Combination treatment of primary central nervous system lymphoma

Abstract

The present invention relates to the combination of R-CHOP therapy with the administration of NGR-hTNF or an analog thereof for the treatment of primary central nervous system lymphoma, preferably relapsed / refractory primary central nervous system lymphoma.

Description

[Technical field]

[0001] The present invention relates to the combination of R-CHOP therapy with the administration of NGR-hTNF or an analogue thereof for the treatment of primary central nervous system lymphoma. [Background technology]

[0002] Primary CNS diffuse large B-cell lymphoma (PCNSL) is an aggressive malignancy with unique clinical behavior that remains localized to the CNS and can rarely disseminate outside the CNS (1). Compared to localized extra-CNS DLBCL, PCNSL patients exhibit inferior survival rates, at least in part due to the ineffectiveness of the agents currently used to treat extra-CNS DLBCL (i.e., the R-CHOP regimen: rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) to cross the blood-brain barrier (BBB) ​​and achieve good CNS bioavailability (2). Currently, PCNSL patients are treated with high-dose methotrexate-based combinations, frequently combined with cytarabine, alkylating agents, and rituximab (3). The widespread use of these current combinations has greatly improved the survival of patients with PCNSL, but these treatments require hospitalization, adequate direct experience, and suffer from associated toxicities (4). In contrast, improved CNS delivery of R-CHOP agents may have the advantage of a well-tolerated treatment that does not require hospitalization and is widely used in oncology-hematology departments. Thus, the use of intravenously administered agents that can induce reversible BBB permeability and improve CNS cytostatic bioavailability is an attractive avenue of investigation in patients with PCNSL.

[0003] Intravenous delivery of tumor necrosis factor (TNF) undergoes selective BBB penetration and improves tumor penetration of chemotherapeutic agents in animal models of brain metastasis (6). TNF is a proinflammatory cytokine with potent antitumor activity, but its use in cancer patients is limited by significant systemic toxicity (7). Growing evidence suggests that the therapeutic index of this cytokine could be improved by vascular targeting approaches (8). This can be achieved, for example, by fusing the N-terminus of human TNF with CNGRCG, a tumor vasculature homing peptide capable of recognizing an isoform of aminopeptidase N (CD13), a membrane-bound metalloproteinase that is up-regulated in angiogenic tumor vessels (9, 10) and is poorly or not expressed in normal vessels (11). The CNGRCG-TNF fusion protein, developed with human TNF (designated NGR-hTNF, developed at the San Raffaele Scientific Institute, Milan, Italy), allows the delivery of extremely low doses of the cytokine, which is not yet pharmacologically active, to the tumor vasculature, thereby avoiding systemic toxic reactions and counterregulatory mechanisms (12). Studies carried out in animal models of melanoma and lymphoma have shown that low doses of NGR-hTNF can locally increase vascular permeability and increase the penetration of chemotherapeutic agents in tumor tissue (8, 10, 12, 14).

[0004] Based on these findings, we hypothesized that low-dose NGR-hTNF could alter the BBB and improve the penetration and activity of R-CHOP in patients with PCNSL.As part of the translational research program of NGR-hTNF, we designed a prospective phase II study aimed at evaluating the feasibility and activity of six courses of R-CHOP chemoimmunotherapy preceded by BBB penetration with low-dose intravenous administration of NGR-hTNF in patients with relapsed or refractory PCNSL. In a per-protocol planned "proof-of-principle" part of the study, changes in BBB permeability of lymphoma lesions and normal-appearing brain parenchyma were investigated in the first 10 enrolled patients by dynamic contrast enhanced (DCE) magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). Changes in the concentration of R-CHOP agent in plasma and cerebrospinal fluid (CSF) samples, as well as changes in the expression of CD13 (the target of NGR-hTNF) in diagnostic biopsies, were investigated as indicators of the specificity of the effect of NGR-hTNF on tumor vasculature. Herein, we report the results of this "proof-of-concept" study as a first step towards the development of a simple, easy-to-perform and active treatment for PCNSL patients, similar to the treatment of DLBCL outside the CNS used worldwide. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Batchelor TT. Primary central nervous system lymphoma. Hematology Am Soc Hematol Educ Program 2016;2016:379-85. [Non-Patent Document 2] Ferreri AJM, Holdhoff M, Nayak L, Rubenstein JL. Evolving Treatments for Primary Central Nervous System Lymphoma. Am Soc Clin Oncol Educ Book 2019;39:454–66

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[0006] Diffuse large B-cell lymphoma (DLBCL) is treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), which has acceptable tolerability and efficacy. However, this chemoimmunotherapy is ineffective in primary CNS DLBCL (PCNSL) due to poor permeability of the blood-brain barrier. NGR-linked tumor necrosis factor-α (NGR-hTNF), a peptide that targets CD13+ tumor vasculature, induces endothelial and drug permeation in lymphoma-bearing mice. In this study, we addressed the safety, activity, and vascular permeability changes of R-CHOP21 preceded by NGR-hTNF in relapsed / refractory PCNSL patients in a phase 2 study. The combination of NGR-hTNF / R-CHOP was well tolerated. Dynamic contrast-enhanced MRI and 99m Tc-DTPA SPECT demonstrated that NGR-hTNF selectively increased vascular permeability in tumor / peritumor regions, where CD13 is consistently expressed. NGR-hTNF did not affect plasma / CSF concentrations of R-CHOP agents. Neuroimaging, histopathology, and pharmacokinetic results were consistent with the activity of NGR-hTNF / R-CHOP and were associated with rapid and significant tumor regression in 7 / 10 patients.

[0007] In the present invention, it was surprisingly discovered that R-CHOP administration preceded by low-dose NGR-hTNF is a safe and active approach with rapid and significant tumor regression in 7 / 10 PCNSL patients.

[0008] Accordingly, the present invention provides a combination of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) and NGR-hTNF or an analogue thereof, for use in treating primary central nervous system lymphoma in a subject, the combination comprising at least one course of R-CHOP preceded by administration of NGR-hTNF or an analogue thereof.

[0009] NGR-hTNF analogs are described in WO 2004 / 041297 and WO 01 / 61017, which are incorporated by reference.

[0010] Preferably, the course of R-CHOP is 375 mg / m 2 of rituximab, 750 mg / m 2 of cyclophosphamide, 50 mg / m 2 of doxorubicin, and 1.4 mg / m 2 It consists of vincristine.

[0011] Preferably, the course of R-CHOP is 375 mg / m on day 1. 2 of rituximab on day 2, 750 mg / m 2 of cyclophosphamide, 50 mg / m 2 of doxorubicin, and 1.4 mg / m 2 It consists of vincristine.

[0012] Preferably, administration of NGR-hTNF or an analog thereof is performed prior to the CHOP agent.

[0013] Preferably, the NGR-hTNF dose is 0.8 ug / m 2 It consists of:

[0014] In a preferred embodiment, the combination comprises six courses of R-CHOP, each preceded by administration of NGR-hTNF or an analog thereof.

[0015] Preferably, the course of R-CHOP preceded by administration of NGR-hTNF or an analog thereof is separated by 18 to 21 days.

[0016] The tumor to be treated is preferably a primary central nervous system lymphoma that is relapsed or refractory.

[0017] Relapsed or refractory refers to tumors that have relapsed or are resistant to standard up-front treatments, such as high-dose methotrexate-based chemotherapy with or without rituximab. Relapsed or refractory primary central nervous system lymphoma refers to lymphomas that have relapsed or are resistant to standard first-line treatments (e.g., high-dose methotrexate-based chemotherapy with or without rituximab). The first-line treatments are any known treatments known to those skilled in the art.

[0018] The term "relapsed" refers to disease that has reappeared or grown after a period of regression. The term "refractory" is used to describe when lymphoma does not respond to treatment (meaning that cancer cells continue to grow) or when the response to treatment does not last very long.

[0019] The term "peptide" as used herein includes polypeptides and proteins. The term "polypeptide" includes single chain polypeptide molecules as well as multi-polypeptide complexes in which individual constituent polypeptides are linked in a covalent or non-covalent manner. The term "polypeptide" includes two or more amino acids in length, typically having more than 5, 10, or 20 amino acids.

[0020] Polypeptide sequences for use in the present invention are not limited to specific sequences and fragments thereof, but are understood to include homologous sequences obtained from any source, such as related viral / bacterial proteins, cellular homologues and synthetic peptides, and variants or derivatives thereof. Polypeptide sequences of the present invention also include polypeptides encoded by polynucleotides of the present invention.

[0021] The term "variant" or "derivative", in relation to the amino acid sequences of the present invention, includes any substitution, variation, modification, replacement, deletion, addition of one (or more) amino acid(s) from or to the sequence, so long as the resulting amino acid sequence preferably has the target activity, preferably has at least 25 to 50% of the activity of the polypeptides presented in the sequence listing, more preferably has at least substantially the same activity.

[0022] Thus, sequences can be modified for use in the present invention. Typically, the modifications maintain the activity of the sequence.

[0023] Thus, in one embodiment, amino acid substitutions can be made, for example, from 1, 2, or 3 to 10, 20, or 30 substitutions, provided that the modified sequence retains at least 25 to 50% activity, or substantially the same activity. However, in another embodiment, modifications to the amino acid sequence of the polypeptide of the invention may be made intentionally to reduce the biological activity of the polypeptide. For example, truncated polypeptides that retain the ability to bind to target molecules but lack functional effector domains may be useful.

[0024] Generally, preferably 20%, 10% or 5% of the amino acid residues of the variant or derivative are altered compared to the corresponding region shown in the sequence listing.

[0025] Amino acid substitutions may include, for example, the use of non-naturally occurring analogues, which increase the plasma half-life of therapeutically administered polypeptides (see below for further details regarding the production of peptide derivatives for therapeutic use). Conservative substitutions may be made, for example, according to the following table: Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

[0026] [Table 1]

[0027] Polypeptides of the present invention also include fragments of the above-mentioned polypeptides and variants thereof, including fragments of sequences. Preferred fragments include fragments that contain an epitope or binding domain. Suitable fragments are at least about 5, e.g., 10, 12, 15, or 20 amino acids in length. Suitable fragments may also be less than 200, 100, or 50 amino acids in length. Polypeptide fragments of proteins and their allelic and species variants may contain one or more (e.g., 2, 3, 5, or 10) substitutions, deletions, or insertions, including conservative substitutions. When substitutions, deletions, and / or insertions are made, for example, by recombinant techniques, preferably less than 20%, 10%, or 5% of the amino acid residues shown in the sequence listing are modified.

[0028] The polypeptides and conjugates of the invention are typically made by recombinant means, e.g., as described below. However, the polypeptides and conjugates of the invention may also be made by synthetic means, e.g., solid-phase synthesis, using techniques well known to those skilled in the art. Various techniques for chemical synthesis of peptides are reviewed by Borgia and Fields, 2000, TibTech 18: 243-251, and are described in detail in the references contained therein.

[0029] The peptide can be directly attached to the cytokine or indirectly attached via a spacer, which can be a single amino acid, an amino acid sequence, or an organic residue, such as 6-aminocapryl-N-hydroxysuccinimide. Methods of attachment are known to those skilled in the art and include genetic engineering or chemical synthesis techniques.

[0030] The peptide ligand is preferably attached to the N-terminus of the cytokine, thus minimizing any interference with the binding of the modified cytokine to the receptor. Alternatively, the peptide can be attached to an amino acid residue that is an amide or carboxyl bond receptor, either naturally occurring on the molecule or artificially inserted by genetic engineering techniques. The modified cytokine is preferably prepared by the use of a cDNA that includes a 5'-contiguous sequence that codes for the peptide.

[0031] According to a preferred embodiment, a conjugate product of TNF and the CNGRC sequence is provided. More preferably, the amino terminus of TNF is linked to the CNGRC peptide via a spacer G (glycine).

[0032] The resulting product (NGR-hTNF) was shown to be more active than TNF in an animal model of RMA-T lymphoma. Furthermore, animals treated with NGR-TNF were able to reject further tumorigenic doses of RMA-T or RMA cells. Increased antitumor activity compared to normal TNF was seen in immunocompetent but not immunocompromised animals. This indicates that the increased antitumor activity of TNF conjugated to the "NGR" peptide is due to an enhanced immune response rather than a direct cytotoxic activity of the conjugate.

[0033] It was also demonstrated that the immune effects induced by NGR-TNF in vivo are directly related to the CD13 receptor: for example, antibodies against the CD13 receptor as well as GNGRC ligands were found to compete with NGR-TNF in vivo, thus suggesting a mechanism for receptor targeting by NGR-TNF.

[0034] The therapeutic index of TNF / CD13 ligand conjugates can be further improved by using mutant forms of TNF that can selectively bind to one of the two TNF receptors (p75TNFR and p55TNFR), which can be obtained by site-directed mutagenesis (Loetscher, H., et al., Human tumor necrosis factor alpha (TNF alpha) mutants with exclusive specificity for the 55-kDa or 75kDa TNF receptors. J. Biol. Chem. 1993.268:26350-7, and Van Ostade, X., et al., Human TNF mutants with selective activity on the p55 receptor. Nature 1993.361:266-9.).

[0035] The pharmacokinetics of the modified cytokines according to the invention can be improved by preparing polyethylene glycol derivatives which make it possible to extend the plasma half-life of the cytokine itself.

[0036] Further embodiments of the invention are provided by bifunctional derivatives in which the CD13 ligand modified cytokine is conjugated to an antibody or fragment thereof against a tumor antigen or other tumor angiogenesis marker (e.g., xv integrin, metalloprotease, or vascular growth factor), or to an antibody or fragment thereof against a component of the extracellular matrix (e.g., anti-tenascin antibody or anti-fibronectin EDB domain). The preparation of a fusion product between TNF and the hinge region of a mAb against the tumor-associated TAG72 antigen expressed by gastric and ovarian adenocarcinomas has recently been reported (Yang, J., et al., A genetically engineered single-chain FV / TNF molecule possesses the anti-tumor immunoreactivity of FV as well as the cytotoxic activity of tumor necrosis factor. Mol. Immunol. 1995.32: 873- 81).

[0037] A further embodiment of the present invention is provided by tumor pretargeting using biotin / avidin system. According to this method, the ternary complex formed by 1) biotinylated mAb, 2) avidin (or streptavidin), and 3) CD13 and bivalent cytokine modified with biotin is obtained on tumor antigen site at different stages. Many papers have demonstrated that the pretargeting approach, in comparison with conventional targeting using immunoconjugates, can indeed increase the ratio of targeted active molecules to free active molecules and, as a result, reduce the toxicity of the treatment (Paganelli, G. , et al., Three-step monoclonal antibody tumor targeting in carcinoembryonic antigen positive patients. Cancer Res. 1991.51: 5960-6; ​​Paganelli, G. , et al., Clinical application of the avidin-biotin system for tumor targeting. In D. Goldenberg Ed. Cancer therapy with radiolabeled antibodies. CRC Press, Boca Raton, 1995. P. 239-253; Modorati, G. , et al., Immunoscintigraphy with three step monoclonal pretargeting technique in diagnosis of uveal melanoma: preliminary results. Br. J. Ophtalm. 1994.78 : 19-23; Colombo, P. , et al., Immunoscintigraphy with anti-chromogranin A antibodies in patients with endocrine / neuroendocrine tumors. J. Endocr. Invest. 1993.16 : 841-3).This approach has yielded beneficial results with biotinylated TNF, which can induce cytotoxicity in vitro and reduce tumor cell proliferation under conditions in which normal TNF is inactive (Moro, M., et al., Tumor cell targeting with antibody-avidin complexes and biotinylated tumor necrosis factor alpha. Cancer Res. 1997.57:1922-8. And 26. Gasparri, A., et al., Tumor pretargeting with avidin improves the therapeutic index of biotinylated tumor necrosis factor alpha in mouse models. Cancer Res. 1999.59:2917-23.). The pretargeting approach can also be carried out using a two-phase method by using bispecific antibodies that simultaneously bind tumor antigens and modified cytokines. The use of bispecific antibodies against carcinoembryonic antigen and TNF has recently been described as a tool for TNF tumor pretargeting (Robert, B., et al., 1996. Cytokine targeting in tumors using a bispecific antibody directed against carcinoembryonic antigen and tumor necrosis factor alpha. Cancer Res. 56: 4758.).

[0038] According to a further embodiment, the invention comprises TNF molecules conjugated on different TNF subunits to both a CD13 ligand and an antibody or fragment thereof (directly or indirectly via a biotin-avidin bridge), the antibody or fragment thereof being directed against an antigen expressed on tumor cells or other components of the tumor stroma (e.g. tenascin and fibronectin EDB domain). This results in a further improvement of the tumor-tropic properties of the modified cytokine and a sustained release of the latter in the tumor microenvironment via a trimer-monomer-trimer transition. As shown in previous studies, indeed, the modified subunits of the TNF conjugates can dissociate from the targeting complex and recombine to form unmodified trimeric TNF molecules that can then diffuse into the tumor microenvironment. Release of biologically active TNF has been shown to occur within 24 to 48 hours after targeting (Corti, A., et al., Tumor targeting with biotinylated tumor necrosis factor alpha: Structure activity relationships and mechanism of action on avidin pretargeted tumor cells. Cancer Res. 1998.58:3866-3872).

[0039] The peptides of the invention can be administered therapeutically to a patient. Rather than consisting solely of natural amino acids, it is preferable to use peptides that have been modified, for example to reduce immunogenicity, increase circulating half-life in the patient's body, improve bioavailability, and / or improve efficacy and / or specificity.

[0040] A number of approaches have been used to modify peptides for therapeutic use. One approach is to conjugate the peptide or protein to various polymers, such as polyethylene glycol (PEG) and polypropylene glycol (PPG) (see, e.g., U.S. Pat. Nos. 5,091,176, 5,214,131, and 5,264,209).

[0041] Substitution of natural amino acids with various uncoded amino acids or modified amino acids (eg, D-amino acids and N-methyl amino acids) can also be used to engineer peptides.

[0042] Another approach is to use bifunctional cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate, succinimidyl hexanoate 6-[3-(2-pyridyldithio)propionamide], and sulfosuccinimidyl hexanoate 6-[3-(2-pyridyldithio)propionamide] (see U.S. Pat. No. 5,580,853).

[0043] It may be desirable to use derivatives of the peptides of the present invention that are conformationally constrained. Conformational constraint refers to the preferred conformation of the stability and three-dimensional shape assumed by the peptide. Conformational constraints include local constraints, which are concerned with restricting the conformational mobility of one residue of the peptide; regional constraints, which are concerned with restricting the conformational mobility of a group of residues that form a secondary structure unit; and global constraints, which are concerned with the entire peptide structure.

[0044] The active conformation of the peptide can be stabilized by covalent modification, e.g., cyclization, or by incorporation of gamma lactam or other types of bridges. For example, side chains can be cyclized into the backbone to create L-gamma lactam moieties on each side of the interaction site. See generally Hruby et al., "Applications of Synthetic Peptides," in Synthetic Peptides: A User's Guide: 259-345 (WH Freeman & Co. 1992). Cyclization can also be achieved, for example, by forming cysteine ​​bridges, linking the amino and carboxy terminal groups of the respective terminal amino acids, or linking the amino group of a Lys residue or related homolog with the carboxy group of Asp, Glu, or related homologs. Linking the alpha-amino group of the polypeptide with the epsilon-amino group of a lysine residue using iodoacetic anhydride can also be accomplished. See Wood and Wetzel, 1992, Int'l J. Peptide Protein Res. 39: 533-39.

[0045] Another approach described in US Pat. No. 5,891,418 includes a metal ion complexing backbone in the peptide structure. Typically, the preferred metal peptide backbone is based on the requisite number of specific coordinating groups required in the coordination sphere of a given complexed metal ion. In general, most metal ions that may prove useful have a coordination number of 4 to 6. The nature of the coordinating groups of the peptide chain includes nitrogen atoms with amine, amide, imidazole, or guanidino functional groups; sulfur atoms with thiols or disulfides; and oxygen atoms with hydroxy, phenol, carbonyl, and carboxyl functional groups. Additionally, the peptide chain or individual amino acids can be chemically modified to include coordinating groups such as oxime, hydrazino, sulfhydryl, phosphate, cyano, pyridino, piperidino, or morpholino. The peptide constructs can be either linear or cyclic, although linear constructs are typically preferred. An example of a small linear peptide is Gly-Gly-Gly-Gly, which has four nitrogens in the backbone (N4 complexing system), which can form complexes with metal ions with a coordination number of four.

[0046] A further technique for improving the properties of therapeutic peptides is to use non-peptide peptidomimetics. A wide variety of useful techniques can be used to reveal the exact structure of a peptide. These techniques include amino acid sequencing, X-ray crystallography, mass spectrometry, nuclear magnetic resonance spectroscopy, computer-assisted molecular modeling, peptide mapping, and combinations thereof. Structural analysis of a peptide generally provides a large amount of data, including the amino acid sequence of the peptide as well as the three-dimensional positioning of the atomic components. From this information, non-peptide peptidomimetics can be designed that have the chemical functional groups required for therapeutic activity, but are more stable, e.g., less susceptible to biological degradation. An example of this approach is provided in U.S. Pat. No. 5,811,512.

[0047] Techniques for chemically synthesizing the therapeutic peptides of the invention are described in the references mentioned above and also reviewed by Borgia and Fields, 2000, TibTech 18: 243-251, and are described in detail in the references contained therein.

[0048] For use in therapy, the modified cytokines of the present invention are suitably formulated into pharmaceutical preparations for oral or parenteral administration. Formulations for parenteral administration are preferred, including injectable solutions or suspensions and liquids for injection. For the preparation of parenteral dosage forms, an effective amount of active ingredient is dissolved or suspended in a sterile carrier, optionally adding excipients such as solubilizers, isotonicity agents, preservatives, stabilizers, emulsifiers or dispersants, and then dispensed into sealed vials or ampoules.

[0049] More specifically, the conjugates of the present invention, including polypeptides and polynucleotides, can be preferably combined with various ingredients to produce the compositions of the present invention. Preferably, the compositions are combined with pharma- ceutically acceptable carriers, diluents, or excipients to produce pharmaceutical compositions, which may be for human or animal use. Suitable carriers and diluents include isotonic saline, such as phosphate buffered saline. Examples of excipients can be found in The Handbook of Pharmaceutical Excipients, 2nd Edn, Eds Wade & Weller, American Pharmaceutical Association. The compositions of the present invention can be administered by direct injection. The compositions can be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, or transdermal administration.

[0050] The compositions can be formulated for daily, weekly or monthly administration to provide the desired daily dose, It is understood that the compositions can be conveniently formulated for less frequent administration, for example, every 2, 4, 6, 8, 10 or 12 hours.

[0051] The polynucleotide / vector encoding the polypeptide component may be directly administered as a naked nucleic acid construct, preferably one which further comprises flanking sequences homologous to the host cell genome.

[0052] Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques, including those that involve the use of transfection agents. Examples of these agents include cationic substances (e.g., calcium phosphate and DEAE-dextran) and lipofectants (e.g., Lipofectam™ and Transfectam™). Typically, the nucleic acid construct is mixed with the transfection agent to produce a composition.

[0053] Preferably, the polynucleotide or vector of the present invention is combined with a pharma- ceutically acceptable carrier or diluent to produce a pharmaceutical composition. Suitable carriers and diluents include isotonic saline, such as phosphate buffered saline. The composition can be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, or transdermal administration.

[0054] The described routes of administration and dosage regimens are intended only as a guide, as a person of ordinary skill in the art can readily determine the optimum route of administration and dosage regimen for any particular patient and condition.

[0055] Preparation of cytokines in the form of liposomes can improve their biological activity. Indeed, acylation of TNF amino groups was found to induce an increase in its hydrophobicity without loss of biological activity in vitro. Furthermore, it has been reported that lipid-bound TNF has unaffected cytotoxicity in vitro, immunomodulatory effects, and reduced toxicity in vivo (Debs, RJ, et al., Liposome-associated tumor necrosis factor retains bioactivity in the presence of neutralizing anti-tumor necrosis factor antibodies. J. Immunol. 1989.143:1192-7; Debs, RJ, et al., Immunomodulatory and toxic effects of free and liposome-encapsulated tumor necrosis factor alpha in rats. Cancer Res. 1990.50:375-80.).

[0056] The maximum tolerated dose of bolus TNF in humans is 218-410 pg / m2 (32. Fraker, DL, Alexander, HR & Pass, HI, 1995. Biologic therapy with TNF: systemic administration and isolation-perfusion. In Biologic therapy of cancer. principles and practice, De Vita, V., Hellman, S. & Rosenberg, S. (eds) pp. 329-345. JB Lippincott Company: Philadelphia.), approximately 10-fold lower than the effective dose in animals.Based on data from mouse models, it is believed that at least 10-fold higher doses are required to achieve antitumor effects in humans (Schraffordt Koops, et al., Hyperthermic isolated limb perfusion with tumour necrosis factor and melphalan as treatment of locally advanced or recurrent soft tissue sarcomas of the extremities, Radiothepray & Oncology 1998.48 : 1-4; Hill, S. , et al., Low-dose tumour necrosis factor alpha and melphalan in hyperthermic isolated limb perfusion. Br. J. Sugr. 1993.80: 995-7; Eggermont, AM , et al., Isolated limb perfusion with tumor necrosis factor and melphalan for limb salvage in 186 patients with locally advanced soft tissue extremity sarcomas. The cumulative multicenter European experience. Ann. Surg. 1996.224 : 756-65.).In the first clinical study of hyperthermic isolated-limb perfusion, high response rates were obtained with a specific dose of 4 mg of TNF in combination with melphalan and interferon-gamma (Lienard, D., et al., In transit metastases of malignant melanoma treated by high dose rTNF alpha in combination with interferon-gamma and melphalan in isolation perfusion. World Journal of Surgery 1992.16:234-40.). Other studies have shown that interferon y can be omitted and even lower doses of TNF may be sufficient to induce a therapeutic response (Hill, S., et al., Low-dose tumour necrosis factor alpha and melphalan in hyperthermic isolated limb perfusion. Br. J. Sugr. 1993.80: 995-7; Eggermont, AM, et al., Isolated limb perfusion with tumor necrosis factor and melphalan for limb salvage in 186 patients with locally advanced soft tissue extremity sarcomas. The cumulative multicenter European experience. Ann. Surg. 1996.224: 756-65.). As the two cytokines exert synergistic effects on endothelial cells, their combined and selective targeting may result in a more potent antitumor activity, thus making it possible to overcome the problems of systemic toxicity that are usually encountered in cancer treatments using the same cytokines in combination. Furthermore, TNF is known to decrease the barrier function of the vascular endothelium, thereby increasing the permeability to macromolecules.Another application taking advantage of the lower toxicity of treatment with the modified TNF molecules according to the invention and their tumor vasculotropic properties is their use to increase the permeability of tumor blood vessels to other compounds, either for therapeutic or diagnostic purposes. For example, modified TNF can be used to increase tumor uptake of radiolabeled antibodies or hormones (tumor imaging compounds) in tumor radioimmunoscintigraphy or radioimmunotherapy. Alternatively, the uptake of chemotherapeutic drugs, immunotoxins, liposomes carrying drugs or genes, or other anti-cancer drugs can be increased to enhance their anti-tumor effects. Thus, the cytokines of the invention can be used in combination, separate or sequential preparations with other diagnostic or therapeutic substances in cancer treatment or diagnosis.

[0057] The present invention relates to the use of a combination of modified TNF and IFNy. This combination can be used in a combined, separate or sequential preparation. Advantageously, the combination is also with other diagnostic or therapeutic substances, such as doxorubicin and mephalan, in the treatment or diagnosis of cancer. Thus, the present invention provides a pharmaceutical composition comprising a combination of modified TNF and IFNy, optionally with another tumor diagnostic or antitumor therapeutic substance. Again, this combination can be used in a combined, separate or sequential preparation.

[0058] In patent application no. PCT / IB03 / 02187, it has been discovered that targeted delivery of picogram doses of cytokines provides a novel and surprising strategy for improving the penetration of chemotherapy drugs and increasing the therapeutic index of chemotherapy drugs. Patent application no. PCT / IB03 / 02187 is incorporated herein by reference in its entirety. More specifically, it has been discovered that delivery of very low doses of cytokines to tumors and tumor-associated environments, including tumor vasculature, represents a novel approach that avoids negative feedback mechanisms and retains the ability to alter drug permeability barriers.

[0059] The compositions of the invention may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, or transdermal administration. In one embodiment of this aspect of the invention, the conjugates of the invention may be administered at a dose in the range of 0.5 to 500 ng / kg, preferably in the range of 1 to 50 ng / kg, more preferably in the range of 5 to 15 ng / kg.

[0060] In another embodiment of this aspect of the invention, a pharmaceutical composition is provided comprising a conjugate of the invention in combination with IFNy, the conjugate being present in an amount such as to provide the conjugate or a metabolite thereof in the plasma of a treated subject in an amount of about 35,000 ng / day or less, preferably about 3,500 ng / day or less, more preferably about 1,000 ng / day or less.

[0061] The above dosages refer to dosages for a subject weighing 70 kg. A person skilled in the art could easily modify the listed dosages for subjects having a mass other than 70 kg.

[0062] The described routes of administration and dosage regimens are intended only as a guide, as a person of ordinary skill in the art can readily determine the optimum route of administration and dosage regimen for any particular patient and condition.

[0063] Another aspect of the present invention relates to a cDNA encoding a conjugated cytokine as disclosed herein, which can be prepared from a cytokine cDNA by the addition of a 5'- or 3'-contiguous DNA sequence encoding a CD13 ligand, preferably a homing peptide as described above. The linked cDNA can be used as such or after insertion into a vector for gene therapy. The preparation and therapeutic application of suitable vectors are disclosed in (Mizuguchi, H., et al., Tumor necrosis factor alpha-mediated tumor regression by the in vivo transfer of genes into the artery that leads to tumor. Cancer Res. 1998.58:5725-30.), which is incorporated herein by reference.

[0064] The polynucleotide for use in the present invention comprises a nucleic acid sequence that encodes the polypeptide conjugate of the present invention.Those skilled in the art will understand that, as a result of the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide.Furthermore, it will be understood that those skilled in the art can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotide of the present invention, to reflect the codon usage of any particular host organism in which the polypeptide of the present invention is expressed.

[0065] The polynucleotides of the present invention may comprise DNA or RNA. The polynucleotides of the present invention may be single-stranded or double-stranded. The polynucleotides of the present invention may be polynucleotides that include synthetic or modified nucleotides within the polynucleotide. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and / or 5' ends of the molecule. For the purposes of the present invention, it is understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications may be performed to improve the in vivo activity or survival time of the polynucleotides of the present invention.

[0066] The polynucleotides of the invention can be incorporated into a recombinant replicating vector. The vector can be used to replicate the nucleic acid in a compatible host cell. Thus, in a further embodiment, the invention provides a method for producing a polynucleotide of the invention by introducing a polynucleotide of the invention into a replicating vector, introducing the vector into a compatible host cell, and growing the host cell under conditions that result in replication of the vector. The vector can be recovered from the host cell. Suitable host cells include bacteria (e.g., E. coli), yeast, mammalian cell lines, and other eukaryotic cells (e.g., insect Sf9 cells).

[0067] Preferably, the polynucleotide of the invention in a vector is operably linked to a control sequence capable of providing for the expression of the coding sequence by the host cell, i.e., the vector is an expression vector. The term "operably linked" means that the components described are in a relationship permitting them to function in their intended manner. Regulatory sequences operably linked to a coding sequence are ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.

[0068] The control sequences may be modified, for example by the addition of further transcriptional regulators, so that the level of transcription directed by the control sequences is more responsive to the transcriptional regulators.

[0069] The vectors of the invention can be transformed or transfected into suitable host cells as described below to provide for expression of the proteins of the invention, which method comprises culturing a host cell transformed with an expression vector as described above under conditions that provide for expression by the vector of a coding sequence that encodes the protein, and, optionally, recovering the expressed protein.

[0070] The vector may be, for example, a plasmid or a viral vector, provided with an origin of replication, optionally a promoter for the expression of the polynucleotide, and optionally a regulator of the promoter. The vector may also contain one or more selectable marker genes, for example, an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. The vector may be used, for example, to transfect or transform a host cell.

[0071] Control sequences operably linked to the sequence encoding the protein of the invention include promoters / enhancers and other expression regulation signals. These control sequences can be selected to be compatible with the host cell in which the expression vector is designed to be used. The term "promoter" is well known in the art and encompasses nucleic acid regions of various sizes and complexities, from minimal promoters to promoters that include upstream elements and enhancers.

[0072] Although prokaryotic promoters and promoters functional in other eukaryotic cells can be used, the promoter is typically selected from promoters functional in mammalian cells. The promoter is typically derived from the promoter sequence of a virus or eukaryotic gene. For example, the promoter may be a promoter derived from the genome of the cell in which expression occurs. With respect to eukaryotic promoters, the promoter may be a promoter that functions in a ubiquitous manner (e.g., promoters of a-actin, b-actin, tubulin) or, alternatively, in a tissue-specific manner (e.g., promoters of genes for pyruvate kinase). Tissue-specific promoters specific for certain cells may also be used. The promoter may also be a promoter that responds to a particular stimulus, for example, a promoter that binds to a steroid hormone receptor. Viral promoters may also be used, for example, the Moloney murine leukemia virus long terminal repeat (MMLV LTR) promoter, the Rous sarcoma virus (RSV) LTR promoter, or the human cytomegalovirus (CMV) IE promoter. It may also be advantageous for the promoter to be inducible so that the level of expression of the heterologous gene can be controlled during the life of the cell.

[0073] Inducible means that the level of expression obtained using the promoter can be controlled.

[0074] Furthermore, any of these promoters can be modified by the addition of further regulatory sequences, such as enhancer sequences. Chimeric promoters, which contain sequence elements from two or more of the different promoters mentioned above, can also be used.

[0075] The vectors and polynucleotides of the invention can be introduced into a host cell for the purposes of replicating the vector / polynucleotide and / or expressing a protein of the invention encoded by a polynucleotide of the invention.

[0076] The proteins of the present invention can be produced using prokaryotic cells as host cells, but it is preferred to use eukaryotic cells such as yeast, insect or mammalian cells, especially mammalian cells.

[0077] The vectors / polynucleotides of the invention can be introduced into suitable host cells using various techniques known in the art, such as transfection, transformation, and electroporation. For administering the vectors / polynucleotides of the invention to animals, several techniques are known in the art, such as infection with recombinant viral vectors, such as retroviruses, herpes simplex viruses, and adenoviruses, direct injection of nucleic acids, and biolistec transformation.

[0078] A host cell comprising a polynucleotide of the present invention can be used to express the conjugate of the present invention. The host cell can be cultured under appropriate conditions to express the polypeptide and conjugate of the present invention. Expression of the product of the present invention can be constitutive so that it is continuously produced, or it can be inducible, requiring a stimulus to initiate expression. In the case of inducible expression, production of the protein can be initiated when necessary, for example, by adding an inducer (e.g., dexamethasone or IPTG) to the culture medium.

[0079] Conjugates of the invention can be extracted from host cells by a variety of techniques known in the art, including enzymatic, chemical, and / or osmotic lysis, as well as physical disruption.

[0080] The invention will now be illustrated, by way of non-limiting examples, with reference to the following figures. [Brief description of the drawings]

[0081] [Figure 1] Overall study design. End points and statistics for phase 2 are reported. The NGR-hTNF / RCHOP regimens differed in the 2 phases for structural and clinical reasons. [Diagram 2]Preliminary design (first 10 patients). Enrolled patients received the first course of R-CHOP not preceded by NGR-hTNF, while the other 5 courses were preceded by NGR-hTNF. Brain MRI indicates gadolinium-enhanced magnetic resonance imaging performed for response assessment, while DCE-MRI is dynamic contrast-enhanced MRI of the brain used to evaluate changes in BBB permeability. SPECT is single-photon emission computed tomography performed before and after the third course to evaluate changes in BBB permeability. Arrows represent collection of CSF and plasma samples. [Diagram 3] Expansion phase design (second 18 patients). Enrolled patients received 6 courses of NGR-hTNF / R-CHOP. Brain MRI shows gadolinium-enhanced magnetic resonance imaging performed for response assessment. Arrows indicate baseline CSF and plasma sample collection. [Figure 4] Changes in BBB permeability assessed by DCE-MRI in responders. Changes in the enhancing area are shown on the left, and in the perilesional area on the right; results are expressed as Ktrans. Values ​​for each patient in the first course (without NGR-hTNF) and in the second course (with NGR-hTNF) are connected by a line. Median and range values ​​for each subgroup are reported below each graph. [Diagram 5] Example of increased 99mTc-DTPA uptake after injection of NGR-hTNF followed by R-CHOP during the third course of treatment. Volumes with 99mTc-DTPA uptake of 30% or more (≧30%) were curved in two SPECT studies performed before (left image - blue line) and after (middle image - green line) administration of NGR-hTNF and R-CHOP. Comparison of the curved volumes is depicted in a gadolinium-enhanced T1-weighted MRI showing the tumor (red image). The volumes of the subject before and after NGR-hTNF / R-CHOP delivery were 22 cm3 and 40 cm3, respectively. [Figure 6]Expression of CD13 by the intima of the tumor vasculature. A) Immunohistochemical analysis of CD13 expression in the lymphoma component of a diagnostic brain biopsy of an enrolled patient. Staining was performed using anti-CD13 monoclonal antibody SP187 alone (brown signal, 400x). B) Immunohistochemical analysis of CD13 and αSMA (marker of pericytes). Co-staining was performed with anti-CD13 monoclonal antibody SP187 (brown) and anti-αSMA monoclonal antibody 1A4 (red). Black arrows indicate CD13-positive vessels, red arrows αSMA-positive perivascular cells (bar 20 μm; 630x). Left panel: Representative picture of an area with large vessels and some microvessels with pericyte coverage (red) showing CD13 staining (brown) even in the absence of pericytes. Right: Representative picture of an area with some CD13-positive microvessels (brown). C-D) Confocal immunofluorescence analysis of tissue sections stained with anti-CD13 antibody (green) and with anti-αSMA antibody 1A4 (red) (400×, bar: 50 μm). Inset: 3D projection of CD13 and αSMA staining of mature blood vessels (asterisk) (400×, bar: 25 μm) showing that CD13 is expressed on the luminal side of the vascular endothelium. [Figure 7] Examples of response to R-CHOP preceded by NGR-hTNF. A) Gadolinium-enhanced T1-weighted scan shows a large homogenously enhancing lesion (arrows) in the right parietal lobe in a 65-year-old man with secondary recurrence after high-dose methotrexate and after salvage high-dose ifosfamide-based therapy and whole-brain radiation. B) Tumor regression after four courses of experimental therapy. C) Gadolinium-enhanced T1-weighted scan shows a large polylobate enhancing lesion (arrows) invading the basal ganglia, diencephalon, and left temporal lobe in a 39-year-old man with PCNSL refractory to prior high-dose methotrexate-based chemoimmunotherapy. D) Tumor regression after four courses of experimental therapy. [Figure 8]Chromogranin A (CgA) plasma levels, PPI therapy, and response. A) Relationship between CgA plasma levels and response to NGR-hTNF / R-CHOP. Baseline plasma levels of CgA are shown for patients who achieved complete remission (n=12) and those who did not achieve complete remission (others, n=16). Median (range) CgA levels were 1.14 nM (0.29-2.72) and 2.10 nM (0.47-5.81), respectively (p= 0.066). B and C) Changes in CgA plasma levels after discontinuation of proton pump inhibitors (PPIs). Comparison of CgA concentrations in plasma samples taken at study entry (baseline) and before the third course (2 months) showed a decrease in levels in some patients after PPI discontinuation (B), whereas in patients who did not receive this drug, values ​​remained constant (C). No differences were detected between patients who achieved a complete response (solid line) or a partial response (dotted line). [Figure 9] Expression of CD13 by endothelial cells and pericytes in primary CNS lymphoma vasculature. High-resolution wide-field co-immunofluorescence analysis of PCNSL tissue sections from two enrolled patients stained with polyclonal anti-CD13 antibody (green) and with polyclonal anti-CD31 antibody (marker for endothelial cells, red). Nuclear staining with DAPI (blue). A-C) Example of a blood vessel with CD13-positive endothelial cells (arrows, yellow in merged figure) and CD13-positive mural cells (arrowheads, green, presumably pericytes). See also Supplemental Movie for z-stack images of blood vessels reported in panel A. B) Middle panel: electronic magnification of the highlighted area (dashed rectangle) of the blood vessel shown in the left panel. B) Right panel: control performed on serial sections with secondary antibody alone, showing the absence of staining. D) Example of a blood vessel with CD13-positive mural cells and CD13-negative endothelial cells (green and red, arrowheads and arrows, respectively, in merged figure). Scale bar is 5 μm, magnifications are indicated in each panel. [Figure 10]Expression of CD13 and PDGFRβ in PCNSL vasculature. High-resolution wide-field co-immunofluorescence analysis of PCNSL tissue sections from enrolled patients stained with polyclonal anti-CD13 antibody (green) and with polyclonal anti-PDGFRβ antibody (marker for pericytes, red). Nuclear staining with DAPI (blue). Representative staining of three different vessels is shown. Magnification 60×, scale bar 10 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] Patients and methods Study population and inclusion criteria The "INGRID" trial is a single-arm phase II study focusing on an experimental treatment consisting of six courses of R-CHOP preceded by NGR-hTNF infused into HIV-negative patients with relapsed or refractory PCNSL (EUDRACT: 2014-001532-11-clinicaltrials.gov NCT03536039). The trial has two distinct parts (Figure 1): the first part focuses on the feasibility and "proof of concept" of the effect of NGR-hTNF on BBB permeability; the second part focuses on the activity and tolerability of the experimental treatment. Inclusion criteria were: 1) histologically proven diagnosis of DLBCL according to WHO criteria (17); 2) disease localized exclusively to the CNS, cranial nerves, meninges, and / or eyes both at the time of initial diagnosis and at study entry; 3) lymphoma that had relapsed after chemotherapy containing high-dose methotrexate or was refractory to prior chemotherapy containing high-dose methotrexate; 4) measurable disease; 5) age 18 to 80 years; 6) ECOG performance status of 3 or less (≤3). Patients who had previous organ donation or other forms of immunosuppression, HBV, HCV, and / or HIV infection, or other malignancies were excluded. Any type of consolidation therapy (i.e., total-field radiotherapy - WBRT -, autologous stem cell transplant - ASCT -, oral drug maintenance) during prior treatment was allowed. Prior to study entry, histopathological specimens and neuroimaging studies performed at diagnosis and recurrence were centrally reviewed, and patients underwent physical and neurological examinations, hemograms and biochemical serum profiles (including viral markers: HIV, HBV, HCV), echocardiography, enhanced neck, chest, and abdominal CT scans, bone marrow biopsy, contrast-enhanced brain MRI, CSF examination, ophthalmologic evaluation, and 18Patients were assessed by FDG-PET. Risk was defined according to the IELSG score both at diagnosis and at study entry (18). Written informed consent was obtained from each patient after eligibility was confirmed and after patient review of the protocol. The study conformed to the Declaration of Helsinki and was approved by the IRB of the San Raffaele Scientific Institute, Milan, Italy.

[0083] Explorative phase: Planning and treatment The preliminary design is summarized in Figure 2. The first 10 enrolled patients received a first course of R-CHOP not preceded by NGR-hTNF, whereas the other 5 courses were preceded by NGR-hTNF (Table 1).

[0084] [Table 2]

[0085] In summary, enrolled patients received six courses of R-CHOP21 preceded by NGR-hTNF. Treatment was given for 2 days: 375 mg / m 2 of rituximab was delivered on day 1 and on day 2, 0.8 μg / m 2A dose of 1.000 mg of NGR-hTNF was delivered by 1-h infusion. By protocol, in the first 10 patients, the first course of R-CHOP was not preceded by NGR-hTNF (14). The rationale for the timing and administration schedule of NGR-hTNF has been reported previously (14). Thus, in the expansion phase (after the first 10 cohorts), enrolled patients received 6 courses of R-CHOP preceded by NGR-hTNF. A dose of 1.000 mg of acetaminophen / paracetamol was delivered orally or intravenously 30–60 min before starting each infusion of NGR-hTNF as prophylaxis for infusion-related reactions. Steroids were avoided, but not for the 5 days of prednisolone, which, if clinically indicated, precluded the day of NGR-hTNF infusion. Treatment with PPIs was avoided and replaced with H2-blockers (i.e., ranitidine) at study entry.

[0086] Patients who completed the six planned courses and achieved a complete or partial response (PR) were evaluated for consolidation therapy, which could include WBRT 30–36 Gy, carmustine-thiotepa-conditioned ASCT, or oral lenalidomide maintenance, according to the protocol and prior treatment.

[0087] Expansion phase NGR-hTNF / R-CHOP (Course #1-6) Day 1: Rituximab 375 mg / m 2 (intravenous injection) NGR-hTNF 0.8μmg / m 2 (1 hour infusion immediately following rituximab) Cyclophosphamide 750 mg / m 2 (IV bolus 1 hour after NGR-hTNF) Doxorubicin 50mg / m 2 (IV bolus immediately after cyclophosphamide) Vincristine 1.4 mg / m 2 (Maximum 2 mg) (IV bolus immediately following doxorubicin) Days 2-6: Prednisone 75 mg / d (orally)

[0088] R-CHOP was delivered at conventional doses: rituximab 375 mg / m as an intravenous (IV) infusion on day 0 2 , followed by cyclophosphamide 750 mg / m2 as an IV bolus on day 1. 2 , doxorubicin 50 mg / m as an IV bolus 2 , and vincristine 1.4 mg / m as an IV bolus. 2 (max 2mg); prednisone 75mg / d orally on days 2-6. NGR-hTNF 0.8μg / m 2 was delivered by 1-h infusion. Treatment with proton pump inhibitors was avoided because these drugs increase chromogranin levels and may ultimately interfere with NGR-hTNF activity. H2 blockers (i.e., ranitidine) were permitted as gastroprotective therapy. Steroids were avoided, but not for 5 days of prednisolone, which prevented the day of NGR-hTNF infusion if clinically indicated.

[0089] Expansion Phase: Planning and Treatment The expansion phase schedule is summarized in Figure 3. The other 18 enrolled patients (after the first 10) received 6 courses of R-CHOP preceded by NGR-hTNF (Table 1). The other difference concerns the daily delivery of all agents: rituximab 375 mg / m as an intravenous (IV) infusion. 2 , followed by a 1-hour infusion of NGR-hTNF 0.8 μmg / m 2 , and 2 hours later, CHOP drugs are delivered (cyclophosphamide 750 mg / m as an IV bolus). 2 , doxorubicin 50 mg / m as an IV bolus 2 , and vincristine 1.4 mg / m as an IV bolus. 2(maximum 2 mg; oral prednisone 75 mg / d on days 2–6). Use of proton pump inhibitors and steroids followed the same recommendations as above. Patients who completed the six planned courses and achieved a complete or partial response (PR) during the preparatory or expansion phase were evaluated for consolidation therapy. WBRT 30–36 Gy, carmustine-thiotepa-conditioned ASCT, or oral lenalidomide maintenance were allowed according to the protocol and prior treatment.

[0090] Drugs Rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone are in any commercially available or otherwise available form.

[0091] NGR-hTNF The sequence expressed in E. coli cells is: CNGRCGVRSSSRTLSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL (SEQ ID NO: 1) A recombinant protein consisting of CNGRCG fused to the N-terminus of human tumor necrosis factor alpha. NGR-hTNF analogs are described in WO 2004 / 041297 and WO 01 / 61017, which are incorporated by reference.

[0092] Toxicity and response evaluation Treatment side effects were evaluated separately for each chemotherapy course and graded according to the NCI-NCIC CTC version 3·0 (19). Worst toxicity per organ and per patient was considered. Regular specialist controls, ECG, determination of troponin levels, and echocardiography were performed before each treatment course to exclude cardiac toxicity. The effect of treatment on cognitive function was not evaluated by laboratory tests. All eligible patients were considered for response evaluation. Response was evaluated by gadolinium-enhanced MRI of the brain performed on a 1.5 Tesla scanner after the first, second, fourth, and sixth courses of treatment (Figures 2 and 3). Examinations were performed after the second, fourth, and sixth courses in the presence of positive CSF and / or vitreous. Response was defined according to the IPCG criteria (20) (Table 3).

[0093] [Table 3]

[0094] Briefly, CR consisted of disappearance of all evidence of lymphoma; PR was a greater than 50% (>50%) decrease in tumor size; progressive disease (PD) was a greater than 25% (>25%) increase in tumor size or the discovery of any new lesions; all other conditions were considered stable disease (SD). Only tumor regression confirmed by two consecutive MRIs as a significant change in IPCG criteria was considered a "response," and therefore each "response" had a minimum duration of 6-8 weeks. Responses after the first course of R-CHOP did not lead to therapeutic decisions, but patients who were in PD at the time of any subsequent MRI were considered "off study" and treated according to institutional guidelines. The best response recorded from the start of treatment was considered for analysis. Response duration was measured from the date of best response to the date of objective progression, death from any cause, or last follow-up visit. After treatment was completed, disease was evaluated every 3 months.

[0095] Biomarker evaluation Plasma levels of CgA and sTNF-R1 and sTNF-R2 were examined by ELISA in samples taken at the same time as the tumor response assessment.

[0096] Plasma levels of CgA and sTNF-R (receptors 1 and 2) were examined in samples taken at the same time as tumor response assessment, i.e., before the first course of treatment and after the second and sixth courses. sTNF-R was examined using a commercially available kit (DuoSet ELISAs, R&D Systems). CgA plasma levels were evaluated by using a home-made ELISA kit based on the use of monoclonal antibody B4E11 in the capture step and anti-CgA rabbit antiserum in the detection step (21).

[0097] The association between PPI therapy (yes vs. not) and plasma levels of CgA (continuous variable) and (continuous variable) was assessed by Mann-Whitney U test. The association between treatment response (CR vs. not CR) and plasma levels of CgA or sTNF-R was assessed by chi-square test, and cut-offs were used to distinguish subgroups with "low" and "high" plasma levels. Cut-offs were selected based on ROC curves.

[0098] The association between CgA or sTNF-R (continuous variables) and treatment response (CR vs. not CR) and PPI therapy (yes vs. not) was assessed by Mann-Whitney U test.

[0099] To clarify the expression of CD13 (a target of NGR) in PCNSL vasculature, double immunofluorescence staining experiments were performed on tumor tissue sections from enrolled patients using anti-CD13 and anti-CD31 (a marker for endothelial cells) antibodies and anti-CD13 and anti-platelet-derived growth factor receptor-β (PDGFR-β; a marker for pericytes) antibodies.

[0100] Immunofluorescence study The expression of CD13, CD31, and platelet-derived growth factor receptor-β (PDGFR-β) in tumor tissues was evaluated by immunofluorescence technique on paraffin-embedded specimens (10 μm thick) of diagnostic samples from enrolled patients. After antigen retrieval, immunofluorescence staining was performed using rabbit polyclonal antibody against CD13 / APN (1051-RP02, SinoBiological, 1:500), sheep polyclonal antibody against CD31 / PECAM-1 (marker of endothelial cells, AF806, R&D System, 15 μg / ml), and goat polyclonal antibody against PDGFR-β (marker of pericytes, AF385, R&D System, 10 μg / ml). The antibodies were diluted in PBS containing 1% BSA, 5% normal horse serum, and 0.1% Triton X-100 and incubated overnight at 4°C. After washing, for double staining of CD13 and CD31, binding of anti-CD13 and anti-CD31 antibodies was detected using a mixture of secondary antibodies consisting of donkey anti-rabbit IgG-Alexa Fluor 488 (green, 4 μg / ml) conjugate and donkey anti-sheep IgG-Alexa Fluor 568 (red, 4 μg / ml) conjugate. For double staining of CD13 and PDGFRβ, binding of anti-CD13 and anti-PDGFR-β antibodies was detected using a mixture of secondary antibodies consisting of donkey anti-rabbit IgG-Alexa Fluor 488 (green, 4 μg / ml) conjugate and donkey anti-goat IgG-Alexa Fluor 546 (red, 4 μg / ml) conjugate. Nuclear staining was performed with DAPI (blue). Fluorescence signals were acquired using a high-resolution wide-field microscope (DeltaVision™ Ultra, GE Healthcare) equipped with 40X / 1.35, 60X / 1.42, and 100X / 1.4 objectives. Z-stack acquisition was performed at 0.2 μm and 0.3 μm intervals (100X objective, and 40X or 60X objective, respectively). Z-stacks were deconvolved and processed as required using built-in software. Images were extracted from Z-stacks using Image J software.

[0101] Statistical analysis Overall response rate (ORR: CR and PR) was the primary end point, and a Simon two-stage minimax design was used. The maximum ORR considered of low interest was 30% (similar to the rates reported in previous prospective studies at our institution focusing on salvage treatment in patients with PCNSL (22, 23)), and the minimum ORR considered of interest was 50%. A total of 28 patients were required to demonstrate the difference (one-sided test; type I error: 10; power: 9). In the first stage, 12 patients were enrolled, and if at least four responses were observed, the study was continued to a total of 28 patients. If at least 12 responses were recorded, the experimental treatment was declared active. Associations between responses and clinical and treatment variables were examined by Fisher's exact test. Variables analyzed included age (<60 years vs. >60 years), LDH serum level (normal vs. high), CSF protein concentration (normal vs. high), location of disease (peripheral vs. deep), previous treatment (1 vs. 2-3), previous consolidation therapy (none vs. WBRT and / or ASCT), and type of failure (relapsed vs. refractory disease).

[0102] Duration of response, PFS, OS, and tolerability were secondary endpoints. Duration of response was defined as the time from the first evaluation documenting a response to the date of recurrence, death from any cause, or last follow-up visit. PFS was defined as the interval between study entry and the date of failure (disease recurrence or disease progression), death from any cause, or last follow-up visit. OS was defined as the time from study entry to the date of death from any cause or last follow-up visit. Tolerability was defined by grade 3–4 AEs according to the NCI CTCAE (19).

[0103] BBB permeability assessed by neuroimaging The location of lymphoma lesions, the location of the area around the tumor (perilesional area), and the NGR-hTNF-induced changes in BBB permeability in normal-appearing brain parenchyma were evaluated by DCE-MRI. DCE acquisition followed a standard protocol (28) including conventional T1, T2, Flair, DWI, and Dynamic Susceptibility Contrast Perfusion (DSC) sequences. As shown in Figure 5, DCE-MRI was performed within conventional MRI examinations on day 0 (pretreatment - baseline data) and on day 1 for the first (R-CHOP only), second (first course of R-CHOP preceded by NGR-hTNF), and sixth (last course of R-CHOP preceded by NGR-hTNF) treatment courses. In cases of multiple lesions, the largest lesion was considered. Post-processing of DCE-MRI was performed using Olea software (La Ciotat, France). All dynamic images were corrected for motion artifacts and coregistered to a volumetric post-contrast T1 sequence. Results were expressed as Ktrans values ​​normalized using the contralateral white matter. Ktrans obtained after the second course (measured after NGR-hTNF infusion) were compared with Ktrans obtained after the first course (without NGR-hTNF) to establish the effect of TNF on BBB permeability. Statistical significance was assessed by Wilcoxon matched pairs test.

[0104] BBB permeability assessed by SPECT The changes in BBB permeability induced by NGR-hTNF were also evaluated by brain scintigraphy. 99m Tc-diethylenetriaminepentaacetic acid ( 99mTc-DTPA) penetrates only the disrupted BBB, spreads into the altered tissue and binds by a mechanism that is still not well understood. Tracer uptake at the location of the brain lesion increases in proportion to the degree of BBB penetration. Brain scintigraphy was obtained twice, at baseline (bBS) and after the end of the third course of treatment (paBS), several days (median 4 days, range 1-6) before the third course of treatment. Approximately 555-740 MBq 99m Tc-DTPA was injected as an intravenous bolus. After 90-120 min, SPECT / CT examinations were performed using a dual-head γ-camera equipped with a low-energy high-resolution collimator pair. Qualitative and semiquantitative evaluations of both bBS and paBS were performed. In particular, for the semiquantitative evaluation, PMOD software (ver. 3.2, PMOD Technologies, Switzerland) was used. 99m The amount of Tc-DTPA uptake was evaluated. The amount of interest, 30% of the maximum uptake, was determined by the automatic isocontour method. 99m The area between the bBS and paBS was depicted around the 19Tc-DTPA positive area. 99m The statistical significance of changes in the amount of Tc-DTPA uptake was assessed by Wilcoxon paired tests.

[0105] Expression of target receptor (CD13) of CNGRCG peptide The CNGRCG moiety of NGR-hTNF is able to recognize the CD13 form expressed on tumor blood vessels, resulting in targeted delivery of TNF to the tumor endothelium. The presence of this CD13 form in the tumors treated was assessed by immunohistological and immunofluorescence techniques on paraffin-embedded samples of diagnostic tissue samples from enrolled patients. In the trials and experimental treatments, CD13 expression was not a condition for enrollment of patients.

[0106] Immunohistological analysis was performed automatically using the Ventana-Roche Ultrabenchmark XT immunostainer, anti-CD13 monoclonal antibody (mAb) SP187, and anti-α-smooth muscle actin (α-SMA, a marker for pericytes) mAb 1A4 (Sigma, 10 μg / ml). Immunofluorescence staining was performed using anti-CD13 polyclonal antibody (1051-RP02, SinoBiological, 1:500) and anti-α-SMA antibody (mAb 1A4, Sigma, 10 μg / ml) in PBS containing 1% BSA, 5% normal horse serum, and 0.1% Triton X-100 after antigen retrieval. After overnight incubation, bound antibodies were detected using a mixture of secondary antibodies consisting of donkey anti-rabbit IgG-Alexa Fluor 488 (green) (4 μg / ml) conjugate and donkey anti-mouse IgG-Alexa Fluor 647 (infrared) (4 μg / ml) conjugate. Nuclear staining was performed with DAPI (blue). Fluorescent signals were acquired using an inverted point-scanning confocal SMD-SP8 Leica Microsystem equipped with a 40x / 0.4 objective, Navigator Module, and LASX acquisition software.

[0107] result Evaluable Study Population Between May 2016 and December 2018, 28 patients were enrolled. All patients were evaluable for activity and tolerability. The median age of the 28 evaluable patients was 58 years (range 26-78), and 14 were male (Table 3).

[0108] [Table 4]

[0109] Most patients had unfavorable characteristics at study entry: 23 patients (82%) had a moderate-to-high IELSG risk score, 15 patients had an ECOG-PS ≥ 2, 11 patients had increased LDH serum levels, 11 patients had high CSF protein concentrations, and 12 patients had deep brain lesions. All patients presented with brain parenchymal involvement, and 3 presented with concomitant intraocular disease. No patients had meningeal disease. Patients were heavily pretreated, with 10 having received 2 or more prior therapies, and 17 patients (61%) had received ASCT, WBRT, or both. In 15 patients (54%), lymphoma was refractory to prior therapy.

[0110] toxicity The experimental treatment was well tolerated (Table 4).

[0111] [Table 5]

[0112] 132 of 168 planned courses (79%) were delivered. There were no cases of unexpected toxicity or interruption due to toxicity, and no patients required dose reduction. Treatment delays were recorded in only 6 courses (4%) due to cytopenias. In 12 patients, 16 severe side effects were recorded: seizures (3), deep vein thrombosis (2), grade 3 infections (5), grade 3 syncope (2), grade 3 constipation, grade 4 febrile neutropenia, pulmonary aspergillosis, and grade 2 left ventricular dysfunction. There were 9 cases that responded to NGR-hTNF infusion; all of them (fever (4), chills (4), and arterial hypertension) were grade 1-2 and resolved after interruption of the infusion for 15 minutes and symptomatic treatment. Per protocol, the infusion was delayed and completed after 1 hour. These patients received another course of NGR-hTNF / R-CHOP with protocol prophylaxis without further infusion reactions. Twelve patients required blood / platelet transfusions (7 of whom had previously undergone ASCT).

[0113] Activity (primary endpoint) The best response to the NGR-hTNF / R-CHOP combination was complete in 11 patients (example in FIG. 7) and partial in 10 patients, with an ORR of 75% (95%CI = 59-91%); 7 patients had PD. A predefined activity threshold of at least 12 responses was mostly achieved. The best response was achieved after the second course in 14 patients and after the fourth course in 7 patients. Responses were evenly distributed in the analyzed subgroups according to IELSG risk variables, site and number of lesions, prior treatment, and refractoriness (Table 5).

[0114] [Table 6]

[0115] Seventeen of the 21 responding patients received consolidation therapy: seven patients with WBRT, five with ASCT, one with lenalidomide maintenance, and four with a combination of these treatments. Two of three patients with intraocular disease achieved tumor regression at that site and were free of intravitreal recurrence at 3 and 28 months of follow-up. In all complete responders, responses lasted longer than 6 months (median 10 months; range 6-19). At a median follow-up of 16 months (range 9-26), five patients are recurrence-free and six patients are alive.

[0116] NGR-hTNF inhibitors (biomarker evaluation) A near-significant association (p=0.066) was found between plasma levels of CgA at study entry and CR rate (Figure 8A). When patients were grouped into those with "low" and "high" CgA levels using a ROC-driven cutoff of 1.4 nM, 8 / 13 and 3 / 15 patients, respectively, were found to achieve CR (62% vs. 20%; p=0.05). Notably, plasma levels of CgA at study entry were associated with receipt of PPIs during prior steroid treatment. In PPI-naive (n=14) and PPI-treated (n=14) patients, respectively, median plasma levels of CgA were 1.05 nM (range 0.29-3.27) and 2.26 nM (range 0.33-7.99; p=0.008). CgA concentrations gradually decreased in some patients after PPI discontinuation (Fig. 8B), whereas levels remained stable in patients who did not receive the drug (Fig. 8C).

[0117] Median plasma levels of sTNF-R1 and sTNF-R2 at study entry were 0.66 (range, 0.32-4.88) and 2.14 (range, 0.98-7.26), respectively. sTNF-R concentrations were not associated with response to NGR-hTNF / R-CHOP, did not change after PPI discontinuation, and remained stable throughout treatment (data not shown).

[0118] Expression of CD13 in PCNSL vasculature Immunohistological staining demonstrated the presence of CD13 in diagnostic brain biopsies of enrolled patients: in most cases, stained vessels showed luminal narrowing with an irregular appearance. An example of a CD13-positive vessel signal within a lymphoma lesion is shown in Figure 6A. Immunohistological and confocal immunofluorescence analysis of tissue sections stained with anti-CD13 polyclonal and with anti-α-SMA (pericyte marker) antibodies showed that most stained vessels lacked a pericyte layer (Figure 6B), suggesting immature vessels. 3D images of more mature vessels showed that CD13 was expressed by the intima of the vessels, while it was much lower expressed or almost absent in pericytes (Figure 6C, D). These findings pointed to the fact that CD13 is expressed on the luminal side of the endothelium of the tumor vasculature and can access NGR-hTNF delivered by the intravenous route.

[0119] Double staining using anti-CD13 (NGR target) and anti-PDGFR-β (pericyte marker) antibodies revealed widespread CD13 expression in almost all lymphoma-associated blood vessels (Figure 10). Furthermore, double staining using anti-CD13 and anti-CD31 (endothelial marker) antibodies also revealed CD13 expression in the intima of tumor blood vessels (Figure 9A-C). Tumor blood vessels with CD13-positive pericytes and CD13-negative endothelial cells were also observed (Figure 9D).

[0120] BBB permeability assessed by neuroimaging DCE-MRI analysis showed that vascular permeability increased after NGR-hTNF infusion (Figure 4). This effect was more evident in the perilesional area. The median (range) Ktrans in the contrast-enhanced area after the first course of R-CHOP (without NGR-hTNF) was 23.5 (6.8-98.8) and increased to 35.3 (23.9-887.7; p=0.39) after the second course (NGR-hTNF / R-CHOP combination). In the perilesional area, the baseline value (R-CHOP alone) was lower (median 2.5; range 0.4-3.9), but increased to 4.7 (2.2-37.7; p=0.01) after the second course of NGR-hTNF infusion (NGR-hTNF / R-CHOP combination).

[0121] BBB permeability assessed by SPECT The ability of NGR-hTNF to increase BBB permeability in the tumor and surrounding areas was confirmed by SPECT studies. Quantitative analysis showed that in all cases examined, 99m The increase in the extent of Tc-DTPA positive areas was shown (example in Figure 5). 99m The median Tc-DTPA uptake volume (volume of interest) was 26 cm 3 (range 5-67) and 40cm 3 The median volume increase was 45%, with a range of 14-87%.

[0122] Consideration The results of the INGRID Phase II study presented herein demonstrated that the NGR-hTNF / R-CHOP combination was well tolerated and highly active in patients with heavily pretreated relapsed / refractory PCNSL. Importantly, most of the treated patients underwent consolidation therapy following tumor regression after NGR-hTNF / R-CHOP, increasing the chances of cure in these poor prognosis patients. The activity of this innovative strategy follows a selective enhancement of vascular permeability in the tumor and in the perilesional area. The specificity of the NGR-hTNF effect is also supported by the absence of changes in the concentration of R-CHOP agents in plasma and CSF samples, as demonstrated in the expansion phase of this study, as well as the high levels of expression of CD13 (the target of NGR-hTNF) in endothelial cells and pericytes of tumor-associated capillaries, as demonstrated in this study. Taken together, the results of this study support that increasing BBB permeability and drug penetration in tumors by non-invasive methods is an attractive therapeutic approach in patients with PCNSL.

[0123] Experience in patients with PCNSL with CHOP in the pre-rituximab era and, more recently, with R-CHOP, has indeed demonstrated that this combination is ineffective in patients with PCNSL at presentation and at relapse (5, 24-26), confirming the conventional wisdom that this inefficacy is mostly due to poor CNS bioavailability of the drugs involved. Indeed, when used as an upfront treatment, CHOP chemotherapy is associated with low response rates and does not contribute to improved disease control in combination with high-dose methotrexate-based chemotherapy or WBRT, with 2-year overall survival rates after CHOP-WBRT being only 20-40% (5, 24-25). There are no studies focusing on CHOP with or without rituximab in patients with relapsed or refractory PCNSL, however, the disappointing results reported as first-line treatment (5, 24-25) suggest that CHOP with or without rituximab should be inactive even as a salvage treatment. This view is also supported by the lack of significant responses after the first cycle of R-CHOP alone in the first 10 enrolled patients, despite an ORR of 75% and the possibility of delivering consolidation therapy in 17 of the 21 patients who responded to the NGR-hTNF / R-CHOP combination treatment. The active results obtained with NGR-hTNF / R-CHOP are associated with an excellent safety profile, without unexpected toxicities and, importantly, with the maintenance of dose intensity in all patients. In agreement with previous studies (27-29), the addition of low doses of NGR-hTNF to chemotherapy agents was associated with good tolerability and, in particular, the combination with doxorubicin was not associated with severe cardiovascular events.

[0124] Previous studies focusing on NGR-hTNF and its synergistic effects with chemotherapeutic agents in both animal models and patients with solid tumors have shown that the selectivity of NGR-hTNF for tumor vasculature requires interaction with specific receptors (11, 12, 30). When delivered at low doses, NGR-hTNF engages in high affinity interactions with endothelial cells expressing CD13, TNF-R1, and TNF-R2 in angiogenic tumor vasculature (7), but not with endothelial cells that do not express CD13, as occurs in non-neoplastic tissues. In fact, specific forms of CD13 are expressed by angiogenic blood vessels in tumors and other tissues, but are not (or are barely) expressed by blood vessels in normal tissues (8, 32). Remarkably, NGR-containing drugs can selectively target the form of CD13 expressed by endothelial cells and pericytes in tumor blood vessels, but not by the form of CD13 expressed by other tissues (10, 32). In an expansion phase of this study (14), we showed that the permeability-promoting effect of NGR-hTNF was more evident in the tumor and perilesional areas, where expression of CD13 by tumor vessels was demonstrated by immunohistochemistry and immunofluorescence techniques. Of note, CD13 was found to be abundantly expressed by pericytes of the PCNSL vasculature and, to a lesser extent, by endothelial cells. Importantly, these cells (especially in the latter case) are highly accessible to intravenously delivered NGR-hTNF. Notably, previous studies on CD13 expression in normal brain showed that pericytes, but not endothelial cells, express this marker (33, 34). Although the ability of NGR-hTNF to recognize the CD13 form expressed by normal brain pericytes remains to be elucidated, the following observations suggest that only tumor vessels are targeted in PCNSL patients: first, in the tumor / peritumoral areas; 99mIncreased uptake of Tc-DTPA was seen by SPECT after NGR-hTNF treatment but not in the remaining normal brain (14); second, no changes in plasma and CSF R-CHOP drug concentrations occurred in patients after NGR-hTNF treatment (14). One possible explanation for the selective drug penetration is the CD13+ endothelium in tumor vasculature. By targeting cells, NGR-hTNF further increases their permeability, thereby facilitating selective local drug penetration. As previously observed in brain metastases (6), it is possible that TNF receptors are overexpressed on tumor vasculature compared to normal vessels, resulting in a multivalent, high-affinity interaction of low-dose NGR-hTNF with CD13 and TNF receptors on tumor vessels but not on normal brain vessels.

[0125] In this study, plasma levels of sTNF-R were not associated with treatment outcome, but a significant association was found between plasma levels of CgA, PPI use, and response to NGR-hTNF / R-CHOP. Plasma levels of CgA are known to increase after treatment with PPIs because these drugs induce hypergastrinemia and stimulation of enterochromaffin cells to secrete CgA (36-38). Accordingly, we found that administration of PPIs to PCNSL patients, performed to prevent gastrointestinal toxicity during steroid treatment and chemotherapy, resulted in a significant increase in circulating CgA. Using a ROC-driven cutoff, we also found that 62% of patients with low CgA plasma levels and 27% of patients with high CgA levels achieved CR to NGR-hTNF / R-CHOP (p=0.066). This finding supports the justification for discontinuing PPIs before NGR-hTNF / R-CHOP treatment. If possible, NGR-hTNF / R-CHOP should be initiated after CgA has reached normal levels following PPI cessation, which may require several days to several weeks (36-38).

[0126] In conclusion, the results of the INGRID study confirm that the permeability-promoting effect of low-dose NGR-hTNF on the BBB can be exploited to improve the activity of R-CHOP in PCNSL. NGR-hTNF / R-CHOP combination therapy is active and safe in heavily pretreated patients with relapsed / refractory PCNSL, and its antitumor activity follows the expression of CD13 on tumor vessels. PPIs should be avoided before and during this treatment, because they may attenuate the effect of NGR-hTNF by increasing plasma levels of CgA. This innovative approach deserves to be treated as a first-line treatment for PCNSL patients.

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Claims

1. 1. A pharmaceutical combination of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) and NGR-hTNF or an analog thereof for use in treating primary central nervous system lymphoma in a subject, comprising: at least one course of administration of NGR-hTNF or an analog thereof is administered after administration of rituximab and before administration of a CHOP agent; A combination pharmaceutical, in which NGR-hTNF or an analog thereof and R-CHOP are administered separately.

2. R-CHOP, 375 mg / m 2 of rituximab, 750 mg / m 2 of cyclophosphamide, 50 mg / m 2 of doxorubicin, and 1.4 mg / m 2 2. The pharmaceutical combination of claim 1, comprising vincristine of

3. The dose of NGR-hTNF was 0.8ug / m 2 The pharmaceutical combination according to claim 1 or 2, comprising:

4. The combination pharmaceutical of any one of claims 1 to 3, comprising 6 courses of administration of NGR-hTNF or an analog thereof following administration of rituximab and prior to administration of a CHOP agent.

5. The combination pharmaceutical of claim 1, wherein the courses of administration of NGR-hTNF or an analogue thereof following administration of rituximab and prior to administration of a CHOP agent are separated by 18 to 21 days.

6. The combination pharmaceutical according to any one of claims 1 to 5, wherein the primary central nervous system lymphoma is relapsed primary central nervous system lymphoma or refractory primary central nervous system lymphoma.

7. 1. A composition for use in the treatment of primary central nervous system lymphoma in a subject in combination with R-CHOP, comprising NGR-hTNF or an analog thereof, The treatment includes at least one course in which administration of NGR-hTNF or an analog thereof is performed after administration of rituximab and before administration of a CHOP agent; The composition, wherein NGR-hTNF or an analog thereof and R-CHOP are administered separately.

8. 1. A kit for use in treating primary central nervous system lymphoma in a subject, comprising R-CHOP and NGR-hTNF or an analog thereof, The treatment includes at least one course in which administration of NGR-hTNF or an analog thereof is performed after administration of rituximab and before administration of a CHOP agent; The kit, wherein NGR-hTNF or an analog thereof and R-CHOP are administered separately.

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