci. USA, 76, 4913
`(1979); Liao et al.1 Eur. J. Immunol., 11, 450 (1981);
`Seeger et al., Cancer Res., 4, 2714 (1981); Kennett et al., 40
`Advances in N euroblastama Research, p . 209, Raven
`Press, N.Y. (Evans ed.) (1980); Seeger et al., J.
`Immwzol., 128, 983 (1982); Kemshead et a)., Pediatr.
`Res., 15, 1282 (1981). Gangliosides GD3 and GD2 are
`among the antigenic determinants detected on neuro- 45
`blastoma cells.
`Gangliosides (sialic acid-bearing glycolipids) are thus
`rapidly being characterized as tumor markers that are
`relevant
`target antigens for monoclonal antibody
`(Mab)-roediated immunotberapy [Dippold et al. ( 1983) 50
`Cancer Res. 44;806-810; Houghten et al. (1985) Proc.
`Natl. Acad. Sci. USA 82:1242- 1246; Hellstrom et al.
`(1985) Proc. Natl. Acad. Scl, USA 82:1499-1502; Che(cid:173)
`resh et al. (1985) Proc. Natl Acad. Sci USA
`82:5155-5159; Honsik et al. (1985) Natural Immunity 55
`and Biological Response 4:253; and Steplewski et al.
`(1985) Proc. Natl. Acad. Sci. USA 82:8653- 8657).
`The disialoganglioside G D 3, is expressed preferen(cid:173)
`tially on human mel.anorna cells [Dippold et al. (1980)
`Proc. Natl. Acad. Sci, USA 77:6114-6118; Cheresh et al. 60
`(1984) Proc. Natl. Acad. Sci., USA 81:5767-5771], and is
`an effective target in vitro for both complement(cid:173)
`medfated tumor cytolysis and antibody-dependent cel(cid:173)
`lular cytoxicity (ADCC) by Mabs of the l gG3 subclass
`[Hellstrom et al. (1985) Proc. Natl. Acad. Sci. , USA 65
`82:1499-1502; Cheresh et al. (1985) Proc. Natl Acad.
`Sci .• USA 82:5155-5159; and Honsik. et al. (1985) Natu-
`ral Immunity and Biological Response 4;253]. Mono-
`
`2
`clonal antibodies of the IgG3 class directed against
`GD3 have been reported to effectively suppress the
`establishment of human melanoma tumors in the xeno-
`transplant nude mouse mode [Hellstrom et al. (] 985)
`Proc. Natl, Acad. Sci.. USA 82:1499-1502; and Cheresh
`et al. (1985) Proc. Natl, Acad, Sci., USA 82:5155-5159).
`Murine mononuclear splenocytes "armed" whh anti(cid:173)
`GD3 Mabs were recently reported to eradicate well
`established and progressively growing human mela-
`10 noma tumors in nude mice [Honsik et al. (1985) Natural
`Immunity and Biological Response 4:253). Additionally,
`Houghten et al. (1985) Proc. N atl. Acad. Sci., USA
`82:1242- 1246, using Mab R24 (IgG3) directed to GD3
`(discussed in U.S. Pat. No. 4,507,391), observed major
`15 tumor regressions in 3 of 11 melanoma patients treated
`with that antibody in a Phase I clinical trial. It was also
`reported in Dippo}d et al., Cancer Res .. 44, 806 (1984),
`that Mab R24 could kill GD3-containing human mela-
`noma cells in vitro after prolonged exposure (greater
`than 24 hours) to the antibody suggesting an additional,
`as yet undefined, mechanism of tumor cell killing.
`T aken together, these findings demonstrate that the
`potential therapeutic efficacy of anti-GD3 Mabs war(cid:173)
`rants further study of ganglioside as immunotherapeutic
`targets.
`The fact that the GD2 antigen was shown to be heav(cid:173)
`ily expressed on most excised melanoma and SCCL
`tumors, as well as on numerous tumor cell lines, Pu.lee!
`et al., J. Exp. Med., 155, 1133 (1982) and yet is virtually
`absent from most normal tissues, suggests that it might
`be a good target antigen for in vivo specific immuno-
`therapy, and tumor imaging.
`In a recent report by Kipps et al., J. Exp. Med., 161,
`1 ( 1985), using isotype switch variants of a Mab directed
`to an epitope on Class I human histocompatibility anti(cid:173)
`gens, an IgG2a isotype variant was shown to be. more
`effective in directing ADCC than the corresponding
`lgG I or an lgG2b variant. Recent work from our own
`laboratory, Schulz et al.
`( 1985) J. Exp. Med.
`161:1315- 1325, also showed specific cytolysis. That
`work, using Mab 9.2.27, an lgG2a monoclonal antibody
`that 1mmunoreacts with a chondritin sulfate proteogly(cid:173)
`can that is preferentially expressed on human melanoma
`cells, illustrated that human melanoma tumors, estab(cid:173)
`lished and progressively growing in nude mice, could
`be eradicated by simultaneous injection of that Mab
`along with a relatively large dose of mononuclear
`s plenocytes. Neither the splenocytes nor the antibodies
`alone achieved significant tumor regression.
`Park et al., Cellular Immunol., 84, 94 (1984), reported
`that a monoclonal antibody of IgG2b isotype could
`sensitize K.562 human erythroleukernia cells to ADCC-
`mediated lysis. In that case, it was reported that the
`Mab accelerated killing of the target cells by large gran(cid:173)
`ular lymphocytes known to be enriched in natural killer
`(NK) cells.
`T aken together, the results of these studies indicate
`that monoclonal antibodies may not only be useful rea(cid:173)
`gents for the immunotherapy of cancer, but also that
`different Mabs can induce tumor killing by several dif-
`ferent or even a combination of effector mechanisms.
`Some additional recent reports suggest that mouse
`monoclonal antibodies are relatively well tolerated in
`humans and pose minimal risks and few, If any, side
`effects, Oldham et al., J. C/i11. Oncol, 2, 1235 (1984). In
`using murine Mab 9.2.27, discussed before, to treat mel(cid:173)
`anoma patients, that antibody was shown by the above
`
`Miltenyi Ex. 1005 Page 7
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`

`

`4,844,893
`
`4
`3
`pressed the Thy-1.1 alloantigen were killed by coating
`workers to localize specifically to the tumor site with
`little if any adverse side effects, but provided no appar-
`the target cells with the bispecific hybrid antibody and
`subsequent admixture of the coated target cells with
`ent clinical improvement of the disease over the period
`various dilutions of cytotoxic T lymphocytes bearing
`of time of their study in stage four patients with large
`5 the antigen recognized by the other paratopic portion of
`tutnor burdens.
`the hybrid. Potential target cells that did not bear the
`Another form of anti-tumor immunotherapy involves
`Thy-1.1 antigen were not killed. Related studies by that
`the exposure of mononuclear lymphocytes to the lym-
`group can be found in Staerz and Bevan ( 1986) Proc.
`phokine interleukin-2 (IL-2) to generate lymphokine
`Natl. Acad. Sci. USA, 83:1453-1457; Staerz. and Bevan
`activated killer (LAK) cells [Yron et al. (1980) J.
`Immunol 125:238-245; Lotze et al. ( 1981) Cancer Res. JO (1986) Eur. J. Jmmunol., 16:263-170; and Staerz and
`Bevan (1985) Eur. J. Immunol., 15:1172-1177.
`41:4420--4425; Odmm et al. (1982) J. Exp. Med.
`155:1823-1841; Grimm et al. (1983) J. Exp. Med.
`Still further, Perez et al. (1986) J. &p. Med.
`158:1356-1361].
`163:166-178 described a bispecific heteroconjugate
`For e~ample, Rosenberg ~t al. reported that the use of
`antibody that reportedly targets tIL-2-activated periph-
`~ecombmant ~1:,-2 (rl~•2),stlmulat~d human LAK ceJls 15 era! blood mononuclear cells (PBMCs) to tissue. Thus,
`m ph_ase _I cltmcal tr1~ls res~ted m mar~ed tumor _re-
`one portion of that heteroconjugate binds through its
`gress1on m several pattents with progressively growmg
`paratope [Fab fragment of Mab OKT3 (ATCC CRL
`neoplasms [Rosenberg et al: (1985) _New Engl. J. Med.
`8001)] to the activated PBMCs, while the other para-
`313:1485-1492]. In those_ clinical tnals, large numbers
`tope of the heteroconjugate (Fab fragment of a target-
`(I0'.0-1011) of human penpheral blood Jeuko~ytes were 20 binding Mab) binds to the target tissue.
`subJ~ted to doses of rIL-2 (1000 (!/1.~~ lO c~ls/ml)
`In their study, Perez et al. depleted the lymphocyte
`ex vtvo ~or up to 9~ hours befor~ being mJect~d ~!rave-
`effector cell population ofmonocytes as well as Leu-11-
`nously (i._v.) back. ~to ~e P~tt_ent~. These mdivtduals
`positive (Leu 11 +)cells.They reported that their IL-2-
`then received addition~ t.v. tnJ:ctmos of rIL-2 {up t_o
`activated effectors exhibited the TS antigen (were TS+)
`
`:oo,~ ~/kg body weight) dunng the course of theu- 25" and that elimination of T8+cells using Mab OKT8
`~~TCC CRL 8014) and complem~nt_eliminated cyiox-
`r~lthonugh some dramatic tumor regressions were
`ictty. Removal of T 4+ cells by a Similar strat~gy [Mab
`observed in several patients with extensive neoplasms,
`the treatment also produced a number of relatively O~!~ (A TCC CRL 8002) plus complement] m~re~sed
`severe clinical problems. Among those clinical prob- 30 lys1s m the presence and absence of IL-2 achvatmn.
`!ems were fluid retention, pulmonary edema and occa-
`Those ~uthor~ a~so reported t~e loss of abo~t one-~alf o(
`sional respiratory distress requiring intubation. Those
`the lyuc act1v1ty when_ ac~1vated, hybnd antibody-
`problems were a result of the direct toxicity effects of
`coated effectors were maintained free of IL-2 between 8
`rIL-2 infusions.
`and 24 hours.
`In a still more recent report, Rosenberg et al. (1986) 35 Bispecific heterobifunctional _ monoclon~l para topic
`Science 233:1318-1321, reported use of a three-part mo-
`~olecules 3?d methods of their preparation are also
`dality consisting of (a) an immunosuppressing drug,
`disclosed m U.S.
`Pat. No.
`4,444, 8_78
`and
`PC1;'/US82/01766 (W~ 83/02285), whose d1sclo~ures
`cyclophosphamide, along with (b) rIL-2-expanded tu-
`mor-infiltrating lymphocytes ('TIL) obtained from re-
`are t~corp?rated herem by reference. The .te~hmques
`sected t\llllors and (c) a relatively low dose of rIL-2 to 40 described m U.S. Pat. No. 4,350,626 for linking Pab
`successfully cure twelve mice with MC-38 colon adeno-
`portions of anti-tumor antibodies to tbe ricin A subunit
`carcinoma of advanced hepatic metastases, and to cure
`call: als<? b~ utilized for !~king F~b po_rtions of_ desired
`up to 50 percent of mice with advanced pulmonary
`antibodies m the preparation of bJSpectfic hybnds.
`metastases. The rIL-2 was reportedly administered sys-
`BRIEF SUMMARY OF THE INVENTION
`temically at 25,000 units three times per day for four 45
`days. Those tumors reportedly djd not respond to LAK
`therapy.
`Several groups have reported the use of bispecific
`heteroconjugate (hybrid) antibodies for mediating tar(cid:173)
`get cell lysis. For example, Jung et al. (1986) Proc. Narl. so
`Acad. Sci. USA 83:4479-4483 reported effective killing
`of human melanoma target cells that were coated with
`a bispecific hybrid antibody composed of one paratopic
`molecule that immunoreacted with an antigen on the
`target cell and a second paratopic molecule (OKT3, 55
`ATCC CRL 8001) that immunoreacted with human T
`cells. Human T cells previously stimulated by contact
`with OKT3 were used as the effector cells.
`Bevan, Staerz and co-workers have published several
`papers dealing with the use ofbispecific hybrid antibod. 60
`ies to kill target cells in murine systems. For example,
`Staerz et al. (1985) Nature 314:628-631 reported use of
`a hybrid antibody one of whose paratopic portions
`immunoreacted with an allotypic epitope on the T cell
`receptor of about 25 percelit of peripheral T lympho- 65
`cytes and the other of whose paratopic portions im(cid:173)
`munoreacted with the Thy-1.l alloantigen. That report
`showed that target S. AKR lymphoma cells that ex-
`
`The present invention contemplates a method for
`killing target cells and a composition useful in the
`method. The method utilizes effector cells that are acti(cid:173)
`vated ex vivo, freed from the activating agent, armed
`with antibodies that immunoreact with the target cells
`and thereafter co-incubated with target cells.
`Thus, one aspect of the invention contemplates a
`method of specifically killing target cells and comprises
`the steps of:
`{a) Activating ex vivo a culture of leucocytes such as
`peripheral blood mononuclear cells (PBMCs) or periph(cid:173)
`eral blood lymphocytes (PBLs) that contain lgGl,
`lgG2a, lgG2b, or lgG3 antibody Fe receptors with an
`amount of interleukin-2 (IL-2) sufficient to enhance the
`natural killer activity of those cells and form IL-2-
`activitated effector cells.
`(b) The IL-2-activated effectors are then separated
`from toxic amounts of IL-2, and the IL·2•freed cells are
`collected.
`(c) The Fe receptors of those IL-2-activated effector
`cells are then bound with monoclonal antibodies (Mabs)
`of class IgGl, lgG2a, IgG2b, or tgG3 Fe portions, to
`form armed, IL-2-activated effector cells. The para-
`
`Miltenyi Ex. 1005 Page 8
`
`

`

`4,844,893
`
`5
`6
`FIG. 1 is a schematic representation of various strate-
`topic portions of useful Mabs bind to (imnmnoreact
`with) an antigen expressed on the surface of target cells.
`gies of target cell lysis mediated by antibodies (the Y-
`(d) A cytotoxic amount of the armed, IL-2-activated
`shaped entity) and an effector cells. In strategy A, a
`effector cells is contacted with target cells.
`monoclonal antibody that immunoreacts with an epi-
`(e) That contact is maintained for a time period suffi- 5 tope on the effector cell such as the T cell receptor is
`cient to kill the target cells. This method is useful both
`depicted as being covalently linked to the surface of the
`in vitro and in vivo. The contact and maintenance steps
`target ceU. Strategy B illustrates a monoclonal antibody
`are carried out out in the substantial absence of exo-
`that immunoreacts with an effector cell epitope com-
`geneously supplied IL-2.
`plexed with the target cell via the Fe receptor of the
`Another embodiment of the above method contem- 10 target cell (T-Fc binding). Strategy C illustrates the use
`plates contacting the target cells with a cytolytic
`of a heterobifunctional hybrid monoclonal antibody
`amount of bispecific hybrid monoclonal paratopic mo!-
`that immunoreacts with an epitope on the target cell
`ecules that contain a first anti-T3 paratopic portion that
`and with an epitope on the effector cell; the two por-
`immunoreacts with the T3 antigen expressed on the
`tions of the hybrid antibody are illustrated by wider and
`surfaces of T cells and a second paratopic portion that 15 narrower lines. Aspects of strategy C can be urilized
`immunoreacts with a second epitope expressed on the
`herein. Strategy D illustrates the use of a monoclonal
`surface of the target cells. That second target cell epi-
`antibody that immunoreacts with an epitope on the
`tape is different from the antigen bound by the above,
`target molecule and is complexed to the effector cell via
`tirst-named Mabs, and the immunoreaction of those
`the Fe receptor of the effector cell (E-Fc binding).
`first-named Mabs does not interfere with the immunore- 20 Strategy Dis the principal strategy utilized in the inven-
`action of the second para topic portion of the bispecific
`tion discussed herein. This figure is adapted from Staerz
`hybrid molecules, or vice versa.
`et al., "Targeting for T-Lymphocyte-Mediated Lysis by
`The bispecific hybrid molecules can be contacted
`Hybrid Antibodies" in Cyrolytic Lymphocytes and Com-
`plement, E. Podack, ed., CRC Press, Boca Raron, Fla.
`with the target cells before, substantually simulta-
`neously, or after the contact with the armed, IL-2- 25 (in press).
`activated effector cells. Preferably, that contact is sub-
`FIG. 2 is a graph illustrating augmentation of anti-
`stantially simultaneous with contact by the armed, IL-2·
`body-dependent cellular cytotoxicity (ADCC) induced
`activated effectors. Most preferably, the bispecific hy-
`by short-term ex vivo activation of human peripheral
`brid molecules are coated on the surfaces of anti-T3-
`blood mononuclear cells (PBMCs) with recombinant
`activated T cells that express the T3 antigen prior to 30 interleukin-2 (rlL-2) on 51Cr-labeled M21 human mela-
`noma cells in vitro. Human PBMCs at 106 cells per
`contacting the target cells, and those hybrid-coated or
`-armed anti-T3-activated T cells are present along with
`milliliter (cells/ml) were activated by admixture with
`the armed, IL-2-activated effector cells.
`250 units/milliliter (U/ml) of rlL-2 at 37 degrees C. for
`Another aspect of this invention contemplates a tar-
`different time periods. The PBMCs were thereafter
`get cell killing composition. That composition com- 35 washed substantially free of toxic amounts of rlL-2 and
`prises an aqueous physiologically tolerable diluent me-
`admixed with Mab 11C64 and target M2 l cells at the
`dium that contains dispersed therein armed, IL-2-
`effector to target cell ratios (E:T) shown on the ab-
`activated effector cells from a leucocyte population in
`scissa. The otdinate is in units of percent specific lysis of
`an amount effective to kill target cells (a cytolytic
`the target cells. Quadruplicate determinations were
`amount). The IL-2-activated effector cells are armed 40 made for each point with standard deviations of less
`with monoclonal antibodies of class lgG1, IgG2a,
`than about 10 percenl The ex vivo activation times
`lgG2b, or IgG3 that are bound to the Fe receptors of
`utilized were as follows: No rIL-2 (O); 15 minutes of
`the activated cells, and whose paratopic portions im-
`rIL-2 activation (
`); 30 minutes of rlL-2 activation
`munoreact with an antigen expressed on the surfaces of
`( ); I hour of activation (A); 2 hours of activation ( );
`45 and 4 hours of activation (0).
`the target cells.
`The composition can also contain a cytotoxic amount
`FIG. 3 is a bar graph showing cytolysis by ADCC of
`51Cr-labeled M21 human melanoma cells challenged
`of bispecific hybrid monoclonal paratopic molecules
`that contain first anti-T3 paratopic portions that im-
`with PBMCs from four melanoma patients and two
`munoreact with the T3 antigen expressed on the sur-
`normal, asymptomatic persons used as controls, using a
`faces of T cells and second paratopic portions that im- 50 4-hour assay time. Patients 1, 2 and 4 were in remission
`munoreact with a second epitope expressed on the sur-
`whereas patient 3 had advanced disease and a large
`face of the target cells, as discussed before. Preferably,
`tumor burden. An effector to target cell ratio of 50: I
`the bispecific molecules are present in the composition
`was utilized with the following effector co-incubations
`coated on the surfaces of anti•T3-activited T cells as
`prior to effector-target admixture: 500 U/ ml rlL-2
`armed, anti-T3-activated T cells.
`55 (closed bars): 25 micrograms per milliliter (ug/rnl) Mab
`The present invention has several benefits and ad van-
`11 C64 (hatched bars); and 500 U /ml rIL-2 plus 25
`tages. Saliem among those benefits and advantages is
`ug/ml Mab 11C64 (open bars).
`that a leucocyte population can be activated and armed
`FIG. 4 is another set of bar graphs that illustrate
`ex vivo and then utilized to kill target cells in the ab,
`augmentation of ADCC by IL-2-activated human
`seoce of exogeneously supplied IL-2, thereby avoiding 60 PBMCs. Here, PBMCs from a normal, asymptomatic
`the toxicity problems associated with administration of
`huUlan donor were admixed with target human 51Cr-
`exogenously supplied IL-2 to a recipient animal.
`labeled M21 melanoma cells at different effector to
`Still further benefits and advantages of the invention
`target cell ratios, as shown. The P13MC treatments
`will be apparent from the description of the invention
`during the 4-hour assay illustrated included: admixture
`that follows.
`65 with 250 U/ml of rIL-2 (horizontallY.-hatched bars);
`admixture with Mab 11C64 at 25 micrograms per milli(cid:173)
`liter (ug/ ml) {diagonally-hatched bars); and co-incuba(cid:173)
`tion with 250 U/ml of rIL-2 plus 25 ug/ml Mab 11C64
`
`BRIEF DESCRIPTION OF THE ORA WINGS
`In the Figures forming a portion of this disclosure:
`
`Miltenyi Ex. 1005 Page 9
`
`

`

`4,844,893
`
`5
`
`8
`7
`duced when an antigen is immunologically bound by an
`(open bars). The percentage of specific lysis shown was
`obtained after subtraction of values for NK Jysis.
`antibody or a molecule containing a paratope. An im(cid:173)
`munoreactant is therefore a specific type of complex
`DETAILED DESCRIPTION OF THE
`formed between molecules.
`INVENTION
`The term "intact antibody" is used herein to distin-
`1. DEFINITIONS
`guish a complete molecule secreted by a cell from
`The term "antibody'' refers to a molecule that Is a
`other, smaller, molecules that also contain the paratope
`necessary for biological activity in an immunoreaction
`member of a family of glycosylated proteins called
`immunoglobulins that can specifically combine with an
`with an antigen.
`antigen. Such an antibody combines with its antigen by lO The paratopic molecules useful in the present inven-
`a specific immunologic binding interaction between the
`tion are monoclonal paratopic molecules. A ··mono-
`antigenic determinant (epitope) of the antigen and the
`clonal antibody" (often referred to herein as a "Mab") is
`antibody combining site (paratope) of the antibody.
`a antibody produced by clones of a hybridoma that
`An "antibody combining site" is that structural por-
`secretes but one kind of antibody molecule, and a mono-
`tion of an antibody molecule comprised of heavy and 15 clonal paratopic molecule is a monoclonal antibody.
`The hybridoma cell is fused from an antibody-produc-
`light chain variable and hypervariable regions that spe-
`cifically binds antigen. Using the nomenclature of
`ing cell and a myeloma or other self-perpetuating cell
`Jerne, Ann. lmmunoL (Inst, Pasteur), 125C, 373 (1974)
`line. Such antibodies were first described by Kohler and
`an antibody combining site is usually referred to herein Milstein, Nature, 256, 495-497 (1975), which descrip-
`20 tion is incorporated herein by reference.
`as a "paratope" •
`1:he word "~tigen'' ~as been used hist?rically to
`The terms "monoclonal paratopic molecule", "para•
`designate ~ entity that. 1s boun_d by an antibody, ~nd
`topic molecule", "monoclonal antibody" and "Mab"
`also to d~1gnate the entity that mduc~s _the produc~on
`are used interchangeably herein to refer to an intact
`of the antibody. More current usage limits the meanmg
`monoclonal antibody.
`of antigen!? that entity.~?und by an antibod_y, wher~ 25 The words "secrete" and "produce" are often used
`interchangeably in the art as to cells from which anti-
`the word. immunogen. is used for the e1:1t1ty _that Ill·
`duc~s ~tlbody productto~. Where_ an ~~ty ~iscussed
`body molecules are obtained. Cells that roduce anti-
`herem IS both 1mmunogemc and antlgemc 1t will gener-
`.
`p
`.
`.
`'
`bodies may, however, not secrete those molecules mto
`all b t
`ti
`d
`f .
`Y e erme an an gen.
`11
`h .
`.
`Th h b .d
`t
`·
`e y rt oma ce s o mterest
`th 30 t ell' environment.
`· d t
`"
`t"
`Tho phraso ,.
`t·
`'b d'
`·
`re,ers o
`..
`.. an 1gemc e ermman
`h
`h ·
`e
`•
`·
`1 1
`erem secrete monoc ona anti o 1es mt~ t eir environ-
`actual structural portion of the antigen that is immuno-
`logically bound by an antibody combining site. The
`men~. Ne~ert~eless, such c~lls ~e sometunes ~eferr~d to
`Jeme nomenclature defines an antigenic determinant as
`~erem as an~body-producmg cells'. an~. their antt~~~-
`1es 3:e so~etunes referred_ ~o as. bemg produced m
`an "epitope".
`The term "biologically active" refers at least to the 35 keepmg Wt~. the phrase ~t!lized m the_ art,
`. T~e tei1;I' .supern~tant'_ 1s us~d herein to refer to the
`ability to specifically bind antigen or specific antibody
`m vitro hqu1d med!um 10 which cells are cultured.
`combining site, although other general or effector capa-
`bility may be present as well. Biological activity of a M?noclonal paratop1<: molecules ~roduced by the .hy-
`paratopic molecule containing an antibody combining
`bn~oma cultures. of mter_est herem are secreted mto
`site is evidenced by the immunologic reaction of the 40 their cul~ure medium env_rronment. Therefore the cul-
`ture medium supe~tant 1s one prefe~ed so~ce of ~e
`antibody paratope (combining site) with its epitope
`(antigenic detenninant) upon their admixture in an
`monoclonal paratop1_c molecules and 1s readily obtatn-
`aqueous medium to fonn an immunoreactant, at )east at
`able free from hybndoma cells by well known tech-
`physiological pH values and ionic strengtbs. Preferably,
`niques. El!;emplary of such techniques is low speed cen-
`biological activity occurs under biologicitl conditions· 45 trifugation to sediment cells out of the liquid medium.
`i.e., those conditions wherein a paratope-containing Monoclonal paratopic molecules can alternatively be
`obtained from ascites tumor fluid (ascites fluid) of labo-
`molecule of this invention binds to its epitope within a
`pH value range of about 5 to about 9, at ionic strengths
`ratory animals into which the hybridoma tissue was
`such as that of distilled water to that of about one molar
`introduced. Both methods are described hereinafter.
`sodium chloride, and at temperatures of about 4 degrees 50
`Il. GENERAL DISCUSSION
`C. to about 45 degrees C. The monoclonal paratopic
`As already noted, several strategies have been devel(cid:173)
`molecules useful herein are biologically active.
`oped to obtain cytolysis. Treatments such as systemic
`"ELISA'' refers to an enzyme-linked immunosorbent
`administration of rIL-2 and the sole use of LAK cells
`assay that employs an antigen or antibody bound to a
`produced by activation with rIL-2 have been shown to
`solid phase and an enzyme-antibody or enzyme-antigen 55
`be too non-specific for generalized use. The more re(cid:173)
`conjugate to detect and quantify the amount of antigen
`cently reported results with TIL cells and IL-2 is re(cid:173)
`or antibody present in a sample. A description of the
`ELISA technique is found in Chapter 22 of the 4th
`portedly effective in mice, but still utilizes systemic
`Edition of Basic and Clinical Immunology by D. P. Sites
`administration of rlL-2. Since IL-2 can be toxic, it
`would be beneficial if such systemic uses could be
`et al., published by Lange Medical Publications of Los 60
`avoided.
`Altos, Calif. in 1982 and in U.S. Pat. Nos. 3,654,090;
`3,850,752; and 4,016,043, which are incorporated herein
`As also pointed out previously, the exquisite specific(cid:173)
`by reference.
`ity of monoclonal antibodies has been brought to bear
`"Enzyme" refers to a protein capable of accelerating
`upon cytolysis of pre-specified target cell populations.
`or producing by catalytic action some change in a sub- 65
`However, when used as the only modality, results with
`strate for which it is often specific.
`monoclonals alone have been uneven.
`"Immunoreactant" as used herein refers to the prod(cid:173)
`Four strategies that utilize target-ceU specific Mabs
`uct of an immunological reaction; i.e., that entity pro-
`along with effector cells are iJlustrated schematically in
`
`Miltenyi Ex. 1005 Page 10
`
`

`

`9
`FIG. 1. Strategy A of that Figure illustrates use ofMabs
`whose paratopic portions immunoreact with an epitope
`Ort the effector cell and whose Fe portions are cova(cid:173)
`lently linked to the target cell. Strategy B utilizes Fe
`receptors on the target cell to bind to the antibody 5
`(T-Fc receptor binding), while the paratope binds to the
`effector cell. Strategy C utilizes a hybrid monoclonal
`antibody, a first paratopic portion of which im(cid:173)
`munoreacts with the effector cell whereas the second
`paratopic po