NZ617770B2 - Combination therapy with an anti - cd19 antibody and a nitrogen mustard - Google Patents

Abstract

Disclosed is the use of a synergistic combination of: an antibody specific for CD19; and bendamustine, for the manufacture of a medicament for the treatment of non-Hodgkin’s lymphoma, chronic lymphocytic leukaemia and/or acute lymphoblastic leukaemia, wherein the antibody comprises an HCDR1 region of sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6). on of sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6).

Description

COMBINATION THERAPY WITH AN ANTI - CD19 ANTIBODY AND A EN Cross reference This application claims the benefit of US. provisional application serial number 61/654,097 filed June 1, 2012, US. provisional application serial number 61/647,539 filed May 16, 2012, and US. provisional application serial number 61/523,861 filed August 16, 2011, which are incorporated by reference in their entireties.

Field of the Invention The present sure is related to a pharmaceutical combination of an anti-CD19 dy and a nitrogen d for the treatment of non-Hodgkin’s ma, chronic lymphocytic leukemia and/or acute blastic ia.

Background B cells are lymphocytes that play a large role in the humoral immune response. They are produced in the bone marrow of most mammals, and represent 5-1 5% of the circulating lymphoid pool. The principal function of B cells is to make antibodies against various antigens, and are an essential component of the adaptive immune system.

Because of their critical role in regulating the immune system, ulation of B cells is associated with a variety of disorders, such as lymphomas, and leukemias. These include non-Hodgkin's lymphoma (NHL), c lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL).

NHL is a heterogeneous malignancy originating from lymphocytes. In the United States (US), the incidence is ted at 65,000/year with mortality of approximately 20,000 (American Cancer Society, 2006; and SEER Cancer Statistics Review). The disease can occur in all ages, the usual onset begins in adults over 40 years, with the incidence increasing with age. NHL is characterized by a clonal proliferation of lymphocytes that accumulate in the lymph nodes, blood, bone marrow and spleen, although any major organ may be involved. The current classification system used by pathologists and clinicians is the World Health Organization (WHO) Classification of Tumours, which organizes NHL into precursor and mature B-cell or T-cell neoplasms. The PDQ is currently dividing NHL as indolent or aggressive for entry into clinical trials. The indolent NHL group is sed primarily of follicular subtypes, small lymphocytic lymphoma, MALT (mucosa-associated lymphoid tissue), and marginal zone; indolent encompasses approximately 50% of newly diagnosed B-cell NHL patients. Aggressive NHL includes patients with histologic diagnoses of primarily diffuse large B cell (DLBL, DLBCL, or DLCL) (40% of all newly diagnosed patients have e large cell), Burkitt's, and mantle cell. The al course of NHL is highly variable. A major determinant of SUBSTITUTE SHEET (RULE 26) al course is the histologic subtype. Most indolent types of NHL are considered to be incurable disease. Patients respond lly to either chemotherapy or antibody therapy and most will relapse.

Studies to date have not demonstrated an improvement in survival with early intervention. In asymptomatic patients, it is acceptable to "watch and wait" until the patient becomes symptomatic or the disease pace appears to be accelerating. Over time, the disease may orm to a more aggressive histology. The median survival is 8 to 10 years, and indolent patients often receive 3 or more treatments during the treatment phase of their disease. lnitial treatment of the symptomatic indolent NHL patient historically has been combination chemotherapy. The most commonly used agents include: cyclophosphamide, vincristine and prednisone (CVP); or cyclophosphamide, adriamycin, stine, prednisone (CHOP). Approximately 70% to 80% of patients will respond to their initial chemotherapy, duration of remissions last on the order of 2-3 years. Ultimately the ty of patients relapse. The discovery and clinical use of the anti-CD20 antibody, rituximab, has provided icant improvements in response and survival rate. The current standard of care for most patients is rituximab + CHOP (R-CHOP) or rituximab + CVP (R-CVP). lnterferon is approved for initial treatment of NHL in combination with alkylating agents, but has limited use in the US.

Rituximab therapy has been shown to be efficacious in several types of NHL, and is currently approved as a first line treatment for both nt cular lymphoma) and aggressive NHL (diffuse large B cell lymphoma). However, there are significant limitations of anti-CD20 onal antibody (mAb), including primary resistance (50% response in relapsed indolent ts), acquired resistance (50% response rate upon re-treatment), rare complete response (2% te resonse rate in ed population), and a continued pattern of relapse. Finally, many B cells do not express CD20, and thus many B-cell disorders are not treatable using anti-CD20 antibody therapy.

In addition to NHL there are several types of leukemias that result from disregulation of B cells. Chronic lymphocytic leukemia (also known as "chronic lymphoid ia" or , is a type of adult leukemia caused by an abnormal accumulation of B lymphocytes. In CLL, the malignant lymphocytes may look normal and mature, but they are not able to cope effectively with infection. CLL is the most common form of leukemia in adults. Men are twice as likely to develop CLL as women.

However, the key risk factor is age. Over 75% of new cases are diagnosed in patients over age 50.

More than 10,000 cases are diagnosed every year and the mortality is almost 5,000 a year (American Cancer Society, 2006; and SEER Cancer Statistics Review). CLL is an incurable disease but sses slowly in most cases. Many people with CLL lead normal and active lives for many years.

Because of its slow onset, stage CLL is lly not treated since it is believed that early CLL intervention does not improve survival time or quality of life. Instead, the condition is monitored over time. lnitial CLL treatments vary depending on the exact diagnosis and the progression of the disease. There are dozens of agents used for CLL therapy. Combination chemotherapy regimens such as FCR (fludarabine, cyclophosphamide and rituximab), and BB (bendamustine and rituximab) are effective in both newly-diagnosed and relapsed CLL. Allogeneic bone marrow (stem cell) transplantation is rarely used as a first-line treatment for CLL due to its risk.

Another type of leukemia is acute lymphoblastic leukemia (ALL), also known as acute lymphocytic leukemia. ALL is terised by the overproduction and continuous multiplication of malignant and re white blood cells (also known as lymphoblasts) in the bone marrow. 'Acute' refers to the undifferentiated, immature state of the circulating lymphocytes ("blasts"), and that the disease progresses y with life expectancy of weeks to months if left untreated. ALL is most common in childhood with a peak incidence of 4-5 years of age. Children of age 12- 16 die more easily from it than . Currently, at least 80% of childhood ALL are considered e. Under 4,000 cases are diagnosed every year and the mortality is almost 1,500 a year (American Cancer y, 2006; and SEER Cancer Statistics Review).

The human CD 19 molecule is a structurally distinct cell surface receptor expressed on the surface of human B cells, including, but not limited to, pre-B cells, B cells in early development {i.e., immature B cells), mature B cells through terminal differentiation into plasma cells, and malignant B cells. CD 19 is expressed by most pre-B acute lymphoblastic leukemias (ALL), non-Hodgkin's lymphomas, B cell chronic lymphocytic leukemias (CLL), mphocytic ias, hairy cell leukemias, common acute lymphocytic leukemias, and some Null-acute lymphoblastic leukemias (Nadler et al, J. l., 131 :244-250 (1983), Loken et al, Blood, 70:1316-1324 (1987), Uckun et al, Blood, 71 :13- 29 (1988), Anderson et al, 1984. Blood, 63:1424-1433 , rmann, Leuk.

Lymphoma, 18:385-397(1995)). The expression of CD 19 on plasma cells further suggests it may be expressed on differentiated B cell tumors such as multiple myeloma, cytomas, Waldenstrom's tumors (Grossbard et al., Br. J. Haematol, 102:509- 15(1998); Treon et al, Semin. Oncol, :248-52(2003)).

Therefore, the CD 19 antigen is a target for immunotherapy in the treatment of non-Hodgkin’s lymphoma (including each the subtypes described herein), chronic lymphocytic leukemia and/or acute lymphoblastic leukemia. n CD19 therapies have been shown. T cells expressing an anti-CD19 chimeric antigen receptor (CAR) including both CD3-( and the 4-BB costimulatory domain were administered to three patients with advanced CLL. Kalos et al., T cells with Chimeric n Receptors Have Potent Antitumor Effects and Can Establish Memory in ts with Advanced Leukemia, Science Translational Medicine, vol. 3, no. 95 (10 August 2011), which is incorporated by reference in its entirety. Sadelain et al., The promise and potential pitfalls of chimeric antigen receptors, Current Opinion in Immunology, Elsevier, vol. 21, no.2, 2 April 2009, which is incorporated by nce in its entirety, also describes anti-CD19 chimeric antigen receptors (CARs). Neither Kalos et al. nor Sadelain et al., r, be the antibody specific for CD19 in combination with bendamustine as ified herein.

Bendamustine as a therapy in the treatment of non-hodgkin’s lymphoma was described in Bremer et al., High rates of long lasting remission after 5-day bendamustine chemotherapy cycles in pre-treated low-grade non-Hodgkin’s lymphomas, Journal of Cancer Research and Clinical Oncology, Springer International, Berlin, DE, vol. 128, no. 11, 1 November 2002, which is incorporated by reference in its entirety, and WO2006065392, which is incorporated by reference in its ty, but neither suggests the antibody specific for CD19 in combination with bendamustine as exemplified herein.

The use of a CD19 antibody in non-specific B cell lymphomas is discussed in WO2007076950 (US2007154473), which are both incorporated by reference in their entireties, along with the cursory mention of bendamustine within a long list of potential ation rs, but fails either to teach the antibody exemplified herein or suggest the istic effects of the combination in the treatment of non-Hodgkin’s lymphoma, chronic lymphocytic ia and/or acute lymphoblastic leukemia as exemplified herein.

The use of a CD19 antibody in CLL, NHL and ALL is bed in Scheuermann et al., CD19 Antigen in Leukemia and Lymphoma Diagnosis and lmmunotherapy, Leukemia and Lymphoma, Vol. 18, 385-397 (1995), which is incorporated by reference in its entirety, but fails to suggest the combination exemplified herein.

Additional antibodies specific for CD19 are described in WO2005012493 (US7109304), WO2010053716 (US12/266,999) (lmmunomedics); WO2007002223 (US US8097703) (Medarex); WO2008022152 7,251) and WO2008150494 r), WO2008031056 (US11/852,106) (Medimmune); WO 2007076950 (US ,505 ) (Merck Patent GmbH); (US12/253,895) (Seattle Genetics); and WO2010095031 (12/710,442) (Glenmark ceuticals), which are all incorporated by reference in their entireties.

Combinations of antibodies specific for CD19 and other agents are bed in WO2010151341 (US 13/377,514) (The Feinstein Institute); US5686072 (University of Texas), and WO2002022212 (PCT/USO1/29026) (lDEC Pharmaceuticals), which are all incorporated by reference in their entireties.

It is clear that in spite of the recent progress in the discovery and development of anti-cancer agents, many forms of cancer involving xpressing tumors still have a poor prognosis. Thus, there is a need for improved methods for treating such forms of cancer.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Summary According to one aspect, the present invention provides use of a synergistic combination of: an antibody specific for CD19; and bendamustine, for the cture of a medicament for the ent of non-Hodgkin’s lymphoma, c lymphocytic leukemia and/or acute lymphoblastic leukemia, wherein the antibody comprises an HCDR1 region of sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of sequence TRVFDY (SEQ ID NO: 3), an LCDR1 region of sequence QNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6).

Unless the context clearly requires ise, throughout the description and the claims, the words ise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

Neither alone nor in combination does the prior art suggest the synergistic effects of the combination of the exemplified antibody and bendamustine in the treatment of non-Hodgkin’s lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia.

In another aspect, the present disclosure relates to a synergistic combination of an dy specific for CD19 and a nitrogen mustard. Such combinations are useful in the treatment of B cell ancies, such as, non-Hodgkin’s lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia.

In vitro and in vivo models are considered indicative of how a certain compound or ation of compounds would behave in . In addition, when compounds are combined either in vitro or in vivo, one expects that the combination has only additive effects. Surprisingly, the inventors found that the combination of a ular antibody specific for CD19 and bendamustine mediated a synergistic level of specific cell killing in a chronic B-cell leukemia cell line (MEC-1) in comparison to the antibody and bendamustine alone. This in vitro model is indicative of how the combination will - 5a - work in the treatment of c id leukemia (CLL) in humans. In addition, and also ctedly, the inventors found that the combination of a particular antibody specific for CD19 and bendamustine inhibited tumor growth and synergistically increased median survival days and median increase in an, both in Burkitt’s lymphoma SCID mouse models, in comparison to the antibody and bendamustine alone. These in vivo models are indicative of how the combination will work in the treatment of non-Hodgkin’s lymphoma in humans. In summary, the ation of the ified anti-CD19 antibody and bendamustine behaved synergistically in models relevant to NHL and CLL. As both NHL and CLL are B cell related disorders and CD19 is highly expressed on B-cells, the exemplified combination would have the same mechanism of action and should also behave synergistically in the treatment of other B cell related disorders, e.g. ALL.

Therefore, the combination of the exemplified antibody specific for CD19 and bendamustine will be effective in the treatment of humans in non-Hodgkin’s lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia. In addition, the antibody ic to CD19 exemplified in the present specification has already entered into clinical , where such combinations can be confirmed in humans.

As the mechanism of action of bendamustine and other nitrogen mustards are similar, as they are alkylating agents that form interstrand cross-links (ICLs) n DNA bases, thus blocking fundamental processes such as replication and transcription, it is believed that synergy should also be seen when treating humans having non- Hodgkin’s lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia with a combination of the ified anti-CD19 antibody and a en mustard other than bendamustine.

As the exemplified anti-CD19 dy and other anti-CD19 dies bind CD19, it is believed that synergy should also be seen when treating humans having non-Hodgkin’s lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia with a combination of any anti-CD19 antibody and a nitrogen mustard, e.g., bendamustine.

As the exemplified anti-CD19 antibody binds a specific epitope of CD19, it is believed that antibodies that cross-compete with the exemplified antibody or bind to the same e as the exemplified antibody should also behave synergistically when treating humans having dgkin’s lymphoma, c lymphocytic leukemia and/or acute lymphoblastic leukemia when used in combination with a nitrogen mustard, e.g., bendamustine.

An aspect of the present disclosure comprises a synergistic combination wherein the antibody specific for CD19 comprises an HCDRI region of sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of ce GTYYYGTRVFDY (SEQ ID NO: 3), an LCDRt region of ce RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of ce RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6) and ustine. In preferred aspects, the combination is used for the treatment of non-Hodgkin’s lymphoma, chronic lymphocytic ia and/or acute lymphoblastic leukemia.

Description of Drawings Figure 1 shows the cytotoxicity effects of MOR00208 and bendamustine alone and in ation on MEG-1 cells.

Figure 2 shows the ADCC dose reponse curves of the combination of MOR00208 and bendamustine in MEG-1 cells.

Figure 3 shows the amino acid sequence of the variable domains of MOR00208.

Figure 4 shows the amino acid sequence of the Fc regions of MOR00208.

Figure 5 shows the normalized specific killing data of Table 2.

Figure 6 shows the s of the human Ramos Burkitt’s B-cell lymphoma survival model in SCID mice as described in Example 3. The figure represents the data shown in Table 6, but excludes treatment related deaths.

Figure 7 shows the statistical analysis of the results of the subcutaneously (SC)-implanted human Ramos Burkitt’s B-cell lymphoma tumor growth model in SCID mice, as described in Example 2.

Figure 8 shows the results of the subcutaneously (SC)—implanted human Ramos t’s B-cell lymphoma tumor growth model in SCID mice, as bed in Example 2.

Figure 9 shows the results of the subcutaneously (SC)-implanted human Ramos Burkitt’s B-cell lymphoma tumor growth model in SCID mice, as described in Example 2. In this figure the BEN dosage is 13mg/kg.

Figure 10 shows the results of the subcutaneously (SC)—implanted human Ramos t’s B-cell lymphoma tumor growth model in SCID mice, as described in Example 2. In this figure the BEN dosage is 16mg/kg.

Detailed description of the invention “Synergy , ism” or “synergistic” mean more than the expected additive effect of a ation. The “synergy”, “synergism” or “synergistic” effect of a ation is determined herein by the methods of Chou et al., Clarke et al. and/or Webb et al. See Ting-Chao Chou, Theoretical Basis, Experimental Design, and Computerized Simulation of ism and Antagonism in Drug Combination Studies, Pharmacol Rev 58:621—681 (2006), which is incorporated by reference in its entirety. See also Clarke et al., Issues in experimental design and endpoint is in the study of experimental cytotoxic agents in vivo in breast cancer and other , Breast Cancer Research and Treatment 46:255-278 (1997), which is incorporated by reference in its entirety. See also Webb, J. L. (1963) Enzyme and Metabolic Inhibitors, Academic Press, New York, which is incorporated by reference in its entirety.

The term ody" means monoclonal antibodies, including any isotype, such as, lgG, lgM, lgA, lgD and lgE. An lgG antibody is comprised of two identical heavy chains and two identical light chains that are joined by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three segments called "complementarity-determining regions" ("CDRs") or "hypervariable regions", which are primarily responsible for binding an epitope of an antigen. They are referred to as CDR1, CDR2, and CDR3, numbered sequentially from the inus. The more highly conserved portions of the variable regions outside of the CDRs are called the "framework regions". An “antibody fragment” means an Fv, scFv, dst, Fab, Fab' F(ab')2 fragment, or other fragment, which contains at least one variable heavy or variable light chain, each ning CDRs and framework regions.

A “nitrogen mustard” is a cific DNA ting agents used as chemotherapy.

Alkylating agents add an alkyl group (CnH2n+1) to nucleic acid bases, e.g., adding an alkyl group to the guanine base of DNA at the number 7 nitrogen atom of the imidazole ring. The alkylation steps result in the formation of interstrand cross-links (lCLs). These lCLs are highly cytotoxic, since they block fundamental metabolic processes such as replication and transcription. Nitrogen mustards include cyclophosphamide, chlorambucil, uramustine, ifosfamide, melphalan and bendamustine.

Cyclophosphamide is marketed as Endoxan, Cytoxan, Neosar, Procytox, and Revimmune, and is also known as cytophosphane. Cyclophosphamide, or combinations including cyclophosphamide, is used in the treatment of lymphomas, leukemia and some solid .

Cyclophosphamide has the following ure: mbucil is ed as Leukeran by GlaxoSmithKline. It is used mainly in the treatment of chronic lymphocytic leukemia. Chlorambucil has the following ure: HOWNWC‘ Uramustine is used in the treatment of non-Hodgkin's lymphoma. tine has the following structure: Cl/VNfNH lfosfamide is marketed as Mitoxana and lfex. mide has the following structure: 0 E? h \P/ \‘/\Cl Melphalan is marketed as Alkeran. Melphalan has the following structure: NH2 \/\Cl Bendamustine is marketed under the names Ribomustin®,and Treanda®, and is also known as SDX-105, by Mundipharma International Corporation | imited (Licensee of Astellas Pharma GmbH) and Cephalon for the treatment of chronic lymphocytic leukemias (CLL), indolent B-cell non-Hodgkin's lymphoma (NHL), and other lymphomas. ustine has the following structure: “BEN” when used herein means bendamustine.

"VH" refers to the variable region of an immunoglobulin heavy chain of an antibody, or antibody fragment. "VL" refers to the le region of the immunoglobulin light chain of an antibody, or antibody fragment.

The term “CD19” refers to the protein known as CD19, having the following synonyms: B4, B-lymphocyte antigen CD19, B-lymphocyte e antigen B4, CVID3, Differentiation n CD19, MGC12802, and T-cell surface antigen Leu-12.

Human CD19 has the amino acid sequence of: MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSL GLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLG GLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPElWEGEPPCLPPRDSLNQSLSQDLTMAPGS TLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGK YYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGlLHLQRALVLRRKRK RMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQA RSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPE DEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLR SIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR. (SEQ ID NO: 7) “MOR00208” is an anti-CD19 antibody. The amino acid sequence of the variable s is provided in Figure 3. The amino acid sequence of the heavy and light chain Fc regions of MOR00208 are provided in Figure 4. “MOR00208” and “XmAb 5574” are used as synonyms to be the antibody shown in Figures 3 and 4. The MOR00208 antibody is described in US patent application serial number 12/377,251, which is incorporated by reference in its entirety.

Additional antibodies specific for CD19 are described in US patent no. 7,109,304 (lmmunomedics), which is incorporated by reference in its entirety; US application serial no. 11/917,750 (Medarex), which is incorporated by reference in its entirety; US application serial no. ,106 mune), which is incorporated by reference in its entirety; US application serial no. 11/648,505 (Merck Patent GmbH), which is incorporated by reference in its ty; US patent no. 7,968,687 (Seattle Genetics), which is incorporated by reference in its entirety; and US application serial no. 12/710,442 (Glenmark ceuticals), which is incorporated by reference in its entirety.

“Fc region” means the constant region of an dy, which in humans may be of the lgG1, 2, 3, 4 subclass or others. The sequences of human Fc regions are available at IMGT, Human IGH C-REGIONs, http://www.imgt.org/lMGTrepertoire/Proteins /protein/human/|GH/lGHC/Hu_lGHCallgenes.html (retrieved on 16 May 2011).

“RefmAb33” is an antibody whose amino acid sequence is as follows: Heavy chain including the Fc region: QVTLRESGPALVKPTQTLTLTCTFSGFSLSTAGMSVGWIRQPPGKALEWLADIWWDDKKH YNPSLKDRLTISKDTSKNQVVLKVTNMDPADTATYYCARDMIFNFYFDVWGQGTTVTVSSASTKG APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMIS RTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKE YKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDlAVEWESN GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8) Light chain including the Fc region: DIQMTQSPSTLSASVGDRVTITCSASSRVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRF SGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC EVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9) RefmAb33 is specific for RSV, and is used as isotype l, as it shares the same Fc region as MOR00208.

A nation” means more than one item, e.g. a compound such as an dy and bendamustine.

The present sure also relates to combinations, pharmaceuticals, and pharmaceutical compositions containing the described combinations. The two components of the synergistic combination of the present invention, e.g. the antibody specific for CD19 and bendamustine, may be administered together, simultaneously or separately. When stered together, the two components may be formulated together in one pharmaceutical composition, which may include a pharmaceutical acceptable carrier or excipient. Alternatively the two components might also be formulated in different pharmaceutical compositions. In this case the two components can be administered simultaneously or subsequently. In an embodiment, ustine, is administered prior to and/or separately from the administration of the antibody specific for CD19, e.g. MOR00208.

A ceutical composition includes an active agent, eg. an antibody for therapeutic use in . A pharmaceutical ition may include acceptable carriers or excipients.

"Administered" or “administration” includes but is not limited to delivery by an injectable form, such as, for example, an intravenous, intramuscular, intradermal or subcutaneous route or mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestable solution, capsule or tablet.

A “therapeutically ive amount” of a compound or combination refers to an amount sufficient to cure, ate or partially arrest the clinical manifestations of a given disease or disorder and its complications. The amount that is effective for a particular therapeutic purpose will depend on the severity of the disease or injury as well as on the weight and general state of the subject. It will be understood that determination of an appropriate dosage may be achieved, using routine experimentation, by ucting a matrix of values and testing different points in the matrix, all of which is within the ordinary skills of a trained physician or clinical ist.

The “CDRs” herein are defined by either Chothia et al or Kabat et al. See Chothia C, Lesk AM. (1987) Canonical ures for the hypervariable regions of globulins. J Mol Biol., 196(4):901-17, which is incorporated by reference in its entirety. See Kabat E.A, Wu T.T., Perry H.M., Gottesman KS. and r C. (1991). Sequences of Proteins of Immunological lnterest. 5th edit., NIH Publication no. 91-3242, US Dept. of Health and Human Services, Washington, DC, which is incorporated by nce in its entirety.

“Cross competes” means the ability of an antibody or other binding agent to interfere with the binding of other antibodies or binding agents to CD19 in a standard competitive binding assay. The ability or extent to which an antibody or other binding agent is able to ere with the binding of another antibody or binding le to CD19, and, therefore whether it can be said to cross-compete ing to the invention, can be determined using standard competition binding assays. One suitable assay involves the use of the Biacore technology (e.g. by using the BlAcore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the extent of interactions using e plasmon resonance technology. Another assay for measuring cross-competing uses an ELISA-based approach. A high throughput process for "epitope binning" antibodies based upon their cross-competition is described in International Patent Application No.

The term "epitope" includes any protein determinant capable of specific g to an antibody or otherwise interacting with a molecule. ic determinants generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be "linear" or "conformational." The term r epitope" refers to an epitope with all of the points of ction between the protein and the interacting molecule (such as an antibody) occur linearally along the primary amino acid sequence of the protein nuous).

The term "conformational epitope" refers to an epitope in which discontinuous amino acids that come together in three dimensional conformation. In a conformational epitope, the points of interaction occur across amino acid residues on the protein that are separated from one another.

“Binds the same epitope as” means the ability of an antibody or other binding agent to bind to CD19 and having the same epitope as the exemplified antibody. The epitopes of the exemplified antibody and other antibodies to CD19 can be determined using standard epitope mapping techniques. Epitope mapping ques, well known in the art. include Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66 (Glenn E.Morris, Ed., 1996) Humana Press, Totowa, New Jersey. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the es corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the ts. Such ques are known in the art and described in, e.g., US. Patent No. 4,708,871 ; Geysen et al, (1984) Proc. Natl. Acad. Sci. USA 8:3998-4002; Geysen et al, (1985) Proc. Natl. Acad.

Sci. USA 82:78-182; Geysen et al, (1986) Mol. lmmunol. 23 :709-715. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., en/deuterium exchange, x-ray crystallography and two-dimensional r magnetic resonance. See, e.g., Epitope Mapping Protocols, supra. Antigenic regions of proteins can also be identified using standard nicity and hydropathy plots, such as those calculated using, e.g., the Omiga version 1.0 software program available from the Oxford Molecular Group. This computer program employs the Hopp/Woods method, Hopp et al, (1981) Proc. Natl. Acad. Sci USA 78:3824-3828; for determining antigenicity es, and the oolittle technique, Kyte et al, (1982) J.Mol. Biol. 157: 105-132; for hydropathy plots.

Embodiments An aspect of the present disclosure comprises a combination of an antibody specific for CD19 and a nitrogen d for use in the treatment of non-Hodgkin’s ma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia. ln ments, the combination is synergistic.

Herein, the combination of the exemplified anti-CD19 antibody and bendamustine behaved synergistically in in vitro and in vivo models relevant to NHL and CLL. As both NHL and CLL are B cell related disorders and CD19 is highly expressed on B-cells, the exemplified combination should have the same ism of action and should also behave synergistically in the treatment of other B cell related disorders, e.g. ALL. Therefore, the combination of the exemplified antibody ic for CD19 and bendamustine will be effective in the treatment of humans in non-Hodgkin’s lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia.

As the mechanism of action of bendamustine and other nitrogen mustards are similar, as they are alkylating agents that form interstrand cross-links (lCLs) between DNA bases, thus blocking fundamental ses such as replication and transcription, it is believed that synergy should also be seen when ng humans having non-Hodgkin’s lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia with a combination of the exemplified anti-CD19 antibody and a nitrogen mustard other than bendamustine, e.g. cyclophosphamide, chlorambucil, uramustine, ifosfamide, and lan.

As the exemplified anti-CD19 antibody and other anti-CD19 antibodies bind CD19, it is ed that y should also be seen when treating humans having non-Hodgkin’s lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia with a combination of any anti-CD19 antibody and a nitrogen mustard, where the D19 antibody is, for example, described in US patent ation serial number 12/377,251 (Xencor), WO2005012493, WO2010053716 (lmmunomedics); WO2007002223 (Medarex); W02008022152 (Xencor); WO2008031056 (Medimmune); (Merck Patent GmbH); (Seattle Genetics); and WO2010095031 (Glenmark Pharmaceuticals), all of which are incorporated by reference in their entireties. ln embodiments, the antibody specific for CD19 comprises an antibody that cross-competes with the antibody comprising an HCDR1 region of sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of ce NPYNDG (SEQ ID NO: 2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6). ln embodiments, the antibody specific for CD19 comprises an dy that binds to the same epitope as an antibody comprising an HCDR1 region of sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of ce NPYNDG (SEQ ID NO: 2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of sequence QNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6). ln embodiments, the antibody specific for CD19 comprises an HCDR1 region of sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of ce RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of sequence PIT (SEQ ID NO: 6). ln embodiments, the antibody specific for CD19 comprises a variable heavy chain of the sequence EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPY NEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWG QGTLVTVSS (SEQ ID NO: 10) and a variable light chain of the sequence DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYR MSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO: 11).

In embodiments, the antibody specific for CD19 comprises a heavy chain constant domain of the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. (SEQ ID NO: 12) In embodiments, the antibody specific for CD19 ses a light chain constant domain of the sequence SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. (SEQ ID NO: 13) In embodiments, the en mustard is bendamustine.

In embodiments, the components of the ation, the antibody specific for CD19 and bendamustine, are administered separately. In an embodiment, bendamustine is administered prior to administration of the dy specific for CD19.

In embodiments the combination is a pharmaceutical composition. In ments, the composition comprises an acceptable carrier. In embodiments, the combination is administered in an effective amount.

In another aspect the synergistic combination of an antibody specific for CD19 sing an HCDR1 region of sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence S (SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6) and bendamustine is able to mediate killing of MEC-1 cells by ADCC in the presence of isolated human PBMCs with an at least two-fold, three-fold, four-fold, or five-fold better efficacy than bendamustine alone.

An aspect of the present disclosure comprises a synergistic combination of an antibody specific for CD19 comprising an HCDR1 region of sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS - 14a - (SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6) and bendamustine for the treatment of non-Hodgkin’s ma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia. In embodiments, the non-Hodgkin’s 2012/065906 lymphoma is selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue, marginal zone, e large B cell, t's, and mantle cell. r aspect comprises a method of treating non-Hodgkin’s lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia in an individual in need thereof, which method comprises administration of an antibody specific for CD19 and a nitrogen mustard. ln embodiments of the method, the antibody specific for CD19 comprises an HCDR1 region of sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region of ce GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of ce MQHLEYPIT (SEQ ID NO: 6). ln ments of the method, the antibody comprises the exemplified antibody specific for CD19. ln embodiments of the method the nitrogen mustard is bendamustine.

Examples Example 1: tion of proliferation of MEC-1 cells using MOR00208 and bendamustine alone and in combination Materials MEC-1 cells: chronic B-cell leukemia cell line DSMZ# ACC497; Cell Medium: lscove's Modified Dulbecco's Medium (lMDM) with GlutaMAXT'V', lnvitrogen, Cat No.: 31980-048, 20% FCS; PBMCs: RPMI1640, with e Glutamine, PAN Biotech GmbH, Cat No.: P04-13500 supplemented with 10% FCS; Biocoll: Biochrome AG CAT No.: L6115 LOT No.: 1050T; Bendamustine: Mundipharma LOT No.: 88018; FCS: PAN CAT No.: 3302-P282403 LOT No.: 3; and RefmAb33 (anti-RSV) with same Fc region as MOR00208.

Methods The cytotoxicity of MOR00208 and bendamustine alone and in combination was tested in MEC-1 cells. BEN is an alkylating agent, therefore, functions via direct cytoxicity in MEC-1 cells.

MOR00208 targets CD19 and additionally functions via ADCC in killing MEC-1 cells. For the following groups MEC-1 cell killing was measured: BEN at 100ug/ml; MOR00208 at 6,6pm and the ation of MOR00208 at 6,6pm and BEN at 100ug/ml. These concentrations were chosen as they are near or at the EC50 for MOR00208 and BEN. The following were used as controls: RefmAb33, or PBMCs alone. In both the BEN group and MOR00208+BEN combination group, MEC-1 cells were pre-incubated with BEN 48 hours prior to the ADCC assay measurements. The MEC-1 cells were stained using 1mg/ml Calcein AM then counted and adjusted to 2X105/ml. The PBMCs were counted and adjusted to 6X106/ml. The cell killing assays were done as follows: using 96 well , a 100ul cell suspension of MEC-1 cells was added per well, then 100ul cell suspension of PBMCs was added to each well resulting in an E:T ratio of 30:1. The antibodies were diluted to 1ug/ml in medium. Cells were centrifuged and re-suspended. To the target:effector cell-pellet, 100ul antibody solution or according control solution was added. The mixture was incubated for 4h in cubator at 37°C. The cell killing ements were taken as follows: the incubated cell solution l) was transfered into FACS tubes and 200ul FACS buffer (DPBS + 3%FCS) and 0,5 ul Pl stock solution was added to each tube. FACS-Calibur was used. Dead MEC-1 cells were stained with propidium iodide. Table 1 and Figure 1 show the raw data.

Table 1 Control MOBOO208 BEN 100 ug/ml BEN+MOROO208 6,6pm combination Experiment 1 , 73,6 83,6 94,0 The values represent % dead cells. Each experiment ents PBMCs from different donors. The controls used for each experiment was RefMab33.

Table 2 shows the raw data of Table 1 normalized for specific killing and the results of the Chou calculations done in the determination of ism.

Table 2 Experiment 1 The values shown in Table 2 are calculated as follows: 1) from the raw data (% dead cells) shown in Table 1, the background (controls) were subtracted, resulting in the specific killing for each treatment group; then 2) the specific killing values were normalized by setting the ation of MOR00208 + BEN to 1. The averages of Table 2 are depicted in Figure 5. Example ADCC dose response curves used in the Chou factor calculations of the MOR00208 + BEN combination are shown in Figure 2.

Chou Index (Cl) calculations were completed in order to determine synergy of the combination of the ified anti-CD19 antibody and bendamustine as compared to MOR00208 and BEN alone. Such calculations are described in Ting-Chao Chou, Theoretical Basis, Experimental , and erized Simulation of Synergism and Antagonism in Drug Combination Studies, Pharmacol Rev 58:621—681 (2006), which is incorporated by nce in its entirety and Chou TC, Talalay P, Quantitative analysis of dose-effect relationships: the combined s of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22: 27-55 (1984), which is incorporated by reference in its entirety. The methods of Chou-Talalay are carried out using the Cl-isobol method.

Median-effect equation The median-effect equation models of the effect of an inhibitor (such as a drug) as Fa/FU =(D/D50)"m, where D is the dose, FE, and FU is the fraction of the system affected and unaffected by the dose D (Fa + FU = 1); D50 is the dose producing the median effect (e.g. lC50, ED50, LD50). The constant m determines the shape of the dose-effect curve.

We used Excel Fit software to carry out a linear regression calculation to estimate the parameters m and D50.

The effects of the combination on MEC-1 cells is ed % cell death as described above.

We define the fraction FU to be the ratio of % cell death of the treated cell line to the % cell death of the cell line d to a control. That is: FL, =% cell death(treated cell line)/ % cell death (non-treated cell line) Then the % cell death of a cell line is the constant D50 in the median effect equation, which can be estimated by the linear regression described above.

Cl-isobol method The Cl-isobol method provides a quantitative assessment of synergism between drugs. A combination index (Cl) is estimated from dose-effect data of single and combined drug treatments.

A value of Cl less than 1 indicates synergism; CI = 1 indicates additive effect; and Cl > 1 indicates antagonism. Drug interaction gism or antagonism) is more pronounced the farther a Cl value is from 1.

Formally, the combination index (CI) of a combined drug treatment is defined as Cl 1 + D2/DX2 Here D1 and D2 are the doses of drug 1 and drug 2 of the combination, tively; and Dx1, and Dx2 is the dose of a treatment with only drug 1 and drug 2 that would give the same effect as that of the combination. The doses Dx1 and Dx2 need to be estimated from the dose-effect data of single drug treatments. Essentially, a median effect equation is fitted to the data of each drug. From the median effect equation of a drug, we can estimate the dose (i.e. D) necessary to produce an effect (i.e. Fa, Fu). The further a point lies from the additive line, the bigger the different between 1 and its Cl, thus the stronger the (synergistic or antagonistic) effect is.

As shown in Table 2, the Chou index values indicate clear synergism of the combination of MOR00208 and bendamustine in the specific killing of MEC-1 cells as compared to 08 and ustine alone. This conclusion is based upon the Chou calculations of 0,2, 0.7 and 0.75 of each of the three experiments, respectively, having an average of 0,6, where a Cl <1 indicates synergism. ore, the combination of MOR00208 and bendamustine will also behave synergistically in the treatment of non-Hodgkin's ma (NHL), chronic lymphoid leukemia (CLL), and acute lymphoblastic ia (ALL) in humans. In order to confirm the results of the above Chou calculations, the normalized data of Table 2 was evaluated for statistical significance using the Bonferroni's Multiple Comparison Test. See James, et al, dy-mediated B-cell depletion before adoptive immunotherapy with T cells expressing CD20-specific chimeric T-cell ors facilitates eradication of leukemia in immunocompetent mice, Blood, 114(27):5454-63 (Epub 2009 Oct 30), which is incorporated by reference in its entirety. The results are shown in Table 3.

Table 3 Bonferroni's Mean Diff. Significant? Summary Multiple (P < 0'05) Comparison Test ustine (100ug/ml) vs.

BEN + MOR 208 combination MOROO208 (6.6pM) vs. BEN + MOROO208 ation p < 0,05 p < 0,001 As shown in Table 3, the Bonferroni’s Multiple Comparison Test shows that the combination treatment of BEN + MOR00208 is statistically more effective in the specific killing of MEC-1 cells than the treatment of BEN and MOR00208 alone.

Example 2: MOR00208 and BEN alone and in combination in aneously (SC)-implanted human Ramos Burkitt’s B-cell lymphoma tumor growth model.

RAMOS human Burkitt’s lymphoma cells (ATCC number CRL-1596, lot# 3953138); Vehicle control: 150 mM NaCl, 25 mg/mL mannitol, pH 5.5-6.0; ted with 0.01 M NaOH).

Ref_mAb_33_lgG_Xen (10 mg/mL in PBS, referred to as Ref_mAb_33). Six-week-old, female, C.B-17 SCID mice (CB17/lcr-Prkdcscid/lcrlcoCrl) were purchased from Charles River Laboratories (Wilmington, MA) and acclimated in the laboratories for nine days prior to experimentation.

Methods SCID mice were implanted sub-cutaneously with RAMOS cells (~5 x 106 cells/mouse).

When the mice had tumors of approximately 150 mm3 in size, or ~14 days after inoculation, they were separated into groups, where each group had tumor volumes of relatively the same size.

Treatments began on Day 15. The treatment regimens are provided in Table 4. The study on was 60 days.

Table 4 No. of Dose Treatment Route TCSt Artlcles Animals (mg/kg) and Schedule Bendamustine 13, and 16 IP, Q1 Dx5 1O MOROO208 IV, 6 mg/kg Q3Dx2; 10mg/kg Q3Dx2/3 wks starting on Day Vehicle/ IP, Q1 Dx5 Ref_mAb_33 IV, 6 mg/kg 03Dx2 ; 10 mg/kg 03Dx2/3 wks starting on day 22 08/ 6 or 10/13 MOROO208 and BendamUSt'm and 6 or 10/16 BEN as above Due to a technician error MOR00208 on Day 18 was not administered.

MOR00208, and bendamustine, were administered in a volume of 0.1 mL/10 g of body weight. MOR00208 and vehicle control/Ref_mAb_33 at a concentration of 0.6/1.0 mg/mL, and bendamustine at concentration of 1.3,and 1.6 mg/mL.

The ts were 1) Median days to reach 4000 mg in size, where the statistical analysis was done using the log rank test and 2) Tumor size on study day 34, where the statistical analysis was done using the One-Way-ANOVA and Bonferroni’s post hoc tests. (Raw data not shown).

Tumor weights were calculated using the equation (I x w2)/2, where l and w refer to the larger and smaller dimensions collected at each measurement. The results are shown in Figures 7-10. The combination therapy was not significantly superior to the respective monotherapies in this subcut model, as ed to the clear synergy shown in the orthotopic survival model below. This is ered to be related to the ineffective MOR00208 dosing n in this model. The orthotopic survival model described below, however, is believed to be more predictive of how well the combination treatment would work in the ent of CLL, NHL, and ALL in humans, as the orthotopic model better mimics the multifocal disease nature, including an involvement of the vascular system, as compared to the subcut, solid tumor model above.

Example 3 MOR00208 and bendamustine alone and in combination in human Non-hodgkin RAMOS tumor in SCID mice, survival model Materials Cyclophosphamide (Baxter, Lot. No.1A548C); Vehicle Control: 0.9% sodium chloride, 25mg/ml mannitol, pH 6.5-6.8 (adjusted with 0.01 M NaOH); SCID Mice (University of Adelaide, Waite Campus, aie, SA, Australia, Strain C.B.lgh-1b-PrkchCid); RAMOS human Burkitt’s lymphoma cells (ATCC number CRL-1596); Ref_mAb_33_lgG_Xen (10 mg/mL in PBS, referred to as Ref_mAb_33); Bendamustine (Mundipharma, Lot No. 83889).

SCID mice were eated with Cyclophosphamide (75 mg/kg, i.p., twice daily) for two days prior to RAMOS cell inoculation (Day -2 and -1). On the day of ation (Day 0), the mice were separated into seven groups of ten mice each, and inoculated with 1 x 106 RAMOS cells each intravenously into the tail vein. The d dosing regimen for each group is shown in Table 5 and ced on Day 3. The study duration was 60 days.

Table 5: Dosing regimen Group Compound Treatment Schedule 2 and 3 Bendamustine 13/16 mg/kg, i.p, in 10 mL/kg Once daily (Days 5-9) Twice weekly for 3 1 MOR00208 3 mg/kg, i.V., in 10 mL/kg weeks (Days 3, 6, 10, 13, 17 and 20) Vehicle l i.p., 10 mL/kg Once daily (Days 5-9) Bendamustine Once daily (Days 5'9) 13/16 mg/kg, i.p; 3 mg/kg, iv. and 6 /MOR00208 in 10 mL/kg; “We ““66le for 3 weeks(Days 3, 6, 10, 13, 17 and 20) 4 Bendamustine 26 mg/kg, i.p, in 10 mL/kg Once daily (Days 5-9) Ref mAb 3 mg/kg, iv. Day 3, 6, 10, 13, 17 and 20 The survival data is shown in Table 6 and Figure 6.

Table 6: Death of mice Group Compound Treatment Death of Mice over the Course of Study [Day post Inoculation] 1 MOR00208 3 mg/kg, iv. 25; 29; 29; 30; 31; 33; 35; 38; 38; 39 2 ustine 13 mg/kg, i.p. 10*; 21; 21; 23; 24; 24; 24; 24; 25; 26 3 Bendamustine 16 mg/kg, i.p. 24; 24; 24; 24; 24; 24; 25; 26; 26; 27 4 Bendamustine 26 mg/kg, i.p. 10*; 10*; 10*; 10*; 10*; 12*; 12*; 14*; 16*; 23 Bendamustine/ 13/3 mg/kg, i.p. 12*. 30. 33. 33. 35. 40. 45. 45. 56' 56 MOR00208 / i.V.

Bendamustine/ 16/3 mg/kg, i.p. 6 33, 35, 38, 39, 40, 40, 45,45,45,45_ _ _ _ _ _ _ _ _ MOR00208 “N. i.p. / 3 mg/kg, Vehicle/ Ref_mAb 24; 24; 25; 25; 25; 26; 26; 26; 26; 29 2012/065906 * Compound ty related death From the raw data shown in Table 6, both the median survival in days and median increase in lifespan were calculated. All treatment related deaths were excluded in the calculations. The results are shown in Table 7.

Table 7 Median Median % Group Survival Increase in Evaluation of Treatment (Days Post- Lifespan combinatorial effects Inoculation) (ILS)§ 1 MOROO208 3281 25_5 n.a.

Bendamustine 13 b n.a. 2 24 -5.88 mg/kg Bendamustine 16 n.a. 3 24c -5.88 mg/kg ustine 26 n.a. mg/kg n.a. n.a.

Bendamustine/ Synergy/Potentiation* MOROO208 13/3 40d 56.86 mg/kg Bendamustine/ Synergy/Potentiation** 6 MOROO208 16/3 40d 56.86 mg/kg Vehicle/ Ref_mAb 3 25.5 mg/kg n.a. n'a' a icantly different to Vehicle control/ Ref_mAb_33 (Group 10) (p<0.001), Bendamustine at 13 mg/kg (Group 2) (p<0.001), Bendamustine/ MOR00208 at 13/3 mg/kg (Group 5) (p<0.05) and Bendamustine/ MOR00208 at 16/3 mg/kg (Group 6) (p<0.001). b significantly different to Vehicle control/ Ref_mAb_33 (Group 10) 5) and Bendamustine/ 08 at 13/3 mg/kg (Group 5) (p<0.001). c significantly different to Bendamustine/ MOR00208 at 16/3 mg/kg (Group 6) (p<0.001). g significantly different to Vehicle Control/ Ref_mAb_33 (Group 10) (p<0.001). § vs. vehicle control/ b_33 :Synergy/Potentiation vs. the respective monotherapy groups as lLSCombo (56.86%) > lLSMOR00208 3mg/kg + lLSBendamustine 13mg/kg (25.5% + )% = 19.62%) : Synergy/Potentiation vs. the respective monotherapy groups as bo (56.86%) > lLSMOR00208 3mg/kg + lLSBendamustine 16mg/kg (25.5% + (-5.88)% = 19.62%).

Median % Increased Lifespan (lLS) is calculated as follows: Mean % Increase in an = (SurvivalTreatmem-Mean Survival Commo/ Mean Survival CoerNOO.

Survival times are measured in days post-inoculation.

Classification of Combinatorial Effects The classification of the MOR000208/Bendamustine combination y (combo) effect was evaluated by comparing the lLS of the combination with the added lLS of the respective monotherapies: Synergy/Potentiation*: lLSCombo > lLSMOR00208 3mg/kg + lLSBendamustine. Synergistic effects are classified as potentiation if at least one of the monotherapies has no effect. Additivity: lLSCombo = lLSMOR00208 3mg/kg + lLSBendamustine. Antagonism: lLSCombo < 00208 3mg/kg + lLSBendamustine.

In addition to an analysis of the data for purposes of identifying synergy, the following tical is was also completed. Statistical analyses were carried out using the median . Any animal that died unexpectedly or was culled prior to Day 17 of the study in the Test e treatment groups was excluded from survival analysis calculation. The death/ culling of these animals was attributed to compound toxicity rather than disease progression as they occurred well in advance of the first deaths in the Vehicle Control animals. A survival curve was created using the product limit of Kaplan and Meier, and survival curves compared using the log-rank (Mantel-Cox) test. Where significant differences were found, All Pairwise Multiple Comparison (Holm-Sidak Test) was performed. ison was done between all groups. In addition the comparison of the ing groups were summarised in separate figures for each test article: Vehicle Control/ Ref_mAb (group 10) against ustine groups (Groups 2, 3 and 4) and Vehicle Control / Ref_mAb (group ) against ation groups (Groups 5 and 6) or respective MOR00208 monotherapy group (group 1). A p value of less than 0.05 was considered icant. Results are shown in Tables 8-10.

Table 8: Vehicle Control, MOR00208 and Bendamustine erapy: Log—rank (Mantel—Cox) Test: There is a significant difference (p<0.001).

All Pairwise Multiple Comparison Procedure (Holm—Sidak method): GFOHP Treatment Group 1 Group 2 Group 3 1 0 \R/ehicle Control/ * * *Yes ef_mAb (3 mg/kg) *Yes No 1 MOR00208 (3 mg/kg) ***Yes ***Yes 2 Bendamustine (13 mg/kg) No 3 Bendamustine (16 mg/kg) >“”kYes: There is a statistically significant difference (p<0.001). >kYes: There is a tically significant difference (p<0.05).

No: There is no statistically significant ence (p20.05).

Table 9: Vehicle Control, MOR00208/ Bendamustine Combination —Therapy and respective Monotherapy: Log—rank (Mantel—Cox) Test: There is a icant difference (p<0.001).

All Pairwise Multiple Comparison Procedure (Holm—Sidak method): GI'OUP Treatment Group 1 Group 5 Group 2 Vehicle Control/ Ref_mAb (3 nag/kg) ***Yes ***Yes *Yes l MOR00208 (3 mg/kg) *Yes ***Yes 08/ Bendamustine * * *Yes (3/13 mg/kg) 2 ustine (13 mg/kg) ***Yes: There is a statistically significant difference (p<0.001).

*Yes: There is a statistically significant ence (p<0.05).

Table 10: Vehicle Control, MOR00208/ Bendamustine Combination —Therapy and respective Monotherapy: Log-rank l-Cox) Test: There is a significant difference (p<0.001).

All Pairwise Multiple Comparison Procedure (Holm-Sidak method): Group Treatment Group 1 Group 6 Grgup Vehicle Control/ Ref_mAb (3 nag/kg) ***Yes ***Yes No 1 MOR00208 (3 mg/kg) ***Yes ***Yes MOR00208/ ustine (3/16 mg/kg) ***Yes 3 Bendamustine (16 mg/kg) >“""“Yes: There is a statistically significant difference (p<0.001).

No: There is no tically significant difference (p20.05).

Results As shown in Tables 7-10 and Figure 6, the combination of MOR00208 and bendamustine behaved synergistically and was statistically significant in the Non-hodgkin RAMOS opic tumor survival model as compared to MOR00208 and bendamustine alone.

It is to be understood that the description, specific examples and data, while indicating exemplary ments, are given by way of illustration and are not intended to limit the present invention. Various changes and modifications within the present invention will become apparent to the skilled artisan from the discussion, disclosure and data contained herein, and thus are considered part of the invention.

Claims (19)

1. Use of a synergistic combination of: an antibody specific for CD19; and 5 bendamustine, for the manufacture of a medicament for the ent of non-Hodgkin’s lymphoma, chronic lymphocytic leukemia and/or acute lymphoblastic leukemia, wherein the antibody comprises an HCDR1 region of sequence SYVMH (SEQ ID NO: 1), an HCDR2 region of sequence NPYNDG (SEQ ID NO: 2), an HCDR3 region 10 of sequence GTYYYGTRVFDY (SEQ ID NO: 3), an LCDR1 region of sequence RSSKSLQNVNGNTYLY (SEQ ID NO: 4), an LCDR2 region of sequence RMSNLNS (SEQ ID NO: 5), and an LCDR3 region of sequence MQHLEYPIT (SEQ ID NO: 6).

2. The use ing to claim 1, for the treatment of non-Hodgkin’s ma.

3. The use according to claim 1, for the treatment of chronic lymphocytic leukemia.

4. The use ing to claim 1, for the ent of acute lymphoblastic leukemia. 20

5. The use according to any one of the ing claims, wherein the antibody comprises a variable heavy chain of the sequence EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDG TKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQG TLVTVSS (SEQ ID NO: 10) and a variable light chain of the sequence 25 DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMS NLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO: 11).

6. The use ing to any one of the preceding claims, wherein the antibody 30 comprises a heavy chain constant domain of the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQ 35 VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12).

7. The use according to any one of the preceding claims, wherein the antibody comprises a light chain constant domain of the sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT 5 EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 13).

8. The use according to any one of the preceding claims, wherein the medicament is formulated such that the antibody specific for CD19 and the ustine are 10 administered separately.

9. The use according to any one of the ing claims, wherein the medicament is formulated such that the bendamustine is administered prior to administration of the antibody specific for CD19.

10. The use according to any one of the preceding claims, wherein the medicament mediates g of MEC-1 cells by ADCC in the presence of isolated human PBMCs with an at least ld better efficacy than bendamustine alone. 20

11. The use according to any one claims 1, 2 or 5 to 10, n the non-Hodgkin’s lymphoma is selected from the group consisting of follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue lymphoma, al zone lymphoma, diffuse large B cell lymphoma, Burkitt's lymphoma, and mantle cell lymphoma.

12. The use according to claim 11, wherein the dgkin’s lymphoma is follicular lymphoma.

13. The use according to claim 11, wherein the non-Hodgkin’s lymphoma is small 30 lymphocytic lymphoma.

14. The use according to claim 11, wherein the non-Hodgkin’s ma is mucosa-associated lymphoid tissue lymphoma. 35

15. The use according to claim 11, wherein the non-Hodgkin’s lymphoma is marginal zone lymphoma.

16. The use according to claim 11, wherein the non-Hodgkin’s lymphoma is diffuse large B cell lymphoma. 5

17. The use ing to claim 11, wherein the non-Hodgkin’s lymphoma is Burkitt's lymphoma.

18. The use according to claim 11, wherein the non-Hodgkin’s lymphoma is mantle cell ma.

19. The use according to claim 1, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.