WO2011139738A2 - Therapies using zanolimumab to enhance the immune response - Google Patents

Abstract

Zanolimumab is provided as an adjunctive therapy for medical conditions where Treg cells, or a high level thereof, interfere with optimal treatment.

Description

THERAPIES USING ZANOLIMUMAB TO

ENHANCE THE IMMUNE RESPONSE Cross Relation to Prior Applications

Statement Regarding Federally Sponsored Research or Development

The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of a Clinical Trial Agreement for PRMC Protocol Number P08459 entitled Phase II Study of Aldesleukin (IL-2) Following the Administration of Zanolimumab (Anti-CD4 mAb) in Metastatic Melanoma and Metastatic Renal Cancer

Field of the Invention

Pharmaceutical compositions, regimens and uses for reducing the level of T_reg cells in mammals. In particular, pharmaceutical presentations, treatment regimens and uses for the treatment of cancerous malignancies such as renal carcinoma and melanoma.

Background of the Invention

Since tumors express antigens on their surface, the host immune system should be capable of finding such antigens and killing the tumors. On that basis, many cancer therapies were developed to effectively increase the number of immune response cells that can kill the tumor. These therapies include boosting the amount of tumor antigen or antigen pulsed APCs (Hsu F.J.et al, Nat. Med 1996 2:52-58; Nestle, F.O. Nat.Med 1998 4 328-332) supplying T cells from tumor infiltrates (Rosenberg SA, Science. 1986 233: 1318-21) adding soluble growth factor (Rosenberg SA. N. Eng. J. Med 1988 319:1676-80) or boosting the amount of co-signaling molecules or cytokines. These strategies have not had great success in the clinic. One possibility for lack of clinical success is that the tumor has developed strategies that allow it to evade normal immune surveillance and therefore simply boosting the immune response might not be sufficient to illicit a clinical response One such mechanism in which the tumor is able to prevent an immune attack is through the recruitment of regulatory T cells (Curiel TJ JCI, 2007. 1 17(5) 1 167-74 ).. Regulatory T cells were initially described as suppressive T cells by

Gershon (Gershon RK. Immunology 1971. 21 903-14) Sehon, et al, later suggested that these cells are partly responsible for lack of tumor immune response (Fujimoto S. Immunology 1975 Immun. Commun. 4: 201-17 ). Sakaguchi, in 1995 charecterized Treg cells as being CD4+ as well as showing that the interleukin receptor alpha chain could serve as a phenotypic marker for Tregs.(Sakaguchi S. J immin. 1995 155: 1 151-1164. More recently the transcription factor foxp3 has been shown to be essential for Treg development and functionality. Several recent publications have demonstrated that Tregs play a central role in failure of the immune systenti to destroy tumors (see for example Zou, W. Nat. Rev. 2006. 6 295-307. While many therapeutics are directed at upregulated the immune system to better fight the tumor, comparatively little has been done in trying to remove these T reg cells from suppressing the immune response to the tumor in the first place, which may be why immunomodulating strategies have met with only modest success. .

An example of a cancer which stimulates the immune system but which has had only modest success is IL-2 which is approved in the United States for the treatment of melanoma and renal carcinoma. IL-2 stimulates the body's inherent immune response to cancer and thereby is used to treat these two cancers. While several biologic agents have modest therapeutic antitumor effects IL-2 is unique in that it is the only immunotherapy with no direct impact on cancer cells. Instead, IL-2 works indirectly to enhance host immunity, presumably by stimulating the body's T cells, white blood cells that orchestrate key immune responses, and by stimulating the production or promoting the effect of lymphokine-activated killer (LAK) cells that can target tumors, but not normal cells.

Although the mechanism of tumor regression mediated by the administration of IL-2 (aldesleukin) is not known, recent studies have demonstrated that one factor that may inhibit the potential for response is the ability of aldesleukin to mediate the in vivo generation of

CD4⁺CD25⁺ , T-regulatory cells that can inhibit immune reactions. See Ahmadzadeh.M., P.A.Antony, and S.A.Rosenberg (2007), "IL-2 and IL-15 each mediate de novo induction of FOXP3 expression in human tumor antigen-specific CD8 T cells" J Immunother. 30:294-302. Aldesleukin thus plays an important role in tolerance to self antigens by inducing the production of T-regulatory cells. The generation of T-regulatory cells may explain why patients who experience an objective response and then recur do not re-respond to a second aldesleukin treatment.

Zanolimumab (HuMax-CD4) is a high affinity human antibody that targets the CD4 receptor on T-lymphocytes. Genmab A/S of Denmark has run two Phase II studies using HuMax-CD4 to treat cutaneous T-cell lymphoma (CTCL), one in early stage patients and the other for patients with advanced disease, both of which achieved positive results. Thirty-eight CTCL patients with mycosis fungoides (MF), the most common form of CTCL, were treated in these studies. Genmab had US Orphan Drug designation for HuMax-CD4 to treat MF patients and made plans for a pivotal study with HuMax-CD4 under an FDA Fast Track designation for patients who have failed available therapies. International Non-proprietary Name (INN): Zanolimumab has reached the status of recommended INN which is the international nonproprietary name (generic name) for HuMax-CD4, as adopted by WHO.

US Patent Application 2009/0317407 to Michael G. LaCelle discloses methods to stimulate, such as enhance or augment, an immune response against a target antigen, such as one or more tumor antigens or pathogen antigens by reducing or depleting CD4+ T cells in a subject at a time subsequent to the subject receiving a first dose of a therapeutically effective amount of an immunogenic composition that includes the target antigen.

Summary of the Invention

Tumor growth is dependent on the recruitment of regulatory T cells to prevent the immune response from ablating the tumor Tregs are further increased by the administration of IL-2 and other immunostimulants which may thus be counterproductive to the therapy afforded by the immunostimulant. The present invention comprises the administration of zanolimumab or a comparable antibody, to lower the amount of Tregs. In one embodiment of the invention, zanolimumab or a comparable antibody is administered with an immunostimulant with such an immunostimulant such as IL-2 to a mammal for a condition such as cancer. One aspect of the invention is based on the transient elimination of CD4⁺ T- regulatory cells with zanolimumab to enhance the clinical effectiveness of another agent administration by decreasing T-regulatory cell generation. One particular aspect of the invention would be the enhancement of anti-cancer agents which operate, at least in part, by stimulation of the immune system of the host although the invention is applicable to counteract the T-regulatory cell interference in the course of therapy with other agents or treatments or because of the generation of excessive Treg cells in particular circumstances or genetic factors.

Detailed Description of the Invention

CD8⁺ T cells are known mediators of anti-tumor responses. The role of CD4⁺ T cells in tumor treatment is less defined. In addition to their known helper function a subset of CD4⁺ T cells, CD25⁺ T regulatory cells, possess the ability to suppress T cells and regulate tolerance to self proteins. See Asano,M., M.Toda, N.Sakaguchi, and S.Sakaguohi (1996) "Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation" J. Exp. Med. 184:387- 396.

T regulatory (T_reg) cells mediate homeostatic peripheral tolerance by suppressing autoreactive T cells. Naturally occurring CD4⁺ CD25⁺ T_reg cells have surfaced as the principal T cells involved in the maintenance of peripheral self tolerance. See Sakaguchi.S (2004)

"Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative control of immune responses" Annu. Rev. Immunol. 22:53 1-562. T_reg cells develop in the thymus and represent 5-10% of the peripheral CD4 T cell compartment in mice and humans. These regulatory cells are characterized by their constitutive expression of CD25, CTLA-4, glucocorticoid-induced TNFR (GITR) and the transcription factor FOXP3. See Hori.S., T.Takahashi, and K.Sakaguchi (2003) "Control of autoimmunity by naturally arising regulatory CD4 cells" Adv Immunol 81 :331-371. The mechanism of suppression is cell contact dependent but how regulatory T cells induce and maintain self tolerance is unknown.

The importance of T_reg cells in mediating tolerance to self tissues is observable in humans and mice. In humans, the immune dysregulation, polyendocrinopathy, enteropathy, X- linked syndrome (IPEX) is a recessive and often fatal disorder of early childhood characterized by autoimmune mediated protracted diarrhea, ichthyosiform dermatitis, insulin-dependent diabetes mellitus, thyroiditis, and hemolytic anemia. Direct sequencing of genomic DNA from patients has demonstrated that IPEX is caused by mutations of FOXP3, a gene that plays a critical role in Tregs. See Bennett,C.L, J.Christie, F.Ramsdell, M.E.Brunkow, P.J.Ferguson, L.Whitesell, T.E.Kelly, F.T.Saulsbury, P.F.Chance, and H.D.Ochs (2001) "The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3." Nat. Genet. 27:20-21.

Multiple mouse studies have demonstrated the role of T_reg cells as master cell regulators of tolerance to self proteins in vivo. One strain, called scurfy mice, harbors a mutation in Foxp3 similar to IPEX patients resulting in the absence of T_reg cells and consequentially the induction of fatal T cell-mediated autoimmunity. See CL Bennett, above, as well as Fontenot.J.D.,

M.A.Gavin, and A.Y.Rudensky (2003) "Foxp3 programs the development and function of CD4+CD25+ regulatory T cells" Nat. Immunol. 4:330-336. In the absence of T_reg cells, autoimmune disease is mediated by unregulated CD4⁺ T helper cells directed against self tissue antigens in scurfy mice. Similar to these mice, the depletion of T_reg cells from normal mice results in autoimmune destruction of a number of tissue organs. Further, transfer of a normal repertoire of CD4⁺ CD25^" T helper cells to lymphopenic mice, results in autoimmune induction, a process inhibited by the cotransfer of T_reg cells. In the absence of CD25⁺ T regulatory cells, organ-specific destruction of tissue expressing a self antigen can be augmented with self antigen vaccination or through the provision of inflammatory signals. See Itoh.M., T.Takahashi, N.Sakaguchi, Y.Kuniyasu, J.Shimizu, F.Otsuka, and S.Sakaguchi (1999) "Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance" J. Immunol. 162:5317-5326. Of importance, however, is that depletion of T_reg cells can enhance tumor protection to tumor- associated antigens expressed as self antigens. See McHugh.R.S. and E.M.Shevach (2002) "Cutting edge: depletion of CD4+CD25+ regulatory T cells is necessary, but not sufficient, for induction of organ-specific autoimmune disease" J. Immunol. 168:5979-5983.

The influence of CD4⁺ T cell subsets on adoptive cell transfer therapy has recently been evaluated in the Surgery Branch, NCI, using a well described murine model of cancer. In this model, adoptive transfer of tumor/self reactive CD8+ T cells with vaccination and aldesleukin leads to regression of large established melanoma in lymphodepleted hosts. See

Overwijk.W.W., M.R.Theoret, S.E.Finkelstein, D.R.Surman, L.A.de Jong, F.A.Vyth-Dreese, T.A.Dellemijn, P.A.Antony, P.J.Spiess, D.C. Palmer, D.M.Heimann, C.A.KIebanoff, Z.Yu, L.N.Hwang, LFeigenbaum, A.M.Kruisbeek, S.A.Rosenberg, and N.P.Restifo (2003) "Tumor regression and autoimmunity after reversal of a functionally tolerant state of self-reactive CD8+ T cells" J. Exp. Med 198:569-580. Although CD4⁺ T cells control CD8⁺ T cell effector function, memory and maintenance, the absence of CD4⁺ T cells augmented adoptive immunotherapy when using CD81 T cells directed against a persisting tumor/self antigen. See Antony.P.A., C.A.Piccirillo, A.Akpinarli, S.E.Finkelstein, P.J. Speiss, D R. Surman, D.C.Palmer, C.C.Chan, C.A.KIebanoff, W.W.Overwijk, S.A.Rosenberg, and N.P.Restifo (2005) "CD8+ T cell immunity against a tumor/self-antigen is augmented by CD4+ T helper cells and hindered by naturally occurring T regulatory cells" J Immunol 174:2591 -2601. However, adoptive transfer of CD4⁺ CD25^" T helper cells with tumor/self reactive CD8⁺ T cells and vaccination into CD4⁺ T cell- deficient recipients resulted in autoimmunity and regression of established tumor. Maintenance of tumor/self CD8⁺ effector cells was dependent on CD4⁺ CD25^" T helper capable of producing IL-2. Transfer of CD25⁺ T_reg cells alone or combined with CD4⁺ CD25^" T helper cells prevented effective immunotherapy.

A host homeostatic environment devoid of T_reg cells and competing cells can influence the efficacy of tumor vaccination as well. Mouse studies have demonstrated that tumor-specific T cells preferentially expand in the lymphopenic environment after a melanoma vaccine given to RAG1 mice reconstituted with naive T cells from normal mice. See Hu.H.M., C.H.Poehlein, W.J.Urba, and B.A.Fox (2002) "Development of antitumor immune responses in reconstituted lymphopenic hosts" Cancer Res. 62:3914-3919. Furthermore, vaccination of reconstituted lymphopenic hosts could elicit superior anti-tumor immunity compared to normal hosts, highlighting the potential clinical benefit of performing tumor vaccination during immune reconstitution of the lymphopenic host.

Similar to mice, the need to override self tolerance mechanisms is thought to be important in mediating tumor regression in humans as well. Immune responses directed against self proteins expressed by both normal tissues and cancers helps explain the frequent occurrence of autoimmune vitiligo in melanoma patients responding to aldesleukin based therapies with absence of any vaccinationj In adoptive transfer studies, prior lymphodepletion of the melanoma patient facilitates the in vivo expansion, function and survival of transferred lymphocytes reactive against tumor/self proteins expressed on melanoma cells. The success of this treatment likely results from the ability to infuse large numbers of activated antitumor lymphocytes into an appropriate host homeostatic environment devoid of T_reg cells.

In ovarian carcinoma, T_reg cells are reported to contribute to the growth of human tumors in vivo by suppressing tumor-specific T cell immunity and are associated with a high death hazard and reduced survival. See Curiel.T.J., G.Coukos, L.Zou, X.Alvarez, P.Cheng,

P.Mottram, M.Evdemon-Hogan, J.R.Conejo-Garcia, L.Zhang, M.Burow, Y.Zhu, S.Wei,

I.Kryczek, B.Daniel, A.Gordon, L.Myers, AXackner, M.L.Disis, K.L.Knutson, L.Chen, and W.Zou (2004) "Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival" Nat. Med. 10:942-949. Study of peripheral blood lymphocytes from melanoma patients has demonstrated the presence of functional CD4⁺ CD25⁺ T_reg cells in the circulation and melanoma antigen-specific T_reg cells have been described. See Dudley.M. (2000) "Cell transfer therapy: Basic principles and preclinical studies". Principles and Practice of the Biological Thereapy of Cancer 3rd ed.:305-321. CD4⁺ CD25⁺ T_feg cells have been reported to be overrepresented in human metastatic melanoma lymph nodes and inhibit the function of infiltrating T cells!. See Viguier.M., F.Lemaitre, O.Verola, M. S.Cho, G.Gorochov, L.Dubertret, H.Bachelez, P.Kourilsky, and L.Ferradini (2004) "Foxp3 expressing CD4+CD25(high) regulatory T cells are overrepresented in human metastatic melanoma lymph nodes and inhibit the function of infiltrating T cells"J Immunol. 173:1444-1453. This could explain the poor clinical response of cancer patients under immunotherapeutic protocols, and provides a new basis for future immunotherapeutic strategies neutralizing T_reg in vivo to strengthen local antitumor immune responses.

Until recently the only available methods for eliminating CD4+CD25+ cells in vivo included the administration of anti-CD25 monoclonal antibody. This antibody is not of use, however, in eliminating regulatory cells because any activation of the immune system results in the up regulation of CD25 on activated effector lymphocytes. The two to three week half-life of monoclonal antibodies in the circulation would thus result in the undesirable elimination of activated effector lymphocytes expressing the CD25 molecule. Regulatory cells are the only cells in the non-activated circulation that express high affinity IL-2 receptors (CD25). Recently, the in vivo depletion of CD4+CD25+ cells in patients via the administration of denileukin difitox which is a recombinant immunotoxin that selectively targets cells that express high affinity IL-2 receptors in vivo has been evaluated. In this study, 13 patients (12 with metastatic melanoma, 1 with kidney cancer) were treated at in the Surgery Branch, NCI, with either the FDA-approved low (9 mcg/kg) or high (18 mcg/kg) dose of denileukin diftitox. See Attia.P. and et al (2005) "Inability of a fusion protein of IL-2 and diphtheria toxin (Denileukin Diftitox, DAB389IL-2, ONTAK) to eliminate regulatory T lymphocytes in patients with melanoma". There were no clinical responses and, furthermore, there was no evidence that T_reg cells were eliminated or even reduced. In contrast, there was evidence that the IL-2 portion of the fusion protein actually increased the number of T_reg cells as determined by Foxp3 quantification and CD4+CD25+ frequency. Thus, further studies utilizing alternative immunotoxins for the selective targeting T_reg cells in vivo may be warranted. Therefore, RFT5-dgA which is a recombinant immunotoxin that selectively targets CD25 expressing cells in vivo was evaluated. It was demonstrated that treatment of human PBMC with RFT5-dgA in vitro results in the preferential depletion of CD25+ Treg cells. Therefore, a clinical study (06-001 37) in metastatic melanoma patients was initiated to determine if treatment with RFT5-dgA may reduce T_reg cells and mediate objective clinical responses in vivo. Of five treated patients tested, two patients developed elevated serum HAMA and human anti-ricin A chain antibody (HARA) levels following therapy. No objective responses were observed in patients treated on this study. Preliminary laboratory results show a transient and substantial albeit incomplete reduction in circulating T_reg cells based upon reduced CD25 and FOXP3 protein expression by CD4+ T cells after RFT5-dgA administration in the first 5 treated patients. Our data show that although CD25 is highly expressed on the surface of many FOXP3+ CD4+ T cells in vivo, a second population of human FOXP3+ CD4+ T cell express low to intermediate levels of CD25. The frequency of low expressing FOXP3+ CD25 T cells did not change during treatment whereas the CD25 high FOXP3+ CD4 T cell frequencies decreased 98.7% for the first 6 days of treatment. These results indicate that the RFT5-dgA CD25-directed immunotherapy was not able to totally eliminate FOXP3+ cells in vivo. See Powell Jr.,D.J., P.Attia, V.Ghetie, J.Schindler, E.S.Vitetta, and S.A.Rosenberg (2008) "Partial reduction of human Foxp3+ CD4 T cells in vivo after CD25-directed recombinant immunotoxin

administration" J. Immunother. 31 :189-198.

While not being bound thereby, it is believed that the use of zanolimumab positively affects or acts synergistically with therapy with an agent which stimulates the production of T- regulatory cells (including an anti-cancer agent), by dampening the increase in T regulatory cells which may also be a side effect of such therapy. That is, while therapy in the treatment of various disease states may have many positive aspects in stimulating production of immune entities, it may also cause an increase in those TReg cells which, paradoxically, limit the effect of such therapy, such as an immunostimulatory therapy. By adding zanolimumab to the regimen before, during or (after administration of such an agent (including IL-2), particularly in those individual patients who exhibit high levels of or increased propensity to TReg increases for any reason, including immunostimulation by IL^L2 or other such immune enhancing therapy can be attenuated.

Monoclonal Antibody

The monoclonal antibody for use in the present invention is zanolimumab or a CD4 binding peptide which is comparable thereto. Zanolimumab is set forth as 6G5 as described in W097/13852. The DNA sequences for synthetic material are set forth as SEQ ID No: 219 and 220 in U.S. Patent 7,084,260. Zanolimumab (GenMab, Denmark) is also known as Humax-OD4 and HM6G (Fishwild et al., Clin Immunol. 92(2), 138-52 (1999) and the same as 6G5 as described in W097/13852),

The preparation of monoclonal antibodies is conventional. See, for example, Kohler & Milstein, Nature 256, 495-497 (1975); Coligan et al., supra sections 2.5.1-2.6.7; and Harlow, et al., in "Antibodies: A Laboratory Manual", page 726, Cold Spring Harbor Pub. (1988).

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well- established techniques. Such isolation techniques include affinity chromatography with Protein- A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for instance Coligan et al., supra sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al.,

"Purification of Immunoglobulin G (IgG)" in: Methods in Molecular Biology, 10, 79-104, Humana Press, NY (1992).

Methods bf in vitro and in vivo manipulation of monoclonal antibodies are well known to those skilled in the art. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler & Milstein, Nature 256, 495-497 (1975), or may be made by recombinant methods. The monoclonal antibodies for use with the present invention may also be isolated from phage antibody libraries using the techniques described in Clackson et al.; Nature 352, 624-628 (1991 ), as well as in Marks et al., J Mol Biol 222, 581-597 (1991 ). Another method involves humanizing a monoclonal antibody by recombinant means to geneitate antibodies containing human specific and recognizable sequences. See, for review, Holmes et al., J Immunol 158, 2192-2201 (1997) and Vaswani, et al., Annals Allergy, Asthma & Immunol 81 , 105-115 (1998).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or epitope. Furthermore, in contrast to conventional polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In additional to their specificity, the monoclorial antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

The monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies; so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567); Morrison et al., Proc Natl Acad Sc/ 81 , 6851-6855 (1984).

In one embodiment, the monoclonal antibodies are "human" antibodies

(immunoglobulins). Human antibodies may for example be prepared using transgenic non- human animals. Such animals are used to produce heterologous ahtibodies and useful methods are well known to those skilled in the art. The construction of transgenic animals harboring a functional heterologous immunoglobulin transgene is one method by which antibodies reactive with self antigens may be produced. First, the immunized animal that serves as the source of B cells must make an immune response against the presented antigen. In order for an animal to make an immune response, the antigen presented must be foreign and the animal must not be tolerant to the antigen. According to the present invention, the antigen is CD4, for instance human CD4 or a fragment thereof or a (poly)peptide comprising at least one epitope of CD4. Examples of suitable methods for preparation of human antibodies are described, for example, in WO 97/13852, page 80 to 98 under "Specific preferred embodiment" and in references contained therein. In one embodiment, the present invention relates to the use of monoclonal, human antibodies capable of binding to CD4.

An antibody for use according to the present invention is capable of binding an epitope of CD4, such as of human CD4 (for sequence of human CD4, see for instance (Maddon P J et al., Cell. 42(1 ), 93-104 (1985)). In one embodiment, the epitope is positioned in the extracellular domain of CD4. In one embodiment, the epitope is positioned in a domain of CD4 involved in TCR binding. In one embodiment, the antibody competes with Leu3A for binding to CD4 (Fishwild et al., Nat. Biotechnol. 14(7), 845-51 (1996)). A non-antibody CD4 binding peptide may also be capable of binding such epitope.

As used herein, specific binding to CD4 refers to the ability of the CD4 binding peptide, such as the anti-CD4 antibody or antigen binding fragment thereof, to bind to CD4, with an affinity of at least 1 x 10^"7 M, for instance with an affinity of at least 1 x 10^"8 M, such as 1 x 10^"9 M, for instance 1 x 10^"10 M.

The term "preferentially binding to CD4" refers herein to the property of the CD4 binding peptide, such as an anti-CD4 antibody or antigen binding fragment thereof, to bind to CD4 with above-mentioned affinity, wherein the affinity for CD4 is at least two-fold, such as at least 5-fold greater, for example at least 10-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than CD4 or a polypeptide closely-related to CD4.

The term "selectively binding to CD4" refers herein to the property of the CD4 binding peptide, such as an anti-CD4 antibody or antigen binding fragment thereof, to bind to CD4 with above-mentioned affinity, wherein the affinity for CD4 is at least two-fold, such as at least 5-fold greater, for example at least 10-fold greater than its affinity for any other polypeptide.

The present invention relates to the use of zanolimumab or a monoclonal antibody which is functionally comparable thereto, eg with respect to CD4 binding and particularly functionally and structurally comparable thereto. Thus, this a) component of the pharmaceutical

presentation is a CD4 binding peptide, such as an anti-CD4 antibody or antigen binding fragment thereof, capable of modulating at least one biological activity of CD4, for instance human CD4, such as an activity associated with cell cycle regulation. In one embodiment, the CD4 binding peptide, such as the anti-CD4 antibody or antigen binding fragment thereof, is capable of one or more of the activities.

Different assays available to the person skilled in the art may be used to determine whether a CD4 binding peptide, such as an anti-CD4 antibody or antigen binding fragment thereof, (also designated test CD4 binding peptide) recognizes the same or an overlapping epitope as a particular monoclonal antibody such as zanolimumab. For instance, the assay involves the steps of:

providing CD4 or a fragment thereof comprising the epitope recognized by the reference antibody adding the test CD4 binding peptide and the reference antibody to the said CD4, wherein either the test CD4 binding peptide or the reference antibody is labelled with a detectable label. Alternatively, both the test CD4 binding peptide and the reference antibody may be labelled with different detectable labels, and

detecting the presence of the detectable label(s) at CD4

hereby detecting whether the test CD4 binding peptide is capable of displacing the reference antibody

If the test CD4 binding peptide is capable of displacing the reference antibody, the test CD4 binding peptide recognizes the same or an overlapping epitope as the reference antibody. Thus if the reference antibody is labeled With a detectable label, then a low detectable signal at CD4 is indicative of displacement of the reference antibody. If the test CD4 binding peptide is labelled with a detectable label, then a high detectable signal at CD4 is indicative of

displacement of the reference antibody. The CD4 fragment may for instance be immobilized on a solid support enabling facile handling. The detectable label may be any directly or indirectly detectable label, such as an enzyme, a radioactive isotope, a heavy metal, a colored compound or a fluorescent compound.

The zanolimumab, or a comparable anti-CD4 antibodies or antigen binding fragments thereof, for use according to the present invention may be administered to patients by any method known in the medical arts for delivery of proteins and antibodies. Peptides, such as antibodies are particularly suited for parenteral administration. Parenteral administration may for example be by subcutaneous, intramuscular or intravenous administration, including infusion or injection. The pharmaceutical compositions of the present invention are suitable for

administration using alternative drug delivery approaches as well (see for instance Langer, Science, 249, 1527-1533 (1990)).

Pharmaceutical compositions for parenteral administration usually comprise a solution of zanolimumab (or comparable as above) dissolved in an acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for instance water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well known sterilization techniques. These compositions may also be subjected to a virus reduction or multiple virus reductions by conventional well known techniques. The compositions may contain

pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH-adjusting and buffering agents, tonicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of zanolimumab (or comparable), in these formulations can vary widely, for example from less than about 0.5%, usually at or at least about 0.1 % to as much as 1.5% or 2.0% or even more by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in; for example, Remington's Pharmaceutical Sciences, 17th Ed., Mack Publishing Company, Easton, Pa. (1985), which is incorporated herein by reference.

The zanolimumab (or comparable CD4 binding peptides, such as an anti-CD4 antibodies or antigen binding fragments thereof) may be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide compound) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, arid the like. Salts formed with the free carboxyl group may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Injectables are usually prepared either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid such as sterile water prior to injection. The preparation may also be emulsified. The active ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like, and combinations thereof. In addition, if desired, the preparation may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, salts, pH buffering agents, or which enhance the effectiveness or transportation of the preparation.

For some embodiments, the zanolimumab (or comparable CD4 binding peptide, such as an anti-CD4 antibody or antigen binding fragment thereof) is administered locally, for example by direct injection to the disease site. In other embodiments, it is administered in a systemic manner.

For zanolimumab (or comparable) the efficacy may be enhanced if the preparation further comprises pharmaceutically acceptable additives and/or carriers. Such additives and carriers will be known in the art. In some cases, it will be advantageous to include a compound, which promotes delivery of the active substance to its target. In addition, the zanolimumab may be modified to provide a cytotoxic, diagnostic or radioactive moiety appended thereto, eg by a covalent bond, for purposes of providing further braking on the activity and/concentration of Treg cells or to assess the progress of a therapy. The dose of such zanolimumab may be from about 0.5 to about 50 mg/kg of body weight per administration, eg intraveneously per week. More specifically, the dose may be about 1 to about 20 mg/kg per week.

Treg Cell Stimulator

As defined by the National Cancer Institute, an immunostimulant is a substance that augments the ability of the immune system to fight infection and disease.

Embodiments of immunostimulants may be both specific and non-specific. Specific immunostimulants, such as vaccines or antigens, are those that provide antigenic specificity in the immune response whereas non-specific immunostimulants, such as an adjuvant or hormones (e.g. IL-2), act irrespective of antigenic specificity to augment immune response of other antigens. The present invention is particularly concerned with non-vaccine

immunostimulants. Among the types of medicaments, agents, treatments, and natural conditions which result in the overstimulation of Treg cells are anti-CTLA-4, anti-PD1 , a vaccine or any other immune stimulant whose activity may be negatively regulated by T regulatory cells. A particular embodiment of the invention is IL-2 as the immunostimulant. Thus, embodiments of immunostimulants may for example be defined as any receptor agonist such as aldesleukin, arbidol, arbidol hydrochloride, autologous melanoma cells, bacillus calmette Guerin

polysaccharide, nucleic acids, bacterial lysate, beta-glucan, birch pollen allergen, tree pollen allergen, bordetella pertussis (inactivated), dipththeria toxoid, haemophilus influenza type b capsular polysaccharide, hepatitis B surface antigen, tetanus toxoid, bordetella pertussis antigens, carrageenan, cholera toxin B subunit, and vibrio cholera.

The term IL-2, as used herein, refers to natural and recombinant IL-2, active fragments, pharmaceutically acceptable analogues, derivatives thereof, and mixtures thereof, in amounts effective to activate their respective high affinity cytokine receptors. For purposes of the present invention suitable agents having IL-2 activity can include but are not limited to natural and recombinant forms of IL-2, IL-2 fusion proteins, and PEG, carbohydrate, lipid, therapeutic agent, reduced, non-glycosylated, and mutated derivatives thereof, and pharmaceutically acceptable analogues and derivatives thereof, and mixtures thereof, where a dose of about 1 ,000 pmole/m² yields a peak plasma concentration about 20 pM 2 hours after a subcutaneous injection.

Further, IL-2 refers to a polypeptide obtained from tissue cultures or by recombinant techniques exhibiting the spectrum of activities characterizing this protein. The word includes not only human IL-2 (hlL-2), but also other mammalian IL-2 such as, e.g., mouse, rat, rabbit, primate, pig and bovine IL-2. Bovine IL-2 is described by Cerretti et al., PNAS, 83:3223-3227 (1986). The term "IL-2" also refers to a protein that is capable of stimulating the proliferation of hlL-2 dependent cytolytic and helper T-cell lines, as set forth in the standard assays of Gillis, S., et al., J. Immunol,. (1978) 120:2027-2032 and of Watson, J., J. Exp. Ned. (1979) 150:1510- 1519. The amino acid sequence of native hlL-2 is shown in FIG. 1 df US Patent 6,060,068. This primary amino adid sequence may be obtained as the native protein from natural sources or may be recombinantly derived. The recombinant hlL-2 is a particular version for use herein.

IL-2 is preferably formulated as a pharmaceutical composition with a pharmaceutically acceptable carrier. Such presentations are described in US Patent 6,060,068. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are

physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly iin humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Sterile water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.

Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Certain adjuvants mentioned above, particularly mineral oils and adjuvants containing mineral oils (e.g., Freund's adjuvant) are not acceptable for use in humans.

Parameters that may be monitored in order to adjust the dose of the agent administered daily are the blood cell count of circulating lymphocytes, monocytes and polymorphonuclear leukocytes, such as T-cells, B-cells, NK cells, monocytes, eosinophils, neutrophils, basophils, antigen-presenting cells, among others. \ri the case of viral infections, particularly in the presence of the human immunodeficiency virus, one of the ways to determine an adjustment in the dose administered to an individual is to monitor the count of circulating microorganisms.

Different preparations of a specific cytokine or related agent, and different formulations may require varied daily doses, which depend on the agent's binding constant to the respective receptors, and the existence and number of different kinds of receptors they selectively bind to. These values may be determined utilizing a ligand-receptor binding assay. The ligand-receptor binding assay may be conducted, for the different agents in accordance with this invention, as described by Robb et al., J. Exp. Med. 154: 1455-1474, 1981. In brief, a cytokine such as IL-2, I must be in native configuration, and substantially free of contamination by other molecules. The ligand or agent may be labeled, for example, using radioisotopes, enzymes, and other markers. Target cells, isolated membranes, cytoplasm, or nuclei, may then be mixed together with the labeled ligand, and the ligand and receptor allowed to reach a steady state, where the rate of association of the ligand with the receptor about equals the rate of dissociation of the ligand from the receptorL Subsequently, any unbound ligand or agent may be separated from the receptor-bound ligand or agent, usually by centrifuging the bound ligand or agent, and the amounts of the bound and unbound ligand or agent measured. The affinity of the ligand- receptor interaction (Kd) may be calculated, as well as the number of receptors per cell, or per weight of cytoplasm or nuclei, from these two experimentally determined values, knowing the number of cells, or amount of membranes, cytoplasm, or nuclei, used. The data may be plotted by the method described by Scatchard, Ann. NY Acad. Sci. 51 : 660^672, 1949. The ratio of bound vs. free ligand or agent may be plotted on the y-axis and the amount of bound ligand or agent on the x axis. The slope of the data points yields the Kd (x y),' whereas the x-axis intercept yields the number of receptors. Knowing the equilibrium dissociation constant (Kd), the % receptors that will be occupied at each ligand or agent concentration may be calculated as described above.

The dosage of the agent having IL-2 activity of the present invention will further depend on the disease state or condition being treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.

Bioequivalent doses of IL-2 can be obtained using formulations from different sources by those with skill iri the art by for example first assessing the biologic activity in vitro using the CTLL assay (Gillis, S. J._; Immunol. 120:2027, 1978), calculating the specific activity of the formulation or performing in vivo pharmacokinetic testing to determine the potency of the formulation and determining the appropriate dosage for use in accordance with the teachings of this invention.

IL-2 has been obtained commercially, eg from Chiron (Chiron Therapeutics Inc.,

Emeryville Calif.) or Amgen (Amgen Corporation.Thousand Oaks, Calif.). As described in US Patent 6,921 ,530, Chiron IL-2 revealed differences in the formulations of the two products. Amgen IL-2 was formulated to an ultimate concentration of 0.4 mg/ml. in low ionic strength sodium acetate buffer (10 mM), pH 4, while Chiron IL-2 was formulated at 1.1 mg/ml, in phosphate buffer at physiological ionic strength (150 mM) and pH 7. This formulation of the Chiron product rendered it less soluble, and the ionic detergent sodium dodecyl sulfate (SDS) was added to the preparation. The Chiron formulation was approximately 6-fold less than the Amgen preparation, due to the decreased available protein concentration in the Chiron preparation. At present, IL-2 may be obtained as Proleukin (aldesleukin) from Novartis Pharmaceuticals Corporation of East Hanover, New Jersey, USA and there are listed on the labeling US Patents RE 33,653; 4,530,787; 4,569,790; 4,748,234; 4,572,798; 4,959,314; and 5,464,939 (revised October 2008).

The methods of the present invention contemplate single as well as multiple

administrations given either simultaneously or over an extended period of time. Given that 1 pmole IL-2 is equivalent to about 250 IU, particular daily doses of IL-2 are about 125,000 lU/kg of patient body weight/day to about 720,000 IU/kg/3 times per day..

Other Aspects and Agents

While an aspect of the present invention include as a first embodiment the method for the treatment of a disease in a patient by decreasing the Treg cell concentration in said patient, a further aspect comprises the sequential steps of:

A) determining the Treg cell concentration in the patient;

B) administering a first dose of zanolimumab or a monoclonal antibody which is comparable thereto to the patient; and

C) redetermining the Treg cell concentration in said patient,

as a second embodiment. The Treg cell concentration can be determined in peripheral blood, a solid tumor or by draining lymph nodes. Further, the Treg cell concentration can be measured by foxp3 expression or CCL21 expression (Natures Reviews Cancer, Vol 10, May 2010 P1038). After step C), one may follow with the sequential following steps D) and/or E): D) administering an additional dose of zanolimumab to said patient; and

E) administering an immunostimulant to said patient.

A third embodiment of the invention includes the use of zanolimumab as above plus IL-2 as above and an antibody against HER2, such as Herceptin or pertuzumab, for treatment of various cancers, such as breast, stomach and ovarian.

A fourth aspect of the present invention is the use of zanolimumab and IL-2, as described above, with adoptive cell therapy (ACT, or cell transfer therapy). ACT includes a process of removing T-cells from a tumor (Tumor Infiltrating lymphocytes or TILs) and expanding or prdliferating (increasing the number of) cells outside of the patient's body in vitro with IL-2 to stimulate the expansion. After expansion, the patient is administered conditioning chemotherapy for lymphodepletion (shown by Mark E Dudley et al in Science, 2002 October 25: 298(5594): 850-854) followed by infusing the expanded cells back into the patient with IL-2 (optionally a high dose) to generate an anti-tumor response. Further ACT descriptions are found in articles by Steven A. Rosenberg et al in Nature Reviews/Cancer, Volume 8, April 2008, pages 299-308 and by Robert Edward Hawkins, David Gilham, Reno Debets, Zelig Eshhar, Naomi Taylor, Hinrich Abken and Ton Schumacher in Human Gene Therapy.- o\ available-, ahead of print.doi: 10.1089/hun.2010.086. Further descriptions of ACT are found in the following:

Gattinoni, L, Powell Jr., D.J., Rosenberg, S.A. and Restifo, N.P. Adoptive immunotherapy for cancer: Building on success. Nat Rev Immunol. 6(5):383-93, (May 2006),

Powell Jr., D.J., de Vries, C, Allen, T., Ahmadzadeh, M. and Rosenberg, S.A. Inability to mediate prolonged reduction of regulatory T cells following transfer of autologous CD25- depleted PBMC and interleukin-2 after lyrriphodepleting chemotherapy. J Immunother.

30(4):438-47, (May-Jun 2007) and Johnson, L.A., Heemskerk, B., Powell Jr., D.J., Cohen, C.J., Morgan, R.A., Dudley, M.E., Robbins, P.F., and Rosenberg, S.A. Gene transfer of tumor- reactive T-cell receptors confers both high avidity and tumor-reactivity to non-reactive PBMC and TIL. J Immunol. 177(9): 6548-6559, (Nov 2006).

Presentation

The term "effective amount," as used herein, refers to that amount of an antibody or an antigen binding portion thereof which is sufficient to effect treatment, prognosis or diagnosis of a disease as indicated herein when administered to a subject. A therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the seventy of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. Dosage regimen may be adjusted to provide the optimum therapeutic response. An effective amount is also one in which any toxic or detrimental effects (i.e., side effects) of an antibody or antigen binding portion thereof are minimized and/or outweighed by the beneficial effects. Additional preferred dosages regimens are described further below in the section pertaining to pharmaceutical compositions.

A useful form of the invention is a unit dosage composition, which may be packaged in a sterile container, having sufficient amounts of one or more forms of the agent to maintain a therapeutic level in blood for a period of about 24 hours. This amount of the agent to be administered may be calculated as described herein for each individual agent. The agent may be administered as known in the pharmaceutical arts, particularly subcutaneously,

transdermal^, intrapulmonarily, transbuccally, intravenously or by implant.

The unit dosage composition may be prepared in a variety of forms for the delivery of the agent. Examples are powder, tablet, capsule, dragee, cream, solution, suspension, emulsion, gel, spray, or liposomal or other micellar forms. Preferred are solid, particularly freeze-dried, and liquid forms. Also preferred are other forms such as those suitable for injection, topical application, controlled release products, inhalation, and others. Examples of controlled release products are transdermal and intradermal devices, slow release oral formulations, patches, skin, mucosal and transbuccal implants, suppositories, and t+ie like. Implants are preferred for long term delivery of the agent. Controlled release products may be prepared as is known in the art and designed for releasing desired amounts of the agent over a predetermined period of timeL In most instances, the amount of the agent contained in the control release product is substantially higher than the desired daily dose. In some cases, the product may contain sufficient amounts of agent for releasing a daily dose over a period of days, weeks, mdnths, and even years. The product may be tested, and the amount of agent to be released adjusted in accordance with the observed absorbed dose. Any of the forms for administration of the agent may also contain other formulation components.

Various forms of the composition are provided herein for administration of the agent of the invention under the conditions prescribed herein. One of them is a systemic composition, also comprising a diluent and/or carrier for system administration, and optionally other additives which are described below or standard in the art. This form may be in the form of a solution, suspension, powder, tablet, an emulsion, and encapsulated particles, among others, or mixtures or combinations of these forms. The agent may be present in the systemic composition in an amount of about 0.0001 to 50 wt % of the composition, more preferably about 0.1 to 30 wt %.

Various simultaneous and sequential administration protocols may be used in the method of the present invention, ie with respect to the two components a) and b):

a) zanolimumab or a monoclonal antibody which is comparable thereto, and

b) an agent Which stimulates the production of T-regulatory cells, including IL-2 or an interleukin which is comparable thereto.

Such dosing regimens may vary according to the identity, severity and stage of the disease being treated as would be apparent to one skilled in the art. Thus, Example 1 provides an initial protocol for assessing and diagnosing the particular effect in a given patent population. In addition, the IL-2 may be given at about 125,000 lU/kg/day for up to about 14 days, while the higher dose of about 720,000 IU/kg/3 times per day would be given up to about 5 days. These two modes of dosing can be administered intravenously while the lower dose may also be administered subcutaneously.

Disease

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, and blastoma. More particular examples of such cancers with known Treg involvement include breast cancer (Knutson KL. J. Immunol. 2006 177(1) 84-91), sarcoma (Knutson KL J. Immunol. 2006 177(1 ) 84-91), chronic lymphocytic leukemia (Beyer M 2005. Blood 106 2018^25, ovarian (Barnet B. 2005 Am. J. Reprod. Immunol. 54 369-377, glioblastoma (Curtin JF. (2008 PLoS ONE 3(4) e1983.doi 10.1371), neuroblastoma (Johnson BD J, Immunother 1997 30:203-214), leukemia (Onizuka S Cancer Res 1999 59- 3128-33), fibrosarcoma (Onizuka S Cancer Res 1999 59-3128-33), myeloma (Onizuka S Cancer Res 1999 59-3128-33), glioma (El Andaloussi A J. Neurooncol 2007 83: 145-52), colon carcinoma (Ko KJ. Exp Med. 2005 202: 885-91 ), colorectal (Golgher D Eur. J. Immunl. 2003 171 5931 -39), plasmacytoma (Onizuka S Cancer Res. 1999 59 3128-33), mastocytoma

(Onizuka S Cancer Res. 1999 59 3128-33), non-small cell lung cancer (Woo EY Cancer Res 2001 61 4766-72), oesophageal cancer (Ichihara F Clin. Cancer Res. 2003. 9 4404-8), gastric cancer (Ichihara F Clin. Cancer Res. 2003. 9 4404-8), hepatocellular carcinoma (Ichihara F Clin. Cancer Res. 2003. 9 4404-8), pancreatic cancer (Liyanage UK . Immunol. 2002 169 2756-61), renal cancer (Ahmadazadeh M Blood 2006 107 2409-14), Ewing sarcoma (Zhang H. Nature Med 2005 1 1238-1243), alveolar sarcoma (Zhang H. Nature Med 2005 1 1238-1243) and rhabdomyosarcoma (Zhang H. Nature Med 2005 1 1 1238-1243). In one preferred embodiment of the invention the cancer is cervical intraepithelial neoplasia, or cervical cancer where relatively strong evidence exists for Treg involvement. (Loddenkemper C. Cancer Sc/.2009. 100 (6) 1 1 12-1 1 17

Other cancers for use with one or more modes of the invention include: squamous cell cancer, small-cell lung cancer, neurofibromatosis, cervical cancer, liver cancer, bladder cancer, hepatoma, colon cancer, melanoma, endometrial carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer. In a particular embodiment, a cancer treated or diagnosed using the methods of the present invention is selected from melanoma, breast cancer, ovarian cancer, renal carcinoma, gastrointestinal/colon cancer, lung cancer, and prostate cancer. Most particularly, the cancer to be addressed is melanoma or renal carcinoma.

The malignant disease may be any malignant disease, for instance primary cancer or metastatic cancer. The term "malignant disease" as used herein is meant to cover also pre- malignant diseases.

By "primary cancer" is meant a group of tumor cells, which have acquired at least one characteristic feature of cancer cells, however have not yet invaded the neighboring tissues and hold together in a tumor localized at the place of primary origin. By ^'metastatic cancer" is meant a group of tumor cells, which originate from the cells of a primary cancer, which have invaded the tissue surrounding said primary cancer, disseminated through the body, adhered at a new distant place and grown to a new tumor.

Pre-malignant and/or malignant conditions may for example be cancer or conditions which may develop into a cancer. The term cancer within the scope of the present invention covers both malignant and benign tumors, as well as leukaemia and lymphoma.

Therapeutic indications for the present invention, e.g. an immunostimulant and zanolimumab combination, include but are not limited to, non-autoimmune diseases where Treg cell reduction is beneficial, such as cancer.

Cancer may for example be adenomas, carcinomas or sarcomas. Cancer may for example be selected from the group consisting of melanoma, brain tumors, neuroblastomas, breast cancer, lung cancer, prostate cancer, cervix cancer, uterine cancer, ovarian cancer, leukaemia, colon cancer, rectum cancer, cancer of the testis, cancer of the kidney, cancer of the liver, cancer of the lip, cancer of the tongue, cancer of the stomach, skin cancer, sarcomas, mesotheliomas, bladder cancer, bone tumors, malignant pleural effusions, ascites, meningeal carcinomatosis, head and neck cancers and cancers of endocrine organs such as: thyroid gland, pituitary gland and suprarenal gland.

In one embodiment the malignant disease wherein the various embodiments of the invention may be used is selected form a group consisting of CD4⁺ nodal T-cell lymphomas, such as peripheral T-cell lymphoma, angioimmunoblastic T-cell lyniphoma or anaplastic large T- cell lymphoma.

Reference I

IL-2 monotherapy

The five year survival of patients With metastatic melanoma and metastatic kidney cancer is less than 10% in most reported series. Combination chemotherapy can induce objective regressions of melanoma but this treatment is rarely, if ever, curative. High-dose aldesleukin is the only treatment known to cure some patients with metastatic renal cell cancer and metastatic melanoma. Of 255 metastatic renal cell cancer patients treated with high-dose aldesleukin, 15% had an objective response, including 7% of patients with a complete response (CR). See Proleukin Packgage Insert (2007) Novartis Pharmaceutical Corporation. East Hanover, NJ. Of 270 patients with metastatic melanoma treated with high-dose aldesleukin,

16% had an objective response, includingi 6% of patients with a CR. Although most patients with partial responses ultimately progress, complete responses tend to be durable. Of 33 patients with complete responses treated in the Surgery Branch of the National Cancer Institute, Rockville, Maryland, USA, 24 have never recurred with ongoing responses from 21 to 257 months and more. Thus it appears that in a subset of patients with metastatic melanoma or kidney cancer, curative responses can be induced by aldesleukin. These results, confirmed in many reports by other investigators, led to the approval of aldesleukin for the treatment of patients with melanoma and kidney cancer by the US Food and Drug Administration.

Reference II

Zanolimumab monotherapy

HuMax-CD4™ (zanolimumab) Phase II studies treating patients with mycosis fungoides (MF) a type of cutaneous T-cell lymphoma (CTCL). Data from all patients in the study showed a median response duration of more than 45 weeks (10.5 months). Furthermore, analysis of the time to response showed that 85% of the responding patients (11/13) obtained clinical response within 8 weeks.

Additionally, previously released data was analyzed to assess the relation between dose and the serum concentration of HuMax-CD4 measured immediately before the next treatment (trough values). Increasing doses of HuMax-CD4 resulted in increased trough values.

Furthermore, a 55% response rate was achieved in patients reaching lesser than or equal to 10 mu g/ml serum concentration compared to only 6% (1/16) in patients not reaching this serum level. In the 280 mg dose group, 25% (5/20) were above 10 mu g/ml and a 15% response rate (3/20 patients) was achieved. In the 560 mg dose group, 93% (13/14) were above 10 mu g/ml and a 50% response rate (7/14) was obtained. In the 980 mg group 100% (4/4) were above 10 mu g/ml and a 75% response rate (3/4) was obtained.

These data were presented by Dr. Y. H. Kim, Professor of Dermatology and Director, Multidisciplinary Cutaneous Lymphoma Clinic, Stanford University at the 63rd Annual Meeting of the American Academy of Dermatology in New Orleans, Louisiana, USA on February 18, 2005 as P2124.

Example 1

Zanolimumab specifically recognizes CD4 protein expressed on a subset of T lymphocytes and on monocytes from humans, and non-human primates. Ongoing clinical studies have identified a 14 mg/kg dose of zanolimumab weekly as a safe and efficacious dose. Toxicities of zanolimumab included headache, influenza-like illness, injection/infusion site reaction, nasopharyngitis, pyrexia, diarrhea, fatigue, and cytokine ilelease syndrome at the time of infusion. This Example is based on the present invention which comprises transient elimination of CD4⁺ T-regulatory cells with zanolimumab to enhance the clinical effectiveness of IL-2 (aldesleukin) administration by decreasing T-regulatory cell generation. This Example demonstrates the ability of a combination of aldesleukin and zanolimumab (anti-CD4 mAb) administration to mediate tumor regression in patients with metastatic melanoma and metastatic kidney cancer. The following procedure also determines the rate of depletion and repopulation of CD4+ cells and CD4+CD25+ T regulatory cells in treated patients and the toxicity of this treatment regimen. Patients in this Example 1 are 18 years of age or older and have measurable metastatic melanoma or metastatic kidney cancer; clinical performance status of ECOG 0, or 1 and have not previously received high dose of aldesleukin.

Patients receive zanolimumab at a dose of 14 mg/kg as an i.v. infusion weekly (± 3 days) for 9 weeks. After the fifth and seventh dose of zanolimumab, aldesleukin will

administered as an i.v. bolus at a dose of 720,000 lU/kg every 8 hours for a maximum of 15 doses.

Zanolimumab is manufactured by GenMab, Copenhagen, Denmark, and provided by Tenx Biopharma, Inc., Philadelphia, PA. Zanolimumab is a clear colourless liquid. It is supplied in 5 mL glass vials containing 20 mg zanolimumab/mL phosphate buffered saline (PBS).

Patients receive 14 mg/kg weekly for 9 wdeks. Treatment is administered on an outpatient basis where possible. Zanolimumab is given as an i.v. infusion and is filtered using an inline filter (0.22 μητι) prior to the infusion. The concentration of zanolimumab iiti the vials is 20 mg/mL (each vial contains 100 mg). One vial of PBS (7 mL) is used to dilute the study drug. For all infusions, the start and stop times, the total volume and the rate of infusion is recorded in the medical record to obtain a complete dosing history. The infusion rate isapproximately 1 mUmin. Actual infusion time varies depending on the weight of the patient who is kept under observation for 2 hours after the fiitst infusion with zanolimumab and for at least 30 minutes after subsequent infusions. Zanolimumab is stored in a refrigerator at 2-8°C.

Aldesleukin IL-2 is manufactured by Novartis Pharmaceuticals Corporation, Florham Park, NJ and provided as single-use vials containing 22 million IU (1.3 mg) aldesleukin as a sterile, white to off-white lyophilized cake plus 50 mg mannitol and 0.18 mg sodium dodecyl sulfate, buttered with approximately 0.17 mg monobasic and 0.89 mg dibasic sodium phosphate to a pH of 7.5 (range 7.2 to 7.8). The vial is reconstituted with 1.2 mL of Sterile Water for Injection, USP, and the resultant concentration is 18 million lU/ml or 1.1 mg/mL. Diluent is directed against the side of the vial to avoid excess foaming and the contents are gently swirled until completely dissolved without shaking: Since vials contain no preservative, reconstituted solution is used with 24 hours.

Intact vials are stored in the refrigerator (2° - 8°C) protected from light. Reconstituted aldesleukin is further diluted with 50 mL of 5% Human Serum Albumin (HSA). The HSA is added to the diluent prior to the addition of aldesleukin. Dilutions of the reconstituted solution over a 1000-fold range (i.e., 1 mg/mL to 1 mcg/mL) are acceptable in either glass bottles or polyvinyl chloride bags. Aldesleukin is chemically stable for 48 hours at refrigerated and room temperatures, 20 - 30°C. The final dilution of aldesleukin is infused over 15 minutes.

Patients undergo complete evaluation of tumor with physical examination, CT of the chest, abdomen and pelvis and clinical laboratory evaluation 2 weeks after zanolimumab administration (approximately 4 weeks after aldesleukin administration. If the patient has stable disease (SD)or tumor shrinkage, repeat complete evaluations are performed every 1-3 months. After the first year, patients continuing to respond continue to be followed with this evaluation every 3-4 months until off study criteria are met.

If patients have stable disease or a partial response to treatment after the initial evaluation, or if a patient recurs or progresses after a clinical response, they may be eligible for re-treatment.

Patients are entered into one of two strata: metastatic melanoma or metastatic renal cancer. Each of the 2 strata are conducted using an optimal two-stage phase II design to rule out an unacceptably low 15% clinical response rate, in favor of a modestly high response rate of 35% (p1 =0.35). With alpha=0.10 and beta = 0.10, the study initially enrolls 19 evaluable patients in each strata. If 0-3 of the 19 patients have a clinical response, then no further patients are accrued to the strata. If 4 or more of the first 19 patients in each strata have a response, then accrual continues until a total of 33 patients enrolls in each strata . To allow for the possibility of a small number of inevaluable patients, the accrual ceiling for the trial is set at 70 patients.

Apheresis is performed, prior to any treatment, prior to the 2^nd cycle of aldesleukin and 1 week after the treatment. At other time points, patient peripheral blood lymphocytes (PBL) are obtained from whole blood by purification using centrifugation on a Ficoll cushion. Lymphocytes are evaluated by a variety of tests including evaluation of specific lysis and cytokine release, limiting dilution analysis of precursor frequency, ELISA-spot assays, and lymphocyte subset analysis may be used to evaluate response to melanoma antigens. In general, differences of 2 to 3 fold in these assays are indicative of true biologic differences. Immunological monitoring will consist of quantifying T cells reactivity including CD4, CD8, CD56 and CD4CD25+ cells. FOXp3 levels are evaluated by TaqMAN and CD4+CD25+ cells by flow cytometry weekly during treatment and at one month after therapy and are repeated at two months.

Response Criteria (Evaluation of target lesions):

Complete Response (CR): Disappearance of all target lesions

Partial Response (PR): At least a 30% decrease in the sum of the longest diameter (LD) of target lesions taking as reference the baseline sum LD.

Progression (PD): At least a 20% increase in the sum of LD of target lesions taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesions.

Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD taking as references the smallest sum LD.

All measurable lesions up to a maximum of 10 lesions representative of all involved organs should be identified as target lesions and recorded and measured at baseline. Target lesions should be selected on the basis of their size (lesions with the longest diameter) and their suitability for accurate repetitive measurements (either by imaging techniques or clinically). A sum of the longest diameter (LD) for all target lesions will be calculated and reported as the baseline sum LD. The baseline sum LD will be used as reference to further characterize the objective tumor response of the measurable dimension of the disease.

Response Criteria (Evaluation of non-target lesions):

Complete Response (CR): Disappearance of all non-target lesions and normalization of tumor marker level.

Non-Corriplete Response: Persistence of one or more non-target lesions

Progression (PD): Appearance of one or more new lesions. Unequivocal progression of existing non-target lesions

All other lesions (or sites of disease) should be identified as non-target lesions and should also be recorded at baseline. Measurements are not required, and these lesions should be followed as "present" or "absent."

Claims

What is claimed is:

1. A method for the treatment of a malignancy in a mammal which comprises administering to said mammal a pharmaceutical therapy which comprises the following components: a) zanolimumab or a monoclonal antibody which is comparable thereto, and b) IL-2 or an interleukin which is comparable thereto.

2. The method of Claim 1 , wherein said component a) is zanolimumab.

3. The method of Claim 1 , wherein said component a) is a monoclonal antibody

comparable to zanolimumab.

4. The method of Claim 3, wherein said component a) is a monoclonal antibody which is functionally comparable to zanolimumab.

5. The method of Claim 3, wherein said component a) is a monoclonal antibody which is functionally and structurally comparable to zanolimumab.

6. The method of Claim 1 , wherein said malignancy is melanoma or renal carcinoma.

7. The method of Claim 1 , wherein said mammal is a human.

8. The method of Claim 1 , wherein said therapy consists essentially of said components a) and b).

9. The method of Claim 1 , wherein said administering is essentially simultaneous.

10. The method of Claim 1 , wherein said administering of component a) takes place at least 1 day before the administering of component b).

11. The method of Claim 1 , wherein the dose of component a) is about 0.1 to 50 mg per kilogram of body weight, and the dose of component b) is about 100,000 to 1 ,000,000 IU per kilogram of body weight.

A method of enhancing the efficacy of IL-2, or an interleukin which is comparable thereto, in a mammal which comprises the administration to said mammal of a pharmaceutical composition comprising zanolimumab.

A method for the treatment of a disease in a mammal susceptible to Treg cell modulation, which comprises administering to said mammal a pharmaceutical therapy which comprises the following components:

i) zanolimumab or a monoclonal antibody which is comparable thereto, and ii) a cytotoxic agent, an agent which stimulates specific elements of the immune system, an agent which stimulates the immune system generally or is a composition comprising autologous or allogenic tumor infiltrating lymphocytes, and

iii) optionally a third therapeutic agent, which third agent augments Treg cell reduction.

The method of Claim 13, wherein said component ii) is a composition containing autologous tumor infiltrating lymphocytes which have been proliferated in vivo.

The method of Claim 13, wherein said disease is renal carcinoma.

The method of Claim 13, wherein said component iii) is cyclophosphamide, a calcineurin inhibitor, cyclosporine, tacrolimus, fludarabine, TLR ligation, an anti-CTLA-4 antibody, an anti-CCR4 receptor or chemokine CCL22.

The method of Claim 13, wherein said component i) is a monoclonal antibody which is functionally and structurally comparable to zanolimumab.

The method of Claim 13, wherein said disease is melanoma.

The method of Claim 13, wherein said method is a method of improving the response of said mammal to said disease.

The method of Claim 13, wherein said therapy consists essentially of said components i) and ii).

21. The method of Claim 13, wherein said administering is essentially simultaneous.

22. The method of Claim 13, wherein said administering of component i) is at least 1 day before the administering of component ii).

23. The method of Claim 13, wherein the dose of component i) is about 0.1 to 50 mg per kg of body weight.

24. A method for the transient elimination of CD4⁺ T-regulatory cells in a mammal which comprises the administration to said mammal of a pharmaceutical composition comprising zanolimumab.

25. A method for the treatment of a disease in a patient by decreasing the Treg cell

concentration in said patient, which comprises the sequential steps of:

A) determining the Treg cell concentration in the patient;

B) administering a first dose of zanolimumab or a monoclonal antibody which is comparable thereto to the patient; and

C) re-determining the Treg cell concentration in said patient.

26. The method of Claim 25, wherein said Treg cell concentration is in the peripheral blood, solid tumor mass or lymph node of said patient.

27. The method of Claim 25, wherein after step C), further comprising the sequential following steps D) and/or E):

D) administering an additional dose of zanolimumab to said patient; and

E) administering an immunostimulant to said patient.

28. The method of Claim 25, wherein said step A) is by measuring foxp3 expression or CCL21 expression.