PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`Internatlonal Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 7 :
`C07K 16/00
`29 June 2000 (29.06.00)
`
`(43) International Publication Date: (11) International Publication Number:
`
`WO 00/37504
`
`(21) International Application Number:
`
`PCT/US99/30895
`
`(74) Agent: HARE, Christopher, A.; Abgenix, Inc., 7601 Dumbar—
`ton Circle, Fremont, CA 94555 (US).
`
`(22) International Filing Date:
`
`23 December 1999 (23.12.99)
`
`(30) Priority Data:
`60/113,647
`
`23 December 1998 (23.12.98)
`
`US
`
`(71) Applicants (for all designated States except US): PFIZER, INC.
`[US/US]; Eastempoint Road, Groton, CT 06340 (US). AB-
`GENIX, INC. [US/US]; 7601 Dumbarton Circle, Fremont,
`CA 94555 (US).
`
`(81) Designated States: AE, AL, AM, AT, AU, AZ, BA, BB, BG,
`BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE,
`ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP,
`KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA,
`MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU,
`SD, SE, SG, SI, SK, SL, TJ, TM, TR, ’I'I‘, TZ, UA, UG,
`US, UZ, VN, YU, ZA, ZW, ARIPO patent (GH, GM, KE,
`LS, MW, SD, SL, SZ, TZ, UG, ZW), Eurasian patent (AM,
`AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent (AT,
`BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU,
`MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI, CM,
`GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): HANSON, Douglas,
`Charles
`[US/US];
`3 Acorn Drive, Niantic, CT 06357
`(US). NEVEU, Mark,
`Joseph [US/US];
`18 Greenbriar
`Court, Mystic, CT 06355 (US). MUELLER, Eileen, Elliott Published
`[US/US]; 4 Butterwick Lane, Old Lyme, CT 06371 (US).
`Without international search report and to be republished
`HANKE, Jeffrey, Herbert
`[US/US];
`1 Jefferson Circle,
`upon receipt of that report.
`Reading, MA 01867 (US). GILMAN, Steven, Christopher
`[US/US];
`118 Sill Lane, Old Lyme, CT 06371 (US).
`DAVIS, C., Geoffrey [US/US]; 1132 Vancouver Avenue,
`Burlingame, CA 94010 (US). CORVALAN, Jose, Ramon
`[CL/US]; 125 Williams Lane, Foster City, CA 94010 (US).
`
`
`
`(54) Title: HUMAN MONOCLONAL ANTIBODIES TO CTLA—4
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`I cr4.1.1
`
`CT11.2.1.4
` 1 0000
`
`
`7
`
`
`
`Enhancement 0! IL-2 Production Induced by Antl-CTLA4 MAbs
`(30 uglml) ln the 72 Hour T Blast/ Hall and
`Superantlgen Assays (6 Donors)
`
`8000
`
`
`
`lL-ZEnhancement(pg/ml)41-SEM
`
`
`T Blast] Hall SEA/ PBMC
`SEAI Blood
`
`
`
`(57) Abstract
`
` In accordance with the present invention, there are provided fully human monoclonal antibodies against human cytotoxic T—lymphocyte
`
`antigen 4 (CTLA—4). Nucleotide sequences encoding and amino acid sequences comprising heavy and light chain immunoglobulin molecules,
`
`
`particularly contiguous heavy and light chain sequences spanning the complementarity determining regions (CDRs), specifically from within
`
`
`FRl and/or CDRI through CDR3 and/or within FR4, are provided. Further provided are antibodies having similar binding properties and
`
`antibodies (or other antagonists) having similar functionality as antibodies disclosed herein.
`
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Zimbabwe
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`Albania
`ES
`Lesotho
`SI
`LS
`Slovenia
`FI
`LT
`Armenia
`Lithuania
`SK
`Slovakia
`Austria
`FR
`LU
`SN
`Luxembourg
`Senegal
`Australia
`GA
`LV
`Latvia
`SZ
`Swaziland
`GB
`MC
`Monaco
`TD
`Chad
`Azerbaijan
`GE
`MD
`TG
`Bosnia and Herzegovina
`Republic of Moldova
`Togo
`Barbados
`GH
`MG
`TJ
`Madagascar
`Tajikistan
`GN
`MK
`TM
`Turkmenistan
`Belgium
`The former Yugoslav
`Burkina Faso
`TR
`Republic of Macedonia
`Turkey
`Mali
`TT
`Bulgaria
`Trinidad and Tobago
`Benin
`UA
`Ukraine
`Mongolia
`Brazil
`Mauritania
`UG
`Uganda
`Belarus
`Malawi
`US
`United States of America
`Canada
`Mexico
`UZ
`Uzbekistan
`VN
`Viet Nam
`Central African Republic
`Niger
`Netherlands
`YU
`Congo
`Yugoslavia
`Switzerland
`ZW
`Norway
`Cate d'Ivoire
`New Zealand
`Cameroon
`Poland
`China
`Portugal
`Cuba
`Romania
`Russian Federation
`Czech Republic
`Sudan
`Germany
`Denmark
`Sweden
`Estonia
`Singapore
`
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People’s
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`
`

`

`wo 00/37504
`
`PCT/US99/30895
`
`HUMAN MONOCLONAL ANTIBODIES T0 CTLA-4
`
`BACKGROUND OF THE INVENTION
`
`1.
`
`Ctoss-Reference to Related Agglications
`
`10
`
`15
`
`20
`
`25
`
`30
`
`The present application claims priority to US. Provisional Patent
`
`Application, Serial No. 60/113,647, filed December 23, 1998, the disclosure of
`
`which is hereby incorporated in its entirety herein.
`
`2.
`
`Summa
`
`o the Invention
`
`In accordance with the present invention, there are provided fully human
`
`monoclonal antibodies against human cytotoxic T—lymphocytc antigen 4
`
`(CTLA-4).
`
`Nucleotide sequences encoding and amino acid sequences
`
`comprising heavy and light chain immunoglobulin molecules, particularly
`
`contiguous heavy and light chain sequences spanning the complementarity
`
`determining regions (CDRs), specifically from within FRl and/or CDRl
`
`through CDR3 and/or within FR4, are provided.
`
`IFurther provided are
`
`antibodies having similar binding properties
`
`and antibodies
`
`(or other
`
`antagonists) having similar functionality as antibodies disclosed herein.
`
`3.
`
`Background of the Technology
`
`Regulation of immune response in patients would provide a desirable
`
`treatment of many human diseases that could lead to a specificity of action that
`
`is rarely found through the use of conventional drugs. Both up—regulation and
`
`down-regulation of responses of the immune system would be possible. The
`
`roles of T cells and B cells have been extensively studied and characterized in
`
`connection with the regulation of immune response. From these studies, the role
`
`of T cells appear,
`
`in many cases,
`
`to be particularly important
`
`in disease
`
`prevention and treatment.
`
`

`

`WO 00/37504
`
`PCT/US99BO895
`
`2
`
`T cells possess very complex systems for controlling their interactions.
`
`Interactions between T cells utilize numerous receptors and soluble factors for
`
`the process. Thus, what effect any particular signal may have on the immune
`
`response generally varies and depends on the particular factors, receptors and
`
`5
`
`counter-receptors that are involved in the pathway. The pathways for down-
`
`regulating responses are as important as those required for activation. Thymic
`
`education leading to T-cell
`
`tolerance is one mechanism for preventing an
`
`immune response to a particular antigen. Other mechanisms, such as secretion
`
`of suppressive cytokines, are also known.
`
`10
`
`Activation of T cells requires not only stimulation through the antigen
`
`receptor
`
`(T cell
`
`receptor
`
`(TCR)), but additional
`
`signaling through co-
`
`stimulatory surface molecules such as CD28. The ligands for CD28 are the B7-
`
`1 (CD80) and B7-2 (CD86) proteins, which are expressed on antigen-presenting
`
`cells such as dendritic cells, activated B-cells or monocytes that interact with T-
`
`15
`
`cell CD28 or CTLA-4 to deliver a costimulatory signal. The role of
`
`costimulatory signaling was studied in experimental allergic encephalomyelitis
`
`(EAE) by Perrin et a1. Immunol Res 142189-99 (1995). EAE is an autoimmune
`
`disorder, induced by Th1 cells directed against myelin antigens that provides an
`
`in vivo model
`
`for studying the role of B7—mediated costimulation in the
`
`20
`
`induction of a pathological immune response. Using a soluble fusion protein
`
`ligand for the B7 receptors, as well as monoclonal antibodies specific for either
`
`CD80 or CD86, Perrin et al. demonstrated that B7 costimulation plays a
`
`prominent role in determining clinical disease outcome in EAE.
`
`The interaction between B7 and CD28 is one of several co-stimulatory
`
`25
`
`signaling pathways that appear to be sufficient to trigger the maturation and
`
`proliferation of
`
`antigen specific T—cells. Lack of co—stimulation, and the
`
`concomitant inadequacy of IL—2 production, prevent subsequent proliferation of
`
`the T cell and induce a state of non—reactivity termed "anergy". A variety of
`
`viruses and tumors may block T cell activation and proliferation, leading to
`
`30
`
`insufficient activity or
`
`non-reactivity of the host's immune system to the
`
`infected or transformed cells. Among a number of possible T—cell disturbances,
`
`

`

`WO 00/37504
`
`PCT/US99/30895
`
`3
`
`anergy may be at
`
`least partly responsible for the failure of the host to clear the
`
`pathogenic or tumorgenic cells.
`
`The use of the B7 protein to mediate anti-tumor immunity has been
`
`described in Chen et al. Cell 71:1093-1102 (1992) and Townsend and Allison
`
`Science 2592368 (1993). Schwartz Cell 71:1065 (1992) reviews the role of
`
`CD28, CTLA-4, and B7 in IL—2 production and immunotherapy. Harding et al.
`
`Nature 356:607-609 (1994) demonstrates that CD28 mediated signaling co—
`
`stimulates murine T cells and prevents the induction of anergy in T cell clones.
`
`See also US. Patent Nos. 5,434,131, 5,770,197,
`
`and 5,773,253,
`
`and
`
`International Patent Application Nos. WO 93/00431, WO 95/01994, WO
`
`95/03408, WO 95/24217, and WO 95/33770.
`
`From the foregoing,
`
`it was clear that T-cells required two types of
`
`signals from the antigen presenting cell (AFC) for activation and subsequent
`
`differentiation to effector function. First, there is an antigen specific signal
`
`generated by interactions between the TCR on the T—cell and MHC molecules
`
`presenting peptides on the APC. Second, there is an antigen—independent signal
`
`that is mediated by the interaction of CD28 with members of the B7 family (B7-
`
`1 (CD80) or 872 (CD86)). Exactly where CTLA-4 fit
`
`into the milieu of
`
`immune responsiveness was initially evasive. Murine CTLA-4 was first
`
`identified and cloned by Brunet et al. Nature 328:267-270 (1987), as part of a
`
`quest
`
`for molecules
`
`that
`
`are preferentially expressed on cytotoxic T
`
`lymphocytes. Human CTLA—4 was identified and cloned shortly thereafter by
`
`Dariavach et al. Eur. J. Immunol. 18:1901—1905 (1988). The murine and human
`
`CTLA-4 molecules possess approximately 76% overall sequence homology and
`
`approach complete sequence identity in their cytoplasmic domains (Dariavach
`
`et al. Eur. J. Immunol. 18:1901—1905 (1988)). CTLA-4 is a member of the
`
`immunoglobulin (1g) superfamily of proteins. The Ig superfamily is a group of
`
`proteins that share key structural features of either a variable (V) or constant (C)
`
`domain of Ig molecules. Members of the Ig superfamily include, but are not
`
`limited to, the immunoglobulins themselves, major histocompatibility complex
`
`(MHC) class molecules (i.e., MHC class I and II), and TCR molecules.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 00/37 504
`
`PCT/US99/30895
`
`4
`
`In 1991, Linsley et al. J. Exp. Med. 174:561—569 (1991), proposed that
`
`CTLA—4 was a second receptor for B7. Similarly, Harper et al. J Immunol
`
`147:1037-44 (1991) demonstrated that the CTLA—4 and CD28 molecules are
`
`closely related in both mouse and human as to sequence, message expression,
`
`gene structure, and chromosomal location. See also Balzano et a1. Int J Cancer
`
`Suppl 7:28-32 (1992). Further evidence of this role arose through functional
`
`studies.
`
`For
`
`example, Lenschow et
`
`al. Science 257 :789—792
`
`(1992)
`
`demonstrated that CTLA-4—Ig induced long term survival of pancreatic islet
`
`grafts. Freeman et al. Science 262:907-909 (1993) examined the role of CTLA—
`
`4 in B7 deficient mice. Examination of the ligands for CTLA-4 are described
`
`in Lenschow et a1. P.N.A.S. 90:11054—11058 (1993). Linsley et al. Science
`
`257 :792—795 (1992) describes immunosuppression in vivo by a soluble form of
`
`CTLA-4. Linsley et al. J Exp Med 176:1595-604 (1992) prepared antibodies
`
`that bound CTLA-4 and that were not cross-reactive with CD28 and concluded
`
`that CTLA-4 is coexpressed with CD28 on activated T lymphocytcs and
`
`cooperatively regulates T cell adhesion and activation by B7. Kuchroo et al.
`
`Cell 802707—18 (1995) demonstrated that the B7-l and B7—2 costimulatory
`
`molecules differentially activated the Thl/Th2 developmental pathways.
`
`Yi—
`
`qun et a1. Int Immunol 8:37-44 (1996) demonstrated that there are differential
`
`requirements for co—stimulatory signals from B7 family members by resting
`
`versus recently activated memory T cells towards soluble recall antigens. See
`
`also de Boer et al. Eur JImmunol 23:3120-5 (1993).
`
`Several
`
`groups
`
`proposed
`
`alternative
`
`or
`
`distinct
`
`receptor/ligand
`
`interactions for CTLA-4 as compared to CD28 and even proposed a third B—7
`
`complex that was recognized by a BB1 antibody. See, for example, Hathcock et
`
`al. Science 262:905—7 (1993), Freeman et al. Science 262:907—9 (1993),
`
`Freeman et al. JExp Med 178:2185-92 (1993), Lenschow et al. Proc Natl Acad
`
`Sci U S A 90:11054—8 (1993), Razi—Wolf et al. Proc Natl Acad Sci U S A
`
`90211182—6 (1993), and Boussiotis et al. Proc Natl Acad Sci USA 90:11059-63
`
`(1993). But, see, Freeman et a1. JImmunol 161:2708—15 (1998) who discuss
`
`finding that BB1 antibody binds a molecule that is identical to the cell surface
`
`form of CD74 and, therefore, the BB1 mAb binds to a protein distinct from B7-
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 00/37504
`
`PCT/US99/30895
`
`5
`
`1, and this epitope is also present on the B7-1 protein. Thus, this observation
`
`required the field to reconsider studies using BB1 mAb in the analysis of CD80
`
`expression and function.
`
`Beginning in 1993 and culminating in 1995,
`
`investigators began to
`
`further delineate the role of CTLA—4 in T—cell stimulation. First, through the
`
`use of monoclonal antibodies against CTLA—4, Walunas et a1. Immunity 1:405—
`
`13 (1994) provided evidence that CTLA—4 can function as a negative regulator
`
`ofT cell activation. Thereafter, Waterhouse et al. Science 270:985-988 (1995)
`
`demonstrated that mice deficient for CTLA-4 accumulated T cell blasts with up-
`
`regulated activation markers in their lymph nodes and spleens. The blast cells
`
`also infiltrated liver, heart, lung, and pancreas tissue, and amounts of serum
`
`immunoglobulin were elevated and their T cells proliferated spontaneously and
`
`strongly when stimulated through the T cell receptor, however,
`
`they were
`
`sensitive to cell death induced by cross-linking of the Fas receptor and by
`
`gamma irradiation. Waterhouse et a1. concluded that CTLA-4 acts as a negative
`
`regulator of T cell activation and is vital
`
`for the control of lymphocyte
`
`homeostasis.
`
`In a comment in the same issue, Allison and Krummel Science
`
`270:932—933 ( 1995), discussed the work of Waterhouse et a1. as demonstrative
`
`that CTLA-4 acts to down regulate T—cell responsiveness or has an inhibitory
`
`signaling role in T—cell activation and development. Tivol et al. Immunity 3:541—
`
`7 (1995) also generated CTLA-4—deficient mice and demonstrated that such
`
`mice rapidly develop lymphoproliferative disease with multiorgan lymphocytic
`
`infiltration and tissue destruction, with particularly severe myocarditis and
`
`pancreatitis. They concluded that CTLA—4 plays a key role in down—regulating
`
`T cell activation and maintaining immunologic homeostasis. Also, Krummel
`
`and Allison J Exp Med 182:459-65 (1995) further clarified that CD28 and
`
`CTLA-4 have opposing effects on the response of T cells to stimulation. They
`
`generated an antibody to CTLA—4 and investigated the effects of its binding to
`
`CTLA-4 in a system using highly purified T cells.
`
`In their report, they showed
`
`that the presence of low levels of B7-2 on freshly explanted T cells can partially
`
`inhibit T cell proliferation, and this inhibition was mediated by interactions with
`
`CTLA—4. Cross-linking of CTLA-4 together with the TCR and CD28 strongly
`
`10
`
`15
`
`20
`
`25
`
`30
`
`

`

`WO 00/37504
`
`PCT/US99/30895
`
`6
`
`inhibits proliferation and IL—2 secretion by T cells. Finally, the results showed
`
`that CD28 and CTLA-4 deliver opposing signals that appear to be integrated by
`
`the T cell in determining the response to antigen. Thus, they concluded that the
`
`outcome of T cell antigen receptor
`
`stimulation is
`
`regulated by CD28
`
`costimulatory signals, as well as inhibitory signals derived from CTLA-4. See
`
`also Krummel et
`
`a1. Int Immunol 8:519—23 (1996) and US. Patent No.
`
`5,811,097 and International Patent Application No. WO 97/20574.
`
`A variety of additional experiments have been conducted further
`
`elucidating the above function of CTLA-4. For example, Walunas et al. J Exp
`
`Med 183:2541—50 (1996), through the use of anti-CTLA-4 antibodies, suggested
`
`that CTLA-4 signaling does not regulate cell survival or responsiveness to IL-2,
`
`but does inhibit CD28—dependent IL-2 production. Also, Perrin et al. J Immunol
`
`157:1333—6 (1996), demonstrated that anti—CTLA-4 antibodies in experimental
`
`allergic encephalomyelitis
`
`(EAE), exacerbated the disease and enhanced
`
`mortality. Disease exacerbation was associated with enhanced production of the
`
`encephalitogenic cytokines TNF-alpha,
`
`IFN-gamrna and 1L—2. Thus,
`
`they
`
`concluded that CTLA-4 regulates the intensity of the autoimmune response in
`
`EAE, attenuating inflammatory cytokine production and clinical disease
`
`manifestations. See also Hurwitz et al. J Neuroimmunol 73:57-62 (1997) and
`
`Cepero et al. J Exp Med 188: 199-204 (1998) (an anti-CTLA—4 hairpin ribozyme
`
`that specifically abrogates CTLA-4 expression after gene transfer into a murine
`
`T-cell model).
`
`In addition, Blair et al. J Immunol 160:12-5 (1998) assessed the
`
`functional effects of a panel of CTLA-4 monoclonal antibodies (mAbs) on
`
`resting human CD4+ T cells. Their results demonstrated that some CTLA-4
`
`mAbs could inhibit proliferative responses of resting CD4+ cells and cell cycle
`
`transition from GO to 61. The inhibitory effects of CTLA-4 were evident within
`
`4 h, at a time when cell surface CTLA-4 expression remained undetectable.
`
`Other CTLA—4 mAbs, however, had no detectable inhibitory effects, indicating
`
`10
`
`15
`
`20
`
`25
`
`30
`
`that binding of mAbs to CTLA—4 alone was not sufficient to mediate down-
`
`regulation of T cell responses. Interestingly, while IL—2 production was shut off,
`
`inhibitory anti—CTLA-4 mAbs permitted induction and expression of the cell
`
`

`

`WO 00/37504
`
`PCT/USQ9/30895
`
`7
`
`survival gene bcl-X(L). Consistent with this observation, cells remained viable
`
`and apoptosis was not detected after CTLA-4 ligation.
`
`In connection with anergy, Perez et al.
`
`Immunity 6:411—7 (1997)
`
`demonstrated that the induction of T cell anergy was prevented by blocking
`
`CTLA-4 and concluded that the outcome of antigen recognition by T cells is
`
`determined by the interaction of CD28 or CTLA-4 on the T cells with B7
`
`molecules. Also, Van Parijs et al. JExp Med 186:1119-28 (1997) examined the
`
`role of interleukin 12 and costimulators in T cell anergy in vivo and found that
`
`through inhibiting CTLA—4 engagement during anergy induction, T cell
`
`proliferation was blocked, and full Th1 differentiation was not promoted.
`
`However, T cells exposed to tolerogenic antigen in the presence of both 1L—12
`
`and anti—CTLA-4 antibody were not anergized, and behaved identically to T
`
`cells which have encountered immunogenic antigen. These results suggested
`
`that
`
`two processes contribute to the induction of anergy in vivo: CTLA—4
`
`engagement, which leads to a block in the ability of T cells to proliferate, and
`
`the absence of a prototypic inflammatory cytokine, IL—12, which prevents the
`
`differentiation of T cells into Th1 effector cells. The combination of IL-lZ and
`
`anti—CTLA-4 antibody was sufficient to convert a normally tolerogenic stimulus
`
`to an immunogenic one.
`
`In connection with infections, McCoy et al. J Exp Med 186:183—7 (1997)
`
`demonstrated that anti—CTLA-4 antibodies greatly enhanced and accelerated the
`
`T cell immune response to szpostrongylus brasiliensis, resulting in a profound
`
`reduction in adult worm numbers and early termination of parasite egg
`
`production.
`
`See also Murphy et al. J.
`
`Immunol. 16124153—4160 (1998)
`
`(Leishmania donovani).
`
`10
`
`15
`
`20
`
`25
`
`In connection with cancer, Kwon et al. PNAS USA 94:8099-103 (1997)
`
`established a syngeneic murine prostate cancer model and examined two
`
`distinct manipulations intended to elicit an antiprostate cancer response through
`
`enhanced T cell costimulation: (i) provision of direct costimulation by prostate
`
`30
`
`cancer cells transduced to express the B7.l
`
`ligand and (ii) in vivo antibody—
`
`mediated blockade of T cell CTLA-4, which prevents T cell down—regulation. It
`
`was demonstrated that
`
`in vivo antibody-mediated blockade of CTLA-4
`
`

`

`WO 00/37504
`
`PCT/US99/30895
`
`8
`
`enhanced antiprostate cancer immune responses. Also, Yang et al. Cancer Res
`
`57:4036-41 (1997) investigated whether the blockade of the CTLA-4 function
`
`leads to enhancement of antitumor T cell responses at various stages of tumor
`
`growth. Based on in vitro and in viva results they found that CTLA—4 blockade
`
`in tumor-bearing individuals enhanced the capacity to generate antitumor T-cell
`
`responses, but the expression of such an enhancing effect was restricted to early
`
`stages of tumor growth in their model. Further, Hurwitz et al. Proc Natl Acad
`
`Sci U S A 95:10067-71 (1998) investigated the generation of a T cell—mediated
`
`antitumor
`
`response depends on T cell
`
`receptor engagement by major
`
`histocompatibility complex/antigen as well as CD28 ligation by B7. Certain
`
`tumors, such as the SMl mammary carcinoma, were refractory to anti—CTLA-4
`
`immunotherapy. Thus, through use of a combination of CTLA—4 blockade and
`
`a vaccine consisting of granulocyte-macrophage colony—stimulating factor—
`
`expressing SMI cells, regression of parental SMl tumors was observed, despite
`
`the ineffectiveness of either treatment alone. This combination therapy resulted
`
`in long-lasting immunity to SMl and depended on both CD4(+) and CD8(+) T
`
`cells. The findings suggested that CTLA-4 blockade acts at the level of a host—
`
`derived antigen—presenting cell.
`
`In connection with diabetes, Luhder et al. J Exp Med 187:427—32 (1998)
`
`injected an anti-CTLA-4 mAb into a TCR transgenic mouse model of diabetes
`
`at different stages of disease. They found that engagement of CTLA-4 at the
`
`time when potentially diabetogenic T cells are first activated is a pivotal event;
`
`if engagement is permitted, invasion of the islets occurs, but remains quite
`
`innocuous for months.
`
`If not, insulitis is much more aggressive, and diabetes
`
`10
`
`15
`
`20
`
`25
`
`quickly ensues.
`
`In connection with vaccine immunization, Horspool et al. J Immunol
`
`160:2706—14 (1998) found that intact anti—CTLA—4 mAb but not Fab fragments
`
`suppressed the primary humoral response to pCIA/beta gal without affecting
`
`recall responses, indicating CTLA-4 activation inhibited Ab production but not
`
`3O
`
`T cell priming. Blockade of the ligands for CD28 and CTLA-4, CD80 (B7-l)
`
`and CD86 (87-2), revealed distinct and nonoverlapping function. Blockade of
`
`CD80 at initial immunization completely abrogated primary and secondary Ab
`
`

`

`W0 (IO/37504
`
`V
`
`9
`
`PCT/US99/30895
`
`responses, Whereas blockade of CD86 suppressed primary but not secondary
`
`responses. Simultaneous blockade of CD80 + CD86 was less effective at
`
`suppressing Ab responses than either alone. Enhancement of costimulation via
`
`coinjection of B7-expressing plasmids augmented CTL responses but not Ab
`
`responses, and without evidence of Th1 to Th2 skewing. These findings suggest
`
`complex and distinct roles for CD28, CTLA-4, CD80, and CD86 in T cell
`
`costimulation following nucleic acid vaccination.
`
`In connection with allograft rejection, Markees et al. J Clin Invest
`
`101:2446-55 (1998) found in a mouse model of skin allograft rejection that
`
`acceptance initially depended on the presence of IFN—gamma, CTLA—4, and
`
`CD4(+) T cells. Addition of anti-CTLA-4 or anti—IFN—gamma mAb to the
`
`protocol was associated with prompt graft rej ection, Whereas anti—IL—4 mAb had
`
`no effect.
`
`In connection with the role of CTLA—4 in relation to CD28, Fallarino et
`
`al. J Exp Med 188:205~10 (1998) generated TCR transgenic/rccombinase
`
`activating gene 2-deficient/CD28—wi1d—type or CD28-deficient mice which were
`
`immunized with an antigen—expressing tumor. Primed T cells fi‘om both types
`
`of mice produced cytokines and proliferated in response to stimulator cells
`
`lacking B7 expression. However, whereas the response of CD28+/+ T cells was
`
`augmented by costimulation with B7—1, the response of the CD28-/- T cells was
`
`strongly inhibited. This inhibition was reversed by monoclonal antibody against
`
`B7—1 or CTLA-4. Thus, CTLA—4 can potently inhibit T cell activation in the
`
`absence of CD28,
`
`indicating that antagonism of a TCR-mediated signal
`
`is
`
`sufficient to explain the inhibitory effect of CTLA-4. Also, Lin et al. J Exp Med
`
`188: 199-204 (1998) studied rejection of heart allografts in CD28-deficient mice.
`
`H—2(q) hearts were transplanted into allogeneic wild-type or CD28-deficient
`
`mice (H-2(b)). Graft rejection was delayed in CD28—deficient compared with
`
`wild-type mice.
`
`Treatment
`
`of wild-type
`
`recipients with
`
`CTLA—4—
`
`immunoglobulin (1g), or with anti—B7—1 plus anti—B7—2 mAbs significantly
`
`prolonged allograft survival. In contrast, treatment of CD28-deficient mice with
`
`CTLA-4-Ig, anti—B7~1 plus anti—B7-2 mAbs, or a blocking anti-CTLA-4 mAb
`
`induced acceleration of allograft rejection. This increased rate of graft rejection
`
`10
`
`15
`
`20
`
`25
`
`3O
`
`

`

`W0 00/37 504
`
`PCT/US99/30895
`
`10
`
`was associated with more severe mononuclear cell infiltration and enhanced
`
`levels of IFN—gamma and lL—6 transcripts in donor hearts of untreated wild—type
`
`and CTLA-4-Ig— or anti-CTLA—4 mAb-treated CD28-deficient mice. Thus, the
`
`negative regulatory role of CTLA-4 extends beyond its potential ability to
`
`5
`
`prevent CD28 activation through ligand competition. Even in the absence of
`
`CD28, CTLA-4 plays an inhibitory role in the regulation of allograft rejection.
`
`Also, further characterization of the expression of CTLA—4 has been
`
`investigated.
`
`For example, Alegre et al. J Immunol 157:4762—70 (1996)
`
`proposed that surface CTLA-4 is rapidly internalized, which may explain the
`
`10
`
`low levels of expression generally detected on the cell surface. They concluded
`
`that both CD28 and IL-2 play important roles in the up-regulation of CTLA—4
`
`expression. In addition, the cell surface accumulation of CTLA-4 appeared to be
`
`primarily regulated by its rapid endocytosis. Also, Castan et a1. Immunology
`
`90:265—71
`
`(1997) based on in situ immunohistological analyses of the
`
`15
`
`expression of CTLA-4, suggested that germinal center T cells, which were
`
`CTLA—4 positive, could be important to immune regulation.
`
`Accordingly, in View of the broad and pivotal role that CTLA-4 appears
`
`to possess in immune responsiveness,
`
`it would be desirable to generate
`
`antibodies to CTLA-4 that can be utilized effectively in immunotherapy.
`
`20 Moreover, it would be desirable to generate antibodies against CTLA-4 that can
`
`be utilized in chronic diseases in which repeat administrations of the antibodies
`
`are required.
`
`BRIEF DESCRIPTION OF THE DRAWING FIGURES
`
`25
`
`Figure 1 provides a series of nucleotide and an amino acid sequences of
`
`heavy chain and kappa light chain immunoglobulin molecules in accordance
`
`with the invention: 4.1.1 (Figure 1A), 4.8.1 (Figure 1B), 4.14.3 (Figure 1C),
`
`6.1.1 (Figure 1D), 3.1.1 (Figure 1E), 4.10.2 (Figure 1F), 2.1.3 (Figure 1G),
`
`4.13.1 (Figure 1H), 11.2.1 (Figure 11), 11.6.1 (Figure 1J), 11.7.1 (Figure 1K),
`
`30
`
`12.3.1.1 (Figure 1L), and 12.9.1.1 (Figure 1M).
`
`Figure 2 provides a sequence alignment between the predicted heavy
`
`chain amino acid sequences from the clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 3.1.1,
`
`

`

`WO 00/37504
`
`PCT/US99/30895
`
`11
`
`4.10.2, 4.13.1, 11.2.1, 11.6.1, 11.7.1, 12.3.1.1, and 12.9.1.1 and the germline
`
`DP—50 (3-33) amino acid sequence. Differences between the DP-SO germline
`
`sequence and that of the sequence in the clones are indicated in bold. The
`
`Figure also shows the positions of the CDRl, CDR2, and CDR3 sequences of
`
`5
`
`the antibodies as shaded.
`
`Figure 3 provides a sequence alignment between the predicted heavy
`
`chain amino acid sequence of the clone 2.1.3 and the germline DP-65 (4—31)
`
`amino acid sequence. Differences between the DP-65 germline sequence and
`
`that of the sequence in the clone are indicated in hold. The Figure also shows
`
`10
`
`the positions of the CDR1, CDR2, and CDR3 sequences of the antibody as
`
`underlined.
`
`Figure 4 provides a sequence alignment between the predicted kappa
`
`light chain amino acid sequence of the clones 4.1.1, 4.8.1, 4.14.3, 6.1.1, 4.10.2,
`
`and 4.13.1 and the germline A27 amino acid sequence. Differences between the
`
`15
`
`A27 germline sequence and that of the sequence in the clone are indicated in
`
`hold. The Figure also shows the positions of the CDRl, CDR2, and CDR3
`
`sequences of the antibody as underlined. Apparent deletions in the CDRls of
`
`clones 4.8.1, 4.14.3, and 6.1.1 are indicated with “OS”.
`
`Figure 5 provides a sequence alignment between the predicted kappa
`
`20
`
`light chain amino acid sequence of the clones 3.1.1, 11.2.1, 11.6.1, and 11.7.1
`
`and the germline 012 amino acid sequence. Differences between the 012
`
`germline sequence and that of the sequence in the clone are indicated in bold.
`
`The Figure also shows the positions of the CDRl, CDR2, and CDR3 sequences
`
`of the antibody as underlined.
`
`25
`
`Figure 6 provides a sequence alignment between the predicted kappa
`
`light chain amino acid sequence of the clone 2.1.3 and the germline A10/A26
`
`amino acid sequence. Differences between the A10/A26 germline sequence and
`
`that of the sequence in the clone are indicated in bold. The Figure also shows
`
`the positions of the CDRl, CDR2, and CDR3 sequences of the antibody as
`
`30
`
`underlined.
`
`’
`
`Figure 7 provides a sequence alignment between the predicted kappa
`
`light chain amino acid sequence of the clone 12.3.1 and the germline A17
`
`

`

`WO 00/37504
`
`PCT/US99/30895
`
`12
`
`amino acid sequence. Differences between the A17 gerrnline sequence and that
`
`of the sequence in the clone are indicated in bold. The Figure also shows the
`
`positions of the CDRl, CDR2, and CDR3 sequences of the antibody as
`
`underlined.
`
`5
`
`Figure 8 provides a sequence alignment between the predicted kappa
`
`light chain amino acid sequence of the clone 12.9.1 and the gerrnline A3/A19
`
`amino acid sequence. Differences between the A3/Al9 germline sequence and
`
`that of the sequence in the clone are indicated in bold. The Figure also shows
`
`the positions of the CDRl, CDR2, and CDR3 sequences of the antibody as
`
`10
`
`underlined.
`
`Figure 9 provides a summary of N-terminal amino acid sequences
`
`generated through direct protein sequencing of the heavy and light chains of the
`
`antibodies.
`
`Figure 10 provides certain additional characterizing information about
`
`15
`
`certain of the antibodies in accordance with the invention.
`
`In Figure 10A, data
`
`related to clones 3.1.1, 4.1.1, 4.8.1, 4.10.2, 4.14.3, and 6.1.1 is summarized.
`
`Data related to concentration,
`
`isoelectric focusing (IEF), SDS-PAGE, size
`
`exclusion chromatography, liquid chromatography/mass spectroscopy (LCMS),
`
`mass spectroscopy (MALDI),
`
`light chain N—terminal sequences is provided.
`
`20
`
`Additional detailed information related to IEF is provided in Figure 10B; related
`
`to SDS—PAGE is provided in 10C; and SEC of the 4.1.1 antibody in 10D.
`
`Figure 11 shows the expression of B7-l and B7-2 on Raji cells using
`
`anti-CD80—PE and anti-CD86-PE mAbs.
`
`Figure 12 shows the concentration dependent enhancement of IL—2
`
`25
`
`production in the T cell blast/Raji assay induced by anti-CTLA-4 blocking
`
`antibodies (BNI3, 4.1.1, 4.8.1, and 6.1.1).
`
`Figure 13 shows the concentration dependent enhancement of IFN—y
`
`production in the T cell blast/Raji assay induced by anti—CTLA-4 blocking
`
`antibodies (BNI3, 4.1.1, 4.8.1, and 6.1.1)(same donor T cells).
`
`30
`
`Figure 14 shows the mean enhancement of IL—2 production in T cells
`
`from 6 donors induced by anti—CTLA—4 blocking antibodies in the T cell
`
`blast/Raji assay.
`
`

`

`WO 00/37504
`
`PCT/U599/30895
`
`13
`
`Figure 15 shows the mean enhancement of IFN—y production in T cells
`
`from 6 donors induced by anti—CTLA-4 blocking antibodies in the T cell
`
`blast/Raji assay.
`
`Figure 16 shows the enhancement of IL-2 production in hPBMC from 5
`
`5
`
`donors induced by anti-CTLA-4 blocking mAbs as measured at 72 hours after
`
`stimulation with SEA.
`
`Figure 17 shows the enhancement of IL—2 production in whole blood
`
`from 3 donors induced by anti-CTLA-4 blocking mAbs as measured at 72 and
`
`96 hours after stimulation with SEA.
`
`10
`
`Figure 18 shows the inhibition of tumor growth with an anti-murine
`
`CTLA-4 antibody in a murine fibrosarcoma tumor model.
`
`Figure 19 shows enhancement of lL—2 production induced by anti—
`
`CTLA4 antibodies (4.1.1 and 11.2.1) of the invention in a 72 h