NZ626680B2 - Uses of immunoconjugates targeting cd138 - Google Patents

Abstract

Disclosed is the use of an immunoconjugate in the manufacture of a medicament for treating a disease associated with target cells expressing CD138 in a human subject, the immunoconjugate comprising at least one engineered targeting antibody targeting CD138 expressing cells, and at least one effector molecule, wherein said engineered targeting antibody is functionally attached to said effector molecule to form said immunoconjugate, wherein at least a part of the engineered targeting antibody confers IgG4 isotype properties, wherein the disease associated with target cells expressing CD138 is a plasmaproliferative disorder or a solid tumour, wherein said treating comprises administering the immunoconjugate in a multiple dose regimen comprising at least two doses, wherein the aggregate dose to be administered within an active treatment cycle is an aggregate maximum tolerable dose (AMTD) or a fraction of the AMTD and wherein said AMTD or said fraction exceeds the dose resulting in dose limiting toxicity (DL T) when the immunoconjugate is administered as a single dose, including as part of a multiple single dose regimen or exceeds the maximum tolerable dose (MTD) when the immunoconjugate is administered as a single dose, including as part of a multiple single dose regimen within said active treatment cycle. molecule, wherein said engineered targeting antibody is functionally attached to said effector molecule to form said immunoconjugate, wherein at least a part of the engineered targeting antibody confers IgG4 isotype properties, wherein the disease associated with target cells expressing CD138 is a plasmaproliferative disorder or a solid tumour, wherein said treating comprises administering the immunoconjugate in a multiple dose regimen comprising at least two doses, wherein the aggregate dose to be administered within an active treatment cycle is an aggregate maximum tolerable dose (AMTD) or a fraction of the AMTD and wherein said AMTD or said fraction exceeds the dose resulting in dose limiting toxicity (DL T) when the immunoconjugate is administered as a single dose, including as part of a multiple single dose regimen or exceeds the maximum tolerable dose (MTD) when the immunoconjugate is administered as a single dose, including as part of a multiple single dose regimen within said active treatment cycle.

Description

USES OF IMMUNOCONJUGATES TARGETING CD133 FIELD OF THE INVENTION The present invention relates to methods and treatment regimens, in particular for human subjects, which include the stration of immunoconjugates that are designed to target cells that express CD138. The present ion is also directed at anticancer combinations, pharmaceutical itions comprising the same, and uses thereof in the treatment of cancers that have target cells that express CD138. The present invention is in particular ed at anticancer combinations that show y or unexpected additive effects in the treatment relative to treatments involving less than all of the components of the combination.

BACKGROUND CD138, which acts as a receptor for the ellular matrix, is overexpressed on multiple myeloma (MM) cells and has been shown to influence MM cell development and/or proliferation. CD138 is also expressed on cells of ovarian carcinoma, cervical cancer (Numa et al., 2002), endometrial cancer (Choi et al., 2007), kidney carcinoma, gall bladder, transitional cell bladder carcinoma, gastric cancer (Wiksten et al. 2008), prostate adenocarcinoma eger et al., 2003), y carcinoma (Loussouarn et al., 2008), non small cell lung carcinoma (Shah et al., 2004), squamous cell lung carcinoma (Toyoshima et al., 2001), colon carcinoma cells and cells of Hodgkin‘s and non—Hodgkin’s lymphomas, colorectal oma (Hashimoto et al., 2008), hepato- carcinoma (Li et al., 2005), chronic lymphocytic leukemia (CLL), pancreatic (Conejo et al., 2000), and head and neck oma (Anttonen et al., 1999) to name just a few.

The publications and other materials, ing patents, used herein to illustrate the invention and, in particular, to provide additional details respecting the practice are incorporated herein by reference. For convenience, the publications are referenced in the following text by author and date and/or are listed alphabetically by author in the appended bibliography. e et al. (2004) reported excellent binding of the murine lgG1 antibody B- B4 to the CD138 antigen expressed on the surface of MM cells. Tassone also reported high cytotoxic activity of the immunoconjugate M1, which comprises the maytansinoid DM1 as an effector molecule. against multiple myeloma cells (see also US Patent Publ. 20070183971). 2012/074867 Ikeda et al. (2008 and 2009) reported promising in vitro results and results in aft models with the immunoconjugate BT062, which is based on 8-84.

While Tassone et al. and lkeda et al. represent contributions to providing an effective ent of MM and a ition of matter that may be employed in such a treatment, there remain a number of needs in the art.

While the use of conjugates, in particular those which have highly toxic effector molecules which are functionally attached to a targeting agent that binds to, e.g., antigens that are not only expressed on target cells, such as tumor cells, but also on non-target cells which perform vital functions in the organism, have been shown to be effective in destroying the target cells, many failed due to their toxicity towards non- target cells. in fact. many immunoconjugates have to be discontinued during clinical trials because a balance between effectiveness and toxicity (therapeutic ) could not be found: at concentrations at which the immunoconjugate can confer ts in terms of combating disease, its toxicity becomes unacceptable. Thus, especially with highly toxic effector molecules, the question often is not only whether the targeting agent of the immunoconjugate can in fact, bring the effector to the target and allow the effector to be released at the target, but also if, on its way to the target cells, the same immunoconjugate will y or attack an unacceptable number of cells or organs that are pivotal to the survival of the organism.

US Patent Publication 20110123554 discloses methods and treatment regimens that include the stration of conjugates targeting CD138 to combat es, in particular in tolerable amounts. However, while these results showed that the immunoconjugate could be effective, while being tolerable, there is a need for further improved treatment regimens.

There remains in particular a need to provide suitable treatment regimens for es associated with CD138 expression, including plasmaproliferative disorders associated with CD138 expression, such as MM. There, more in particular, remains a need for treatment regimens that ensure that toxicities towards non tumor cells, which also express CD138 are kept to a clinically acceptable level, either by employing only certain tolerable amounts of immunoconiugate at levels that balance toxicities with effectiveness to combat es and/or by combining the immunoconjugate with cytotoxic agents known to be effective against the disorder in question. There is also a need for treatment regimens that reduce the need for medications that are used to alleviate other symptoms of the disease and for maintenance therapy to maintain a patients health in a disease-free or limited-disease state after a certain grade of disease control was achieved with the most recent prior treatment.

This ion fulfills, in certain ments, one or more of these needs as well as other needs in the art which will become more apparent to the skilled artisan once given the following disclosure.

SUMMARY OF THE INVENTION In one aspect, the present invention provides use of an immunoconjugate in the manufacture of a medicament for ng a disease associated with target cells expressing CD138 in a human subject, the immunoconjugate comprising at least one engineered targeting antibody targeting CD138 sing cells, and at least one effector molecule, wherein said engineered targeting antibody is functionally attached to said effector molecule to form said immunoconjugate, wherein at least a part of the engineered targeting antibody confers lgG4 isotype properties, wherein the disease associated with target cells expressing CD138 is a plasmaproliferative disorder or a solid tumor, n said treating ses administering the immunoconjugate in a multiple dose regimen comprising at least two doses, wherein the aggregate dose to be administered within an active ent cycle is an aggregate maximum ble dose (AMTD) or a fraction of the AMTD and n said AMTD or said fraction exceeds the dose resulting in dose limiting toxicity (DLT) when the immunoconjugate is administered as a single dose, including as part of a multiple single dose regimen or exceeds the maximum tolerable dose (MTD) when the immunoconjugate is administered as a single dose, including as part of a multiple single dose n within said active treatment cycle. ln'a second aspect, the t invention provides use of an immunoconjugate in the manufacture of a medicament for treating a disease ated with target cells expressing CD138 in a human subject, wherein the conjugate comprises at least one engineered targeting antibody targeting CD138 expressing cells, and at least one effector molecule, wherein said engineered targeting antibody is functionally attached to said or molecule to form said immunoconjugate, wherein at least a part of the engineered targeting antibody confers lgG4 isotype properties, wherein the engineered targeting antibody comprises: (i) a heavy chain having at least 85% sequence identity with SEQ ID NO: 1, wherein the heavy chain comprises a variable region comprising amino acid residues 31 to (CDR1), 51 to 68 (CDR2) and 99 to 111 (CDRB) of SEQ lD NO: 1, and (ii) a light chain having at least 85% sequence identity with SEQ ID NO: 2, wherein the light chain comprises a variable region comprising amino acid residues 24 to 34 (CDR1), 50 to 56 (CDR2) and 89 _ 3 - (followed by page 3a) to 97 (CDR3) of SEQ lD NO: 2; wherein the effector molecule is at least one maytansinoid, wherein the disease associated with target cells expressing CD138 is a proliferative disorder or a solid tumor, wherein said treating comprises administering the medicament in an active treatment cycle of 21 days, at least three times within said 21 days, wherein said active treatment cycle comprises administering at least once a week, wherein the dose of the immunoconjugate to be administered at least once a week is between 80 mg/m2 and 120 mg/mz. in another aspect, the present invention es use of an conjugate in the manufacture of a medicament for treating a e associated with target cells expressing CD138 in a human subject, wherein the immunoconjugate comprises at least one engineered targeting antibody ing CD138 expressing cells, and at least one effector molecule, wherein said engineered targeting antibody is functionally attached to said effector molecule to form said immunoconjugate, wherein the engineered targeting dy comprises a heavy chain having SEQ lD NO: 1 and a light chain having SEQ ID NO: 2, wherein the effector molecule is DM4, wherein the disease associated with target cells expressing CD138 is multiple myeloma, wherein said treating comprises administering the medicament in an active treatment cycle of 21 days, at least three times within said 21 days, wherein said active treatment cycle comprises administering at least once a week, wherein the dose of the immunoconjugate to be administered at least once a week is between 80 mg/m2 and 120 mg/mz.

The invention is directed at a method for treating a disease associated with target cells expressing CD138, sing: stering to a patient in need thereof a pharmaceutical ition an immunoconjugate and a ceutically able carrier at least once a week for at least three weeks, wherein each three week period is optionally ed by a resting period, wherein the immunoconjugate comprises at least one targeting agent targeting CD138 expressing cells, and at least one effector molecule, wherein said targeting agent is functionally attached to said effector molecule to form said immunoconjugate, and n the dose of the immunoconjugate administered at least once a week is about 20mg/m2 to about 280 mg/mz, e.g. once a week at a dose from about 4Omg/m2to about 140mg/m2, and the pharmaceutical composition is administered for at least three weeks alone or in combination with a cytotoxic agent. (followed by page 3b) The invention is also directed at a method for ng a disease associated with target cells expressing CD138 comprising: administering to a subject, in ular a human subject, in need thereof an immunoconjugate sing at least one engineered targeting antibody targeting CD138 expressing cells, and at least one effector molecule, wherein said engineered targeting antibody is onally attached to said effector molecule to form said immunoconjugate, wherein preferably at least a part of the engineered targeting antibody confers lgG4 isotype properties, wherein [FOLLOWED BY PAGE 4] -3b- the immunoconjugate is stered in a multiple dose regimen comprising at least two doses, n the aggregate dose administered within an active treatment cycle, such as an active treatment cycle comprising 21 days, is an aggregate maximum tolerable dose (AMTD) or a fraction of the AMTD and wherein said AMTD andlor said fraction exceeds the ddse resulting in dose limiting ty (DLT) when the immunoconjugate is administered once, preferably on day 1, within said active treatment cycle and/or exceeds the maximum tolerable close (MTD) when the immunoconjugate is stered as a single dose, including a repeated single dose.

The AMTD may exceed the dose of said DLT by at least 20% and said MTD by at least %. The AMTD may be at least 240mg/m2, preferably 300 mg/mz, more preferably 360 mg/m2 or 420 mg/mp‘and the dose resulting in said DLT may be 180 mglm2 or 200 mg/mz. The AMTD may be at least 240mg/m2, preferably 300 mg/mz, more preferably 360 mg/m2 or 420 mg/m2 and said MTD may be at least 160 mg/m2 or at least 180 mg/mz.

The immunoconjugate may be administered at least three times within 21 days, preferably in equal doses.

Said multiple dose regimen may last 3 weeks and may be followed by a resting period.

During this resting period progression free survival or stable disease may be maintained. A level of conjugate in a body fluid of a subject, during said resting period may be at least or up to 0.5 pg/ml. 1 pg/ ml or 2pg/ml, 3 pg/ml, 4 pg/ml, 5 pg/ ml or 6ug/ml.

The "receptor occupancy" of target cells expressing CD138, in particular isolated target cells expressing CD138, ably in target cells isolated from non-solid tumors, such as myleloma cells in bone marrow aspirates, e.g., within 24 hours, preferably within eighteen, twelf, eight or four hours after completition of administration of an immunconjugate according to the present invention is, in one embodiment, more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90% or more than 95%. The "receptor occupancy" of target cells expressing CD138 o a subsequent administration or, respectively, more than 48 hours, more than 72 hours, more than 96 hours (4 days), more than 120 hours (5 days) or more than 144 hours (6 days) after completition of administration is less than 70%, less than 60%, less then 55%, less than 50%, less than 45% or less than 40%.

In one embodiment, the difference in "receptor occupancy" of target cells expressing CD138 twentyfour, eighteen, twelf, eight or four hours after completition of adminstration of the immunoconjugate and the "receptor occupancy" of said target cells more than 48 hours, more than 72 hours, more than 96 hours (4 days), more than 120 hours (5 days) or more than 144 hours (6 days) after completition of stration, is at least 5%, at least, 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least %, at least 40%, at least 45% or at least 50%, preferably between 10% and 50% or % and 40%.

In a further ment, the "receptor occupancy" of target cells expressing 001 38 24 hours, preferably within eighteen, twelf, eight orfour hours after completition of adminstration of the conjugate is high, that is, more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90% or more than 95%, even when the immunoconjugate is administered at realitvely low trations, e.g., at concentrations that constitute less than 50%, less than 60%, less than 70%, less than 80%, but generally more than 10%, more than 20% or more an 30% of the determined DLT of the immunoconjugate when administered once in a 21 day treatment cycle. in yet a further embodiment, the "receptor occupancy" of target cells sing CD138 prior to a subsequent administration or, respectively, more than 48 hours, more than 72 hours, more than 96 hours (4 days), more than 120 hours (5 days) or more than 144 hours (6 days) after completition of administration is less than 70%, less than 60%, less then 55%, less than 50%, less than 45% or less than 40%, even when the immunoconjugate is administered at realitvely high concentrations, e.g., at trations that constitute more than 50%, more than 60%, more than 70%, more than 80% of the determined DLT of the immunoconjugate when administered once in a 21 day treatment cycle.

The invention is also ed at administering a total amount of maytansinoid, in particular 0M4 to a patient within 21 days of more than 2mg/m2, more than 3mg/m2, more than 4mg/m2, more than 5mg/m2, more than 6mg/m2, more than 7mg/m2, more than 8mg/m2 , more than 9mg/m2 or more than 10 mg/m2 preferably in accordance to any one of the methods referred to herein.

The administering may be performed at least once a week, at preferably equal doses for at least three weeks followed preferably by a g period of, e.g., one week.

"Resting period" means in this context a period after a point in time, at which, according to the treatment schedule established for a patient, the next dose should, but was not, administered. For example, in an stration scheme that involves weekly administrations on days 1, 8 and 15, the resting period defines the time after day 22, when there was no administration. in this example, this g period result in a treatment free interval of two weeks. The at least three weeks followed by the resting period may define a treatment cycle of at least 28 days, and wherein, after two or more treatment cycles, at least stable disease may be achieved. The immunoconjugate may, e.g., be administered every 3rd day, every 4‘" day, every S'h day or every 6‘" day during said three weeks period. At least stable e may be maintained during three, four, five, six, seven treatment cycles. After reaching at least stable disease, the immunoconjugate may be administered as a maintenance therapy less than three times or less than twice within said 21 days, preferably once in said 21 days, preferably as a repeated single dose of n 60 mg/m2 and 200 mg/mz, including about 70 mg/m2 about 80 , about 90 mg/m2, about 100 mg/m2 , about 110 mg/mz, about 120 mg/mz. about 130 mg/mz, about 140 mg/mz, 150 mg/m2 about 160 mg/mz, about 170 mg/m2, about 180 mglmz, about 190 mg/m2 and about 200 mg/mz. At least progression free al, stable disease and or a minor response may be obtained for more than 3 months during a maintenance therapy. stration of said immunoconjugate as a repeated multiple dose in treatment cycles lasting at least 21 days may result, after the last administration in each cycle, in an aggregate effective amount and a first level of the immunoconjugate in a body fluid of the subject and wherein, when an amount equivalent to said aggregate effective amount is administered as a single dose or repeated single dose in said treatment cycle, it may result in a second level of the immunoconiugate in a body fluid of said subject, wherein the first level may be equal or below the second level, eg more than %, more than 20 % or more than 30% below the second level.

The treatment cycle may last 21 days and/or the repeated multiple dose may consist of 3 equal, preferably equidistant doses, more ably administered on days 1, 8 and . The aggregate effective amount may be more than/ up to 200mg/m2, about 220mgfm2, about 240mg/m2, about 260mg/m2, about 280 mg/mz, about 300 mg/mz, about 360 mg/m2 or about 420 mg/mz.

The immunoconjugate or pharmaceutical composition may be administered for at least two 21 day treatments cycles with a one week resting period between each treatment cycle. An administration may be followed, after at least two 21 day treatment , each optionally followed by a resting period and/or by a further stration of the conjugate or pharmaceutical composition as a maintenance therapy. The maintenance therapy may comprise administering the immunoconjugate or a ceutical ition comprising the same (i) once every three to six weeks or (ii) at repeated multiple doses, wherein each individual dose of immunoconjugate is about 10mg/m2, about 20mg/m2. about 30mg/m2, about 4Omg/m2, about 50mg/m2, about 60mg/m2, 70 mg/m2, about 80mg/m2, about 90mg/m2, about m2 , about 110mg/m2 or about m2 lower than the individual dose of a primary therapy and/or wherein individual closes may be administered in intervals exceeding the interval of the individual doses, e.g., by 1, 2, 3, 4, 5, 6, 7 days. Any administration of said immunoconjugate as a multiple dose regime may result, 0-2 hours after completion of administration, in a mean plasma level of at least 7 ug/ml , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,40, 50, 60, 70, 80, 90 or 100 ug/ml.

The methods of the invention may further include, determining 0—4 hours, including at about 1, 2,3 or 4 hours, following an completion of administering said immunoconjugate or a pharmaceutical composition comprising the same, a reference level of an said immunoconjugate or of an efficacy blood parameter in a body fluid of a patient and determining in a subsequent stration of said immunoconjugate, at 0-4 hours following an completion of said subsequent administration, a uent level of said immunoconjugate or efficacy blood parameter, wherein, when the reference level is higher than the subsequent level, the aggregate dose in a ent cycle ing said subsequent administration may be sed by 5» 100%, including 10-50% or 20-30% and/or when the reference level is lower than the subsequent level, the aggregate dose in a treatment cycle following said subsequent administration may be lowered by 5- 100%, including 10-50% or 20-30%.

The methods of the present invention may also further comprise ining, 02 hours following an completion of administering an individual dose of said immunoconjugate or a pharmaceutical composition sing the same, a level of said immunoconjugate in a body fluid, wherein, if said level is below 7 ug/ml , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 pg/ml, the individual dose may be increased in the next treatment cycle by at least 10mg/m2, 20mg/m2, about 30mg/m2, about 4Omg/m2, about 50mg/m2, about 60mg/m2, 70 mg/m2, about 80mg/m2, about 2 or about 100mg/m2.

The methods of the t invention may also further comprise determining, 0-2 hours following an completion of administering an individual dose of said immunoconjugate or a pharmaceutical composition comprising the same, a level of an said immunoconjugate in a body fluid, wherein, it said level is above 50 pg/ml, 60, 70, 80 or 100 ugfml, the individual dose may be decreased in the next treatment cycle by at least 1Omg/m2, 20mg/m2, about 30mg/m2, about 4Omg/m2, about 50mg/m2, about 60mg/m2, 70 mgz’mz, about 80mg/m2, about /m2 , about 100mg/m2 , about 110mg/m2 or about 120mg/m2.

In any of the methods of the present invention at least one cytotoxic agent, including two or three, may be administered at least once a week or once in a treatment cycle.

Said cytotoxic agent may be lenalidomide and/or dexamethasone. The said subject to which the drug combination is administered may or may not have previously been d to an immunoconjugate sing an dy targeting CD138 expressing cells, to lenalidomide and/or to dexamethasone. The subject may have responded to an exposure to an immunoconjugate comprising an antibody targeting CD138 expressing cells, lenalidomide and/or dexamethasone. Target cells expressing CD138 may be refractory to exposure to an immunoconjugate sing an antibody targeting CD138 expressing cells, lenalidomide and/or thasone. The subject may have relapsed after said previous exposure. Lenalidomide may be administered at a dose of 5 to 35 mg, preferably at about 25 mg, or at a dose of less than 25, 20, 15 or 10 mg, more preferably orally once a day in a treatment cycle of, e.g., 21 or 28 days and/or dexamethasone may be administered at a close of 20 to 50 mg, preferably at about 40 mg, or at a dose of less than 40 or 30 mg, e.g., orally once a day in a ent cycle of, e.g., of 21 or 28 days or, e.g., on days 1-4, 9-12, 17-20 within 28 days or e.g., on day1,8, 15 and 22.

The t may suffer from a solid tumor comprising target cells which express CD138 and said solid tumor may be refractory to cancer hormone y or chemotherapy or the subject may have ed after hormone therapy or chemotherapy, wherein said administration may result in at least tumor growth delay or tumor . Said immunoconjugate may be administered in a repeated multiple dose regime with individual doses of 20mg/m2 to 160mg/m2. The solid tumor may be estrogen receptor negative and/or progesterone or negative and/or Her2/neu negative, ing triple negative with all of three, e.g., triple—negative breast cancer.

An administration of said immunoconjugate or pharmaceutical composition may also be preceded by an administration of different targeting agent, e.g., an unconjugated antibody targeting CD138 sing cells. wherein said immunoconjugate is administered 1-6, preferably 2-4, hours after completion of the administration of said unconjugated antibody. The unconjugated antibody may be administered at a dose corresponding to a level of 10 to 30 ug/ml immunoconjugate in a body fluid of the t, in particular a plasma level of the subject. This dose administered may correspond to about a difference between a theoretical and actual level of said immunoconjugate in a body fluid, 0-2 hours after tion of an administration of said immunoconjugate to said subject. The targeting agent may be administered at a dose of 10 to 40 mg/m2, preferably 20-30 mg/mz. As a result, the immunoconjugate may be administered at an individual dose that is up to 10mg/m2, up to 20mg/m2 or up to 30mg/m2 lower than the dose administered without said administration of said unconjugated antibody.

The features for the methods of treatment of the invention described herein also apply in the context of a medical use, for example as in the following: a) Use of an immunoconjugate for the manufacture of a medicament for treating a disease ated with target cells expressing CD138 in a subject, in particular a human t, in need thereof (or an conjugate for use in treating a disease associated with target cells expressing C0138 a subject, in particular a human subject, in need thereof), the immunoconjugate comprising: at least one engineered targeting antibody targeting CD138 expressing cells, and at least one effector molecule, wherein said engineered targeting antibody is functionally attached to said or molecule to form said immunoconjugate, wherein preferably at least a part of the engineered targeting antibody confers lgG4 isotype properties, wherein the immunoconjugate is to be administered in a multiple dose regimen comprising at least two doses, wherein the aggregate dose to be administered within an active treatment cycle is an ate m tolerable dose (AMTD) or a fraction of the AMTD and wherein said AMTD and/or said fraction exceeds the dose resulting in dose limiting toxicity (DLT) when the immunoconjugate is administered as a single dose, including as part of a multipie single dose regimen and/or exceeds the maximum tolerable dose (MTD) when the immunoconiugate is administered as a single dose, ing as part of a multiple single dose n within said active treatment cycle; b) Use of a pharmaceutical composition comprising an immunoconjugate and a pharmaceutically acceptable carrier in the manufacture of a medicament for ng a disease associated with target cells expressing 00138 in a patient in need thereof (or a pharmaceutical composition comprising an immunoconjugate and a pharmaceutically acceptable carrier for use in treating a disease associated with target cells expressing CD138 in a patient in need thereof), the immunoconjugate comprising at least one targeting agent targeting CD138 expressing cells, and at least one effector molecule, wherein said targeting agent is functionally attached to said effector molecule to form said conjugate, wherein the pharmaceutical composition is to be administered in an active treatment cycle which is optionally ed by a g , and wherein the dose of the immunoconjugate to be administered at least once a week is about 20mg/m2, about 30mg/m2, about 4Omglm2, about 50mg/m2, about 60mg/m2. 70 mg/m2, about 80mg/m2, about 90mg/m2, about 100mg/m2, about m2, about 120mglm2, about 130mg/m2, about 140mg/m2. about ’l50mg/m2 or about 160mg/m2 , about 170 mg/mz, about 180 mg/mz, about 190 mg/mz, about 200 mg/mz, about 210 mg/mz, about 220 mg/mz, about 230 mg/mz, about 240 mg/mz, about 250 mg/mz, about 260 mg/m2 , about 270 mg/m2 or about 280 mg/m2 and the pharmaceutical composition is to be administered for at least three weeks alone or in combination with a cytotoxic agent .10- The invention is also directed at a kit comprising an antibody against the immunooonjugate and, in a separate container, instructions how to determine, a level of said immunoconjugate in a body fluid obtained from said t by addition of said antibody to said body fluid. The kit may further comprise an conjugate comprising at least one engineered ing antibody targeting CD138 expressing cells, and at least one effector molecule, wherein said engineered targeting antibody is functionally attached to said effector molecule to form said immunoconjugate.

The engineered targeting antibody may comprise an antigen binding region (ABR) against CD138, and a r dy region, wherein at least part of said further antibody region is of a human antibody and confers said lgG4 isotype properties.

The disease may be multiple myeloma, in particular relapsed or refractory multiple myeloma. Refractory multiple myeloma includes "primary tory myeloma" and "relapsed and tory myeloma." Said disease sing CD138 on target cells may be also selected from the group consisting of renal cell oma, endometrial cancer, cervical cancer, te adenocarcinoma, pancreatic carcinoma, gastric cancer, bladder cancer, mammary carcinoma, —carcinoma, colorectal carcinoma, colon carcinoma, squamous cell oma, lung cancer in particular squamous cell lung carcinoma, non Hodgkin lymphoma, thymus, uterus, urinary or ovarian carcinoma.

In preferred embodiments, the immunoconjugate homogenously targets CD138 expressing target cells. in certain embodiments, the engineered targeting antibody of the present invention may (i) consist essentially of antigen binding region (ABR) against CD138 of a non- human antibody, or (ii) comprise an antigen binding region (ABR) against CD138, wherein said antigen binding region is of a non—human antibody, and a r antibody region, wherein at least part of said further antibody region is of human antibody.

The ABR may comprise: (a) heavy chain variable region CDR3 comprising amino acid residues 99 to 111 of SEQ lD NO: 1, and (b) light chain variable region CDRS sing amino acid residues 89 to 97 of SEQ ID NO: 2, respectively.

The AER may further comprise: (a) heavy chain variable region CDR1 and CDR2 comprising amino acid residues 31 to and 51 to 68 ofSEQ lD NO: 1, and/or (b) light chain variable region CDR1 and CDR 2 comprising amino acid es 24 to 34 and 50 to 56 of SEQ lD NO: 2, respectively.

The further antibody region may comprise: (a) amino acid residues 123 to 448 of SEQ ID NO: 1, and/or (b) amino acid residues 108 to 214 of SEQ lD NO: 2, respectively and mutations thereof that (i) maintain or lower the antibody-dependent cytotoxicity and/or complement— dependent cytotoxicity of the engineered targeting dy and/or (ii) stabilize the engineered targeting antibody.

The antibody may comprise a light chain having at least about 70 "/0, more preferably 80%, 85% or 90%, sequence identity with SEQ lD No: 2 and a heavy chain having at least about 70%, more ably 80%, 85% or 90%, sequence identity with SEQ lD No: 1, and comprising the antigen binding regions specified above.

The effector molecule may be attached to said engineered targeting antibody via a linker. The linker may comprise a disulfide bond. The effector molecule (e.g., DM4) may provide sterical nce between the targeting antibody and the effector le. The effector molecule may be at least one maytansinoid (e.g., DM’l, 0M3, or DM4), taxane, another microtubule ting agent or DNA targeting agent such as 001065, or an analog thereof.

The immunoconjugate may bind CD138 with a targeting variation of less than 150%, 140%, 130%. 120%, 110%. 100%, 90%, 80%, 70%. 60% or 50%.

The immunoconjugate may, in certain ments of the methods disclosed herein, comprise: a targeting agent targeting CD138 comprising an isolated polypeptide comprising an amino acid sequence of an immunoglobulin heavy chain or part thereof, wherein said immunoglobulin heavy chain or part thereof has at least 70% sequence identity with SEQ lD NO:1. A constant region of said immunoglobulin heavy chain or said part thereof may be an lgGL‘r isotype constant region.

The targeting agent of the immunoconjugate may se a light chain sequence having at least about 70% sequence identity with SEQ lD N02. The ing agent of the immunoconjugate may also comprise a heavy chain sequence having at least about 70% sequence identity with SEQ ID NO:1.

The present invention is also directed at a pharmaceutical composition comprising any of the immunoconjugates ed herein for the inhibition, delay and/or tion of the growth of tumors and/or spread of tumor cells, and one or more ceutically acceptable excipients.

The pharmaceutical composition may include cytotoxic agents as specified herein.

The present ion is also directed at a kit comprising, in separate containers, said pharmaceutical ition in one or more dosage forms and, in a separate container, instructions how to administer the one or more dosage forms to a subject, in particular a human subject in need thereof, e.g., as repeated single dose or other treatment regime discussed herein. in particular, in certain embodiments, the present invention also provides the immunoconjugate described herein for use in treating a disease ated with target cells expressing CD138. wherein the immunoconjugate is to be administered in the schedules and/or at the dosages bed herein. The immunoconjugate for use in this manner can be sed in a pharmaceutical composition. The immunoconjugate or pharmaceutical composition may also be comprised in a kit. where the kit further comprises the cytotoxic agent and/or the unconjugated antibody targeting CD138, also described herein, in separate containers. The immunoccnjugate/ ceutical composition and the cytotoxic agent and/or the unconjugated antibody are to be simultaneously, separately or sequentially administered as described herein. Similarly, the immunoconjugate/pharmaceutical composition, the cytotoxic agent and/or the unconjugated antibody targeting 00138 can be in the form of a combined preparation for simultaneous, separate or tial use in the manner described herein.

In one aspect of the invention the administration of any of the immunoconjugates disclosed herein is to a t or cells of such a t, in particular a human subject, benefiting from such administration. The immunoconjugate can also be used for the manufacture of a medicament for the treatment of such a disorder.

Use of an immunoconjugate for the cture of a medicament for the treatment of a disease in a subject associated with target cells expressing C0138, wherein the immunoconjugate comprises: (i) at least one targeting agent targeting CD138 expressing cells, and (ii) at least one effector molecule, optionally in combination with one or more cytotoxic agents wherein the targeting agent is functionally attached to the or moiecule to form the immunoconjugate, n the subject does not respond (refractory disease), or responds poorly or is relapsed from, to ent with one or more cytotoxic agents including immunomoduiators and/or proteasome inhibitors, and wherein the immunoconjugate is to be administered to the t, preferably intravenously.

A combined preparation of an immunoconjugate and an agent for treating adverse side effects, for simultaneous, separate or sequential use in treating a disease in a t associated with target cells expressing CD138, wherein the immunoconjugate comprises: (i) at least one targeting agent targeting CD138 expressing cells, and (ii) at least one effector molecule, wherein the targeting agent is functionally attached to the or molecule to form the immunoconjugate, wherein the subject does not respond to, responds poorly to or is relapsed from, treatment with one or more xic agents including immunomodulators and/or proteasome tors, and wherein the conjugate is to be administered to the subject, preferably enously, in a pharmacokinetic equivalent of 5mg/m2 to 140mg/m2 of the immunoconjugate when administered alone.

Use of an immunoconjugate and an agent for treating adverse side effects for the manufacture of a combined ation for aneous, separate or sequential use in treating a disease in a subject associated with target cells expressing CD138, wherein the immunoconjugate comprises: (i) at least one targeting agent targeting CD138 expressing cells, and (ii) at least one effector molecule, wherein the ing agent is functionally attached to the effector molecule to form the immunoconjugate, wherein the subject does not respond to, or responds poorly to or is relapsed from, treatment with one or more cytotoxic agents including immunomodulators and/or proteasome inhibitors, and wherein the immunoconjugate is to be administered to the subject, preferably intravenously, in a pharmacokinetic equivalent of 5mg/m2 to 840mg/m2‘0f the conjugate when administered alone.

The invention is also directed at an anticancer combination comprising at least one cytotoxic agent and at least one immunoconjugate comprising a targeting agent targeting CD138 sing cells, and at least one effector molecule, wherein said targeting agent is functionally ed to said effector molecule to form said immunoconjugate, wherein (a) the combination has a synergy ratio of more than 1. more than 1.1, more than 1.2, more than 1.3. more than 1.4, or (b) the ation has a synergy ratio of about 1 and the effector molecule and the cytotoxic agent have overlapping modes of action, and wherein said anticancer combination is a pharmaceutical composition or a kit comprising the at least one cytotoxic agent and the at least one immunoconjugate in separate containers The cytotoxic agent may be a proteasome inhibitor, an immunomodulatory or an anti- angiogenic agent, a DNA alkylating agent, a histone deacetylase, or a mixture of two or more thereof.

The cytotoxic agent may be omib or carfilzomib, thalidomide, lenalidomide or pomalidomide, melphalan or a mixture of two or more thereof.

The effector molecule and the xic agent of the anticancer combination may have pping modes of action and wherein these modes of action e preferably inhibition of microtubule or induction of cell cycle arrest (melphalan, bortezomib and lenalidomide or thalidomide are cytotoxic agents that induce cell cycle arrest).

Alternatively, they may have non-overlapping modes of action. if the anticancer combination is part of a pharmaceutical composition, the pharmaceutical composition may comprise at least one pharmaceuticaliy acceptable excipient.

The anticancer combination may also be part of a kit in which the at least one cytotoxic agent and the at least one immunoconjugate are stored in separate containers.

The invention is also directed at a method for treating a disease associated with target cells expressing C0138, comprising: administering to a patient in need thereof an effective amount of the anticancer combination mentioned herein or an ncer combination comprising at least one cytotoxic agent and at least one immunoconjugate comprising a targeting agent targeting CD138 expressing cells and at least one or molecule, wherein said targeting agent is functionally attached to said effector le to form said immunoconjugate, and wherein the immunoconjugate overcomes a refractory phenotype of a patient against said cytotoxic agent.

The invention is also directed at a method for treating a disease associated with target cells expressing CD138, comprising: administering to a patient in need thereof an effective amount of an anticancer combination discussed herein and wherein the immunoconjugate overcomes a refractory phenotype.

The ion is also directed at a method for treating a non-plasmaproliferative disease associated with target cells sing 00138, comprising: administering to a subject in need thereof or to cells affected by said non- plasmaproliferative e an effective amount of an immunoconjugate comprising at least one targeting agent targeting CD138 expressing cells, and at least one effector molecule, wherein said targeting agent is functionally attached to said effector molecule to form said immunoconjugate, wherein said CD138 is, in said subject, sed on said target cells and on non—target cells at comparable levels or wherein said CD138 is, in said subject, expressed on said target cells at levels below that of said non— target cells expressing CD138.

Said rget cells expressing CD138 may be epithelium cells.

The invention is also directed at a method for ng a non-plasmaproliferative disease associated with target cells expressing CD138, comprising: administering to a subject in need thereof or to cells affected by said non- plasmaproliferative e an effective amount of an immunoconjugate comprising at least one targeting agent targeting C0138 expressing cells, and at least one effector molecule, wherein said targeting agent is onally attached to said effector molecule to form said immunoconjugate, wherein the target cells of said disease shed 00138 over a period of 24 hours, 2, 3, 4, . 6 days or permanently.

Said disease may be mammary carcinoma.

A combined preparation of at least one cytotoxic agent and at least one immunoconjugate, for simultaneous, separate or sequential use in ng in a t a disease associated with target cells expressing 00138, wherein the immunoconjugate comprises: (i) a targeting agent targeting 00138 expressing cells, and (ii) at least one effector molecule, n the targeting agent is functionally attached to the at least one effector molecule to form the immunoconjugate, and wherein the subject has a tory ype. relapsed after treatment or has not one treatment before.

Use of at least one cytotoxic agent and at least one immunoconjugate for the manufacture of a combined preparation for simultaneous, separate or sequential use in treating in a subject a disease associated with target cells expressing C0138, wherein the immunoconjugate comprises: (i) a ing agent targeting CD138 expressing cells and (ii) at least one effector molecule wherein the targeting agent is functionally attached to the at least one effector molecule to form the immunoconjugate, and wherein the subject has a refractory phenotype, relapsed after treatment or has not undergone treatment before. in a preferred embodiment the combination of the at least one cytotoxic agent and at least one immunoconjugate has a y ratio of more than 1, more than 1.1, more than 1.2, more than 1.3 or more than 1.4 . Alternatively, the combination of the at least one cytotoxic agent and the at least one immunoconjugate has a synergy ratio of about 1 and the effector molecule and the cytotoxic agent have overlapping modes of action.

In a red embodiment the combination of at least one cytotoxic agent and at least one immunoconjugate has a higher efficacy compared to each of the agents alone. A higher y is defined by changes in efficacy blood ters, for example M-Protein levels, Free kappa light chain, and other relevant parameters, which positively change relative to each single agent. in particular, the higher efficacy can be defined by eg. % decline in M-Protein level, the extent of the decline in the M-Protein level, or of the duration of the se in M-Protein.

An immunoconjugate for treating a non-plasmaproliferative disease in a subject associated with target cells expressing CD138, wherein the conjugate comprises: (i) at least one targeting agent targeting CD138 expressing cells, and (ii) at least one effector molecule, wherein the targeting agent is functionally attached to the effector molecule to form the immunoconjugate, and wherein in the subject CD138 is expressed on the target cells at levels comparable (equivalent) to or below the levels at which 00138 is expressed on non-target cells.

Use of an immunoconjugate for the cture of a medicament for treating in a subject a asmaproliferative disease associated with target cells expressing CD138, n the immunoconjugate comprises: (i) at least one targeting agent targeting CD138 expressing cells, and (ii) at least one or molecule, wherein the targeting agent is functionally attached to the effector molecule to form the immunoconiugate, and wherein in the subject C01 38 is expressed on the target cells at levels comparable (equivalent) to or below the levels at which CD138 is expressed on non-target cells.

The invention is also directed at a method for treating a asmaproliferative disease associated with target cells expressing CD138, comprising: administering to a subject in need thereof or to cells of said non- plasmaproliferative disease an effective amount of an immunoconjugate comprising at least one ing agent targeting CD138 expressing cells, and at least one effector molecule, wherein said targeting agent is functionally attached to said effector molecule to form said immunoconjugate, wherein immunoconjugate induces at least tumor stasis, preferably remission of a solid tumor.

This remission may be a remission followed by a time interval which is free of re-growth of said tumor (complete remission). This time interval may be more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, half a year or 1 year or more.

The solid tumor may be a pancreatic carcinoma or a mammary carcinoma.

The disease may renal cell carcinoma, endometrial . cervical cancer, prostate adenocarcinoma, pancreatic oma, gastric cancer, bladder cancer, mammary carcinoma, hepato-carcinoma, colorectal oma, colon oma, squamous cell carcinoma, lung cancer in particular squamous cell lung carcinoma, non Hodgkin lymphoma, thymus, uterus, urinary or ovarian carcinoma, both in form of primary tumors as well as metastatic tumors derived from primary tumors.

The solid tumor may be a mammary carcinoma, which are estrogen receptor negative and/or progesterone receptor negative and/or HerZ/neu negative. A solid tumor according to the present invention may also be a mammary carcinoma, which does not or poorly d to taxane therapy or is hormone refractory.

The receptor occupancy at target cells, such as bone marrow cells, may be more than 70%, more than 80%, more than 90% or more than 75%, i, 2, 3, 4, 5, 6, 7, 8, 9, 10, it or 12 hours after completition of an administration of the immunoconjugate.

BRIEF DESCRIPTION OF THE FIGURES provides a schematic entation of nBT062 having effector molecules attached. is a chemical representation of BT062. shows the conversion of ansamitocin P-3 to sinol ochemistry is omitted for simplicity). shows a representative synthesis scheme of DM4.

FIG. Sis a schematic representation of an antibody conjugation (nBTO62 to 0M4). shows an analysis of the binding of nBT062-SPDB-DM4, nBTOGZ-SPP—DMi, nBTOSZ—SMCC—DM‘i and nBT062 antibody to OPM—2 cells. Different concentrations of nBTOSZ and conjugates were given to the cells and mean fluorescence was measured by FACS analysis.

A)-(D) depict in vitro cytotoxicity of nBTOGZ-DMX conjugates towards MOLP-8 (C01 38") and BJAB (CD138) cells. The cells were ed in flat bottom plates and incubated with the indicated concentrations of immunoconjugates for 5 days. WST reagent was added for further 3 hours to assess cell viability. in (D) cytotoxic activity of nBTOGZ-SPDB-DM4 was analyzed in the presence or absence of blocking antibody (1 uM nBT062). shows the complete ion of a xenograft pancreas carcinoma in mice treated with BT062 vs. a control. Complete remission is maintained: in the treatment free observation period, no tumor re-growth was observed. shows the complete remission of a aft mammary carcinoma in mice treated with BT062 vs. a control. Complete remission is maintained, since in the treatment tree observation period, no tumor re-growth was observed.

Fig. 10 shows the complete remission of a xenograft mammary carcinoma in mice treated with 2 mg/kg or 4 mg/kg BT062 (once ) vs. a control or Taxane. At 1 mg/kg BT—O62 once weekly, tumor stasis is achieved. This is defined as the minimal effective dose.

Fig. 11 shows the complete remission of a xenograft primary lung adenocarcinoma in mice treated with 4 mg/kg and 23.85 mg/kg BT062 (once ) vs. a e control.

Fig. 12 shows the complete remission of a aft r itional cell) oma (metastatic sample) in mice treated with 4 mg/kg and 23.85 mg/kg BT062 (once weekly) vs. a vehicle control. illustrates the rapid plasma clearance for dosages ranging from 40 mg/m2 to120 mg/mz, while higher doses as rated here by a dose of 160 mg/mz, showed plasma clearance closer to the expected value. shows the measured Cmax values of BT062 compared to the theoreticai Cmax vaiues. and 16 show that the Cmax values are generally similar over several treatment cycles in a repeated single dose regime as indicated.

WO 83817 clarifies that the rapid plasma clearance cannot be attributed to a ing effect caused by soluble 00138.

Fig. 18 depicts the progression free survival for human subjects treated with different dosages of BT062 administered in the course of the indicated treatment cycles, wherein each active treatment cycle lasted 21 days and the respective dosage was administered on days 1, 8 and 15 of each cycle. Each cycle of 21 days was followed by a 7 day g period (28 indicate the 21+? days, per ,cycle") As can be seen 14 patients were on study ent for more than 3 months. For two of these patients progression free survival of at least 300 days (about 10 months) has been reported.

Fig. 19 shows in (A) the course of Cmax values with different dosages administered weekly for three weeks followed by a week long resting period and in (B) the Cmax values, 0-2 hours after completion of the administration, for different doses. The theoretical Cmax values are also shown.

Fig. 20 shows the level of serum M-protein measured for a patient receiving 50 mg/m2 weekly for three weeks, followed by a 7 day g period. Days -111 to 169 are shown. Arrows indicate treatment with BT062.

Fig. 21 shows the level of lambda-kappa FLC (strong se before first treatment, strong decrease from day 1 to 57) measured for a patient (oligo-secretory multiple myeloma) receiving 65 mg/m2 weekly for three weeks, followed by a 7 day resting period. Days ~83 to 163 are shown.

Fig. 22 shows the level of lambda-kappa FLC g increase before first treatment, stabilization for two cycles) measured for a patient (oligo-secretory multiple myeloma) receiving 80 mg/m2 weekly for three weeks, followed by a 7 day resting period. Days - 111 to 85 are shown.

Fig. 23 shows the level of lambda-kappa FLC (decrease for three months) measured for a t (oligo~secretory multiple myeloma) receiving 100 mg/m2 weekly for three weeks, followed by a 7 day resting period. Days -83 to 141 are shown.

Fig. 24 shows the level of urine M- n measured for a patient ing 3x 120 mg/m2 weekly for three weeks, followed by a 7 day resting . Days -27 to 337 are shown.

Fig. 25 shows the level of serum M- protein measured for a patient receiving 3x 160 mg/m2 weekly for three weeks, followed by a 7 day resting period. Days -20 to 57 are shown, which indicate a minor response.

Fig. 26 shows the level of kappa FLC measured for a patient ing 160 mg/m2 at three weeks intervals. Days -21 to 101 are shown.

Fig. 27 shows a comparison of plasma levels of BT062 stered as a repeated single dose of 160 mg/m2 in comparison to a multiple dose of 100 mg/m2 and 120 mg/m2 administered three times in an active treatment cycle of equal length (21 days).

Fig. 28 shows serum M-protein levels during an ed administration of BT062 as repeated single doses BTOBZ of 160 mg/m2 , which lead to minor response with manageable side effects.

Fig. 29 shows serum M protein levels and Cmax values over time in a repeated single dose administration for a patient treated with a repeated single dose of BT062 of 160 mg/m2 (see also Fig. 28).

Fig. 30 shows the effect of the combination therapy on median tumor volume (TV) in a xenograft mouse model (MOLP-8 MM xenograph model). The results show the effects of the combination of BT062 and domide.

Fig. 31 shows the effect of the combination therapy on median tumor volume (TV) in a xenograft mouse model. The result shows the effects of the combination of BT062 and VELCADE.

Fig. 32 shows the effect of lenalidomide on different CD138 expressing cells in vitro, in ular MOLP-A cells (A), RPMl8226 cells (B), NCl-HQZQ cells (C) and U266 cells (D). Notably C301 38 expression was not affected in vivo (L363 MM xenograft model) by the treatment of the combination of lenalidomide and dexamethasone (data not shown).

Fig. 33 shows the results of an in vivo (L363 MM xenograft model) drug ation study wherein BT062 g, 4mg/kg) was administered intravenously on days 1, 8, , 22 and 29; lenalidomide was administered orally on days 0-4, 7-11, 14-18, 21- 2528-32 and dexamethasone was administered subcutaneously on days 0, 7, 14, 21 and 28. A considerable reduction in tumor volume relative to the simple combination of lenalidomide and dexamethasone can in particular be seen in the context of the 4 mg/kg BT062 dosage scheme. The s are shown in terms of the effect on the median relative tumor volume in the model relative to an intravenous administration of a e control. The median relative tumor volume on day X was, here and in the subsequent figures calculated as follows: The relative volumes of individual tumors (Individual RTVs) for Day X were calculated by dividing the individual tumor volume on Day X (TX) by the individual volume of the same tumor on Day 0 (TO) multiplied by W0 2013(083817 100%. Group tumor volumes were expressed as the median or mean (geometric) RTV of all tumors in a group (group median/mean RTV).

Fig. 34 shows the level of serum M- protein measured for a patient scheduled to receive 80 mg/m2 of BT062 weekly for three weeks, followed by a 7 day resting period.

BT062 was administered in combination with domide and Dexamethasone. Days -13 to 106 are shown, which indicate a minor response.

Fig. 35 shows the results of an in vivo (human derived breast cancer model in NMRI nude mice) study n BT062 (0.5mg/kg, 1mg/kg, 2mg/kg, 4mg/kg) was administered intravenously on days 0,7,14, 21, 28 and 35 and taxol (1 Omg/kg) was administered intravenously on days 1, 8, 15 and 22. BT062 showed at higher concentrations superior results. The results are shown in terms of the effect on the mean relative tumor volume in the model relative to an enous administration of PBS. For the calculation of the median relative tumor volume on day, see Fig. 33.

Fig. 36 shows the results of an in vivo (human derived breast cancer model with CD138 lHC score 2-3 in NMRl nude mice) study wherein BT062 (1mg/kg, , 4mg/kg, 8mg/kg) was administered intravenously on days 07,14, 21, 28 and 35 and Docetaxel (10mg/kg) was administered intravenously on days 0, 7 and14. BT062 showed at higher concentrations or results. Dooetaxel was as effective as the highest concentration of BT062. The results are shown in terms of the effect on the mean relative tumor volume in the model relative to an intravenous administration of PBS. For the calculation of the median relative tumor volume on day, see Fig. 33.

Fig. 37 shows the results of an in vivo (human derived breast cancer model with CD138 lHC score 1—2 in NMRI nude mice) study wherein BT062 (1mg/kg, , 3mg/kg, 4mg/kg) was administered intravenously on days 0,7,14, 21. 28 and 35 and Docetaxel kg) was administered intravenously on days 0, 7 and14. No difference in the treatment regimens was observed. The results are shown in terms of the effect on the mean relative tumor volume in the model relative to an intravenous administration of PBS. For the calculation of the median ve tumor volume on day. see Fig. 33.

Fig. 38 shows the s of an in vivo (human derived prostate cancer model in NMRl nude mice) study wherein BTOSZ (1mg/kg, . 4mg/kg. 8mg/kg) was administered intravenously on days 0,7,14, 21. 28 and 35 and Docetaxel (10mgikg) was administered intravenously on days 0, 7 and 14. BT062 showed at higher concentrations superior results. The results are shown in terms of the effect on the mean relative tumor volume.

Docetaxei was as effective as the highest concentration of BT062 and allowed for maintenance of the low tumor volume over time.

DETAILED DESCRIPTION OF S AND PREFERRED EMBODIMENTS OF THE INVENTION The t invention relates to the administration to subjects, in particular human ts (patients), in need thereof, of conjugates comprising CD138 targeting agents described herein and the delivery of the effector moiecule(s) of the immunoconjugates to target sites and the release of effector(s) moiecuie in or at the target site, in particular target celIs, tissues and/or organs. More particularly, the present invention s to immunoconjugates comprising such C0138 targeting agents and potent effector molecules that are attached to the targeting agents. The effector molecules may be ted by cleavage and/or dissociation from the targeting agent portion of the conjugate in or at a target site. The immunoconjugates may be administered alone or as part of an anticancer combination that includes a cytotoxic agent such as, but not limited to, a proteasome inhibitor (e.g., bortezomib, carfilzomib), immunomodulatory agentJanti-angiogenic agent (e.g., thalidomide, lenalidomide or pomalidomide), DNA aIkyiating agent (eig., lan) or corticosteroid (e.g., dexamethasone), wherein the anticancer combination has synergistic effects or unexpected additive effects in the treatment of cancer over the immunoconjugate used alone in monotherapy. the cytotoxic agent used alone in monotherapy or both.

The immunoconjugates according to the present invention may be administered to a t in need of treatment or to cells isolated from such a t in need of treatment. The effector molecule or molecules may be reieased from the immunoconjugate by cieavage/dissociation in or at a target cell, tissue and/or organ. in one example, the immunoconjugate BTOSZ, which targets CD138 expressing cells via the nBT062 dy and comprises DM4 as an effector molecule, was administered to a patient with relapsed/refractory multiple a 14 times in an amount of 40 mg/m2 as in a ed multiple dose regime, wherein the length of each active treatment cycle was 21 days with three doses/per cycle being administered on days, 1, 8, and 15 of the cycle and an resting period of one week was inserted before WO 83817 the next active treatment cycle was started. Expressed differently, the treatment cycle was 28 days with three doses/per cycle being administered on days, 1, 8, and 15 of the cycle and none administered on day 22, resulting, in this example, in a ent free period of about two weeks. in this example, the immunoconjugate was administered intravenously to the patient so that it could better trate in and/or at tumor cells.

Measurements of the plasma tration of BTO62 showed that in an initial measurement phase (up to 2 hours after the end of administration) Cmax values for BT062 were significantly below the theoretically calculated value while no DLTs (dose limiting toxicities) were observed, suggesting that BT062 concentrates at the tumor target rather than randomly attaching to target and non-target CD138. A "buffer effect" resulting from sCD138 (soluble CD138) could be excluded (compare Fig. 17). As will be discussed below in the context of administrations at 80mg/m2, a rapid concentration at the target cells could be confirmed.

An active treatment cycle is a treatment cycle that is defined by a regular administration of the active agent, here generally the immunoconjugate, and es any resting periods. An active treatment cycle includes lly three weeks of active treatment and is considered to end not with the last dose administered, but at the time when a further administration would be due. Thus an active treatment cycle inciuding a dose of 120mg/m2 on day 1, 65mglm2 on both days 8 and 15, would be considered to end on day 21 and to be 21 days long. While an active ent cycle generally lasts 21 days, it may range from at least two weeks (14 days) to four weeks (28 days). in the latter case an active treatment cycle and a "full" or ete" treatment cycle are the same. Within the period of an active treatment cycle, the active agent, is regularly administered. This includes, e.g., in alternating 2 and 3 day intervals, in 4 day intervals, in progressive increasing intervals such as on day 1, 3, 6, 10, 15. A treatment cycle may in addition to the active ent further se a g period. E.g. in the example above, the above administration scheme in a ent cycle of 28 days wouid be considered to comprise no administration of day 22. Such a treatment cycle, including a resting period, is also referred to herein as "full" or "complete" treatment cycle. A treatment free period describes the time during which no treatment is given.

Thus, in the above example, the treatment free period would start at day 16. At the beginning of the resting period, no immunoconjugate is administered to the patient. in a preferred embodiment no treatment of any sort is administered during this period. The W0 20131083817 resting period may lasts, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more typical is one week. A treatment free period may last '14, 15, 16, 17, 18, 19, 20, 21 days or more.

In r example, the immunoconjugate BT062 was administered to a patient with relapsed/refractory multiple a 18 times in an amount of 50mgi‘m2 each as repeated multiple doses, wherein the length of each treatment cycle was 21 days with three per cycle being administered on days, 1, 8, and 15 of the cycle and an resting period of one week was inserted before the next treatment cycle was started.

Expressed differently, the treatment cycle was 28 days with three doses/per cycle being administered on days, 1, 8, and 15 of the cycle and none administered on day 22. in this e, the immunoconjugate was administered intravenously to the patient so that it could better concentrate in and/or at tumor cells. No additional means were provided to release the effector molecule from the conjugate. Six treatment cycles were well tolerated and at least stable disease could be achieved over six cycles, with a decrease of serum ein by nearly 25% during after the 3rd and 5‘h treatment cycle (Figure 20). in yet another example, the immunoconjugate BT062 was administered to a patient with ed/refractory multiple myeioma 19 times in an amount of 55mg/m2 as repeated multiple doses, wherein the length of each treatment cycle was 28 days with three doseslper cycle being administered on days, 1, 8, and 15 of the cycle and none administered on day 22. The treatment free period was thus 14 days before the next treatment cycle started. At this concentration plasma levels were still below the theoretical Cmax (mean tage from theoretical Cmax= 60%; Table 11a), but not to the degree observed with lower doses, e.g., ciOmg/m2 or 50 mg/m2 (mean percentage from theoretical Cmax=33% Table 11a). However, a strong decrease of the serum FLC level could be observed afterjust a single treatment cycle and could be maintained for two months (Figure 21). Besides the higher percentage from theoretical Cmax reached at does level of 65 mg/m2, the total plasma tration missing to the theoretical Cmax (here mean total 17.7 mg/m2 , Table 11b) was similar to the one ones observed at lower concentrations of 40 mg/mzor 50 mg/m2(mean total 18.6 mg/m2 and 23.0 mg/mz, Table 11b). Thus, the total plasma concentrations missing to the tical Cmax may stay at different concentrations, despite an increase of the mean percentage from the theoretical Cmax by more than 10% more than 20% or more than 25%, preferably n 15 and 25%, stayed within the range of15—25 mg/mz, namely around 20 mg/m2. For 14 patients (out of 32 on the study) progression free survival of at least 3 months has been reported (Fig. 18), for four of these patients progression free survival of at least 168 days has been reported. One of these four patients showed clear ion of serum M n after 9 treatments (Patient No. 6, please see also Fig. 20) and for another patient a strong decrease in FLC could be observed within the first 2 months (Patient No. 19, see also Fig. 24). The first DLT was observed in the 140 mg/m2 cohort (Patient No. 23), but no DLT was reported for the six other patients at this dose level. For two out of the four patients (Patient Nos. 30 and 32) that were treated with weekly doses of 160 mg/m2 DLT was ed and prompted a reduction of the dose to of 140 mg/mzin uent cycles. in yet another example, the immunoconjugate BT062 was administered to a patient with non—secretory relapsed/refractory multiple myeioma (Patient No. 12 in Fig. 18) for 15 cycles in an amount of 80 mg/m2 as repeated le doses, wherein the length of each treatment cycle was 28 days with three doses/per cycle being administered on day 1, 8, and 15 of the cycle and none administered on day 22. in this e, the immunoconjugate was administered intravenously to the patient so that it could better concentrate in and/or at tumor cells. At this concentration plasma levels were still below the theoretical Cmax, but not to the degree observed with lower doses, eg. 40 mg/m2 (mean percentage from theoretical Cmax-33%; Table 11a). After three administrations at 8Omgfm2, totaling an stration of 240mgim2 (aggregate dose) within three weeks, the immunoconjugate remained weli ted. A rapid concentration at the tumor target could be confirmed at this dosage. Table 12 shows the s of receptor occupancy (R0) measurements. Here the binding of BT062 to the receptor (CD138) was measured on multiple myeloma cells in the bone , ergo the site of the tumor, in the Multiple Myeloma patient. Receptor (CD138) bound BT062 was stained with anti-May antibodies (Sampie 1). Total CD138 was measured with anti—May antibodies after receptor saturation with BT062 (Sample 2). incubation with an lgG1 isotype determined unspecific binding to the sample (Sample 3). The first row in Table 12 shows the results of a measurement within four hours after completition of the administration. As can be seen, the receptor occupancy within 4 hours after end of administration is, in this case, 99%. The patient showed a l response. The duration of an administration (administration time) obviously differs with the mode of administration.

Administration times in intravenous (lV) administrations are generally defined by mg/min (1 mg/min for first 15 min and if ted 3mg/min for the rest) and therefore increase with the dose levels assigned to the patient. The times for flushing the administration line after administration vary as well. in the t study, for doses between 10 mtg/m2 and 200 mg/mz, the shortest infusion time was 18 minutes and m infusion time was 3 hours and 2 minutes with a mean of 1 hour and 36 min.

It 200 mg/m2 are administered completely at 1 mg/min this could result in an stration up to 8 hours. in an alternative embodiment, the immunoconjugate may be administered as iv bolus within a minute.

Thus, an administration according to the present invention is "completed" any time between 0 and 8 hours after start of an administration. generally within 0 and 4, often within 2 hours from the start of an administration.

Figure 22 shows a t (13 in Figure 18) subjected to the same administration scheme (80mgim2 as repeated multiple doses, wherein the length of each treatment cycle was 28 days with three per cycle being administered on day 1, 8, and 15 of the cycle and none administered on day 22) which was administered to a relapsed/refractory patient. The strong increase of lambda-kappa before the first treatment day could be stabilized for 2 cycles.

Patient 12 in Figure 18 (80mg/m2 as repeated multiple doses as above), showed a partial response for about 8 months.

In a further example. the immunoconjugate BT062 was administered to a patient with relapsed/refractory multiple myeloma six times in an amount of /m2 as repeated multiple doses, wherein the length of each active treatment cycle was 21 days with three doses/per cycle being stered on day 1, 8, and 15 and a resting period of 1 week (no administration at day 22 leading ively to a two weeks break of administration). in this example, the immunoconjugate was administered intravenously to the patient so that it could better concentrate in and/or at tumor cells.

Figure 23 shows the result of this dosage scheme with patient 15 (Fig. 18, relapsed refractory with oligo-secretory MM), who showed ssion free survival for more than 3 months.

At this concentration plasma ievei was only below the theoretical Cmax during the first two strations (Table 11a) pointing towards an accumulation of the immunoconjugate after weekly dosing at this dose. However, in the equivalent experiments with 120mg/m2 as repeated multiple dose, these values went down, indicating that the 100mg/m2 outcomes might be a deviation in a single patient and also indicating that at even higher dosages no significant lation might take place.

After three administrations at m2. at 120mg/m2 and, for the most part, at 140mglm2 and totaling an administration of 300mg/m2, 3':30mg/m2 and 420mg/m2, respectively within three weeks, the immunoconjugate remained well tolerated. No DLTs were observed after three 21 day cycles of 3x 100mglm2 (300mg/m2) or 3x m2 (360mg/m2) in each cycle (3x300mg/m22900mg/m2 in 12 weeks and 3x36£imglm2=1OSOmg/m2 in 12 weeks) compared to m2 (tour 21 day cycles of 160mg/m2 each). in a further example, the immunoconjugate BT062 was administered to a patient with edlrefractory multiple myeloma six times in an amount of m2 as repeated multiple doses, wherein the length of each active treatment cycle was 21 days with three doses/per cycle being administered on day 1, 8, and 15 and a resting period of 1 week. In this example, the immunoconjugate was administered intravenously to the patient so that it could better concentrate in and/or at tumor cells.

Figure 24 shows the result of this dosage scheme with patient 19 (Fig. 18, relapsed tory with oligo-secretory MM), who showed an unconfirmed minor response, despite a number of a number of treatment delays (x).

At this concentration the plasma level was still beiow the theoretical Cmax (Table 11a) indicating no relevant accumulation of the conjugate after weekly dosing at this dose. After three administrations at 120mg/m2, totaiing an administration of 360mg/m2 within three weeks. the immunoconjugate ed well tolerated. No DLTs were observed after three 21 day cycles of 3x 12Clmg/m2 (360mgfm2) in each cycle. in a further example, the immunoconjugate BT062 was administered to a patient with relapsed/refractory multiple myeloma seven times in an amount of 16Cimg/m2 as repeated multiple doses, wherein the length of each active treatment cycle was 21 days 2012/074867 with three doses/per cycle being administered on day 1, 8, and 15 and a resting period of 1 week. In this example, the conjugate was administered intravenously to the patient so that it could better concentrate in and/or at tumor cells. Figure 25 shows the results (M-protein decreased by more than 25% qualifying for minor response) for patient 31, which as can be seen from Fig. 18 did not y DLT at this tration.

As indicated in Fig. 18, 2 out of 4 patients displayed DLTs at 160 mg/m2 (elevated liver enzymes, neutropenia) but could resume treatment at 160 mg/mz. in this administration scheme, MAD was 160 mg/mz, while 140 mg/m2 was ined to be the MTD (1 out of 6 patients displayed DLT at this concentration) Single dose every three Single dose every three Repeated single dose weeks weeks 240, 300, 360, 420 Drug -related e No serious drug-related events such as eye toxicity toxicities (up to now), one DLT (palmar—plantar erythrodysaethesia Lsyndrome) at 420 out of six g Table 1: Total amount of BT062 red within 3 weeks results in different tolerability of the drug. A single dose of 200 mglmz in a 3 week2period resulted in DLTs (target related toxicities).

Similar total doses (3x80 mg/mz, 3x 100mg/m , 3x 120mg/m2, 3x 140mg/m2) administered in 3 intervals during a 3 week period did not result in any serious drug related toxicities in ts.

In yet another example, the immunoconjugate BT062 was co—administered to a patient with relapsed multiple myeloma for four cycles in an amount of 80mglm2 as repeated multiple doses, wherein the length of each treatment cycle is 28 days with three per cycle being administered on days, 1. 8, and 15 of the cycle and none administered on day 22. At the same time a 25 mg daily oral dose of lenalidomlde is administered at 1 to 21 and 40 mg of dexamethasone is administered weekly (days 1, 8, 15, 22). In this example, the immunoconjugate is administered intravenously to the patient so that it can better concentrate in and/or at tumor cells. Despite delayed start of treatment cycle 2 and 3 and skipping the dose of BT062 at day 15 of Cycle 3 and lenalidomlde on day 15 to 21 in cycle 3, a minor response achieved after the first cycle was maintained (Fig. 34).

WO 83817 In another example, the immunoconjugate BT062 is co-administered to a t suffering from a pancreatic tumor as repeated multiple dose of 220mg/m2, as solid tumors trap immunoconjugate more y than malignancies not associated with solid masses wherein the length of each treatment cycle is 28 days with three dosesiper cycle being administered on days 1, 8, and 15 of the cycle and none administered on day 22.. At the same time a 10 mg daily oral dose of the lmmunomodulatory agent lenalidomide is administered. In this example, the immunoconjugate is administered intravenously to the patient so that it could better concentrate in and/or at tumor cells.

The administration is ed by a maintenance treatment consisting of a repeated single dose of m2 of the immunoconjugate at day 1 of a 21 day cycle for 4 CD138 or syndecan—1 (also described as SYNDt; SYNDECAN; SDC; SCD‘l; CD138 N, SwissProt accession number: P1882?7 human) is a membrane glycoprotein that was originally described to be present on cells of epithelial origin, and subsequently found on hematopoietic cells (Sanderson, 1989). CD138 has a long extracellular domain that binds to soluble molecules leg, the growth factors EGF, FGF, HGF) and to insoluble molecules (e.g., to the extracellular matrix components collagen and fibronectin) through heparan sulfate chains (Langford, 1998; Yang, 2007) and acts as a receptor for the extracellular matrix. CD138 also mediates cell to cell adhesion through heparin-binding molecules expressed by adherent cells. lt has been shown that CD138 has a role as a co-receptor for growth factors of myeloma cells (Bisping, 2006).

Studies of plasma cell differentiation showed that CD138 must also be considered as a differentiation n (Bataille, 2006).

In malignant hematopoiesis, CD138 is highly expressed on the majority of MM cells, ovarian carcinoma, kidney oma, gall bladder carcinoma, breast carcinoma, prostate cancer, lung cancer, colon carcinoma cells and cells of Hodgkin’s and non- Hodgkin’s lymphomas, chronic lymphocytic ia (CLL) (Horvathova, 1995), acute lymphoblastic leukemia (ALL), acute myeloblastic ia (AML) (Seftalioglu, 2003 (a); Seftalioglu, 2003 (b)), solid tissue sarcomas, colon carcinomas as well as other hematologic malignancies and solid tumors that express CD138 (Carbone et al., 1999; Sebestyen et al.,1999; Han et al., 2004; Charnaux et al., 2004; ell et al.,2004; Orosz and Kopper, 2001 ). sion of CD138 is also associated with different types 2012/074867 of gastrointestinal malignancies (Conejo et al., 2000). As shown in Table 2, a number of tumorgenic cell lines exist which are associated with CD138 expressionloverexpression. cell line mt CD138 Ex ression leo nM RFl* rece utors/cell 29 MM 0.38 502 788,752 PC-3 urostate cancer 0.79 541 195,671 MM 1.59 617 782,987 MM 1.78 425 4 SK-BR—3 breast carcinoma 2.72 485 444,350 LNCaP uostate cancer 7.39 179 23,388 i CAPAN-Z pancreas 15.51 328 carcinoma pancreas 36.38 34 18,085 carcinoma T470 42,264 Jurkat T cell l m ohoma . .

Table 2: CD138 expression on different cell lines. in the context of MM it was shown that the sensitivity s BT062 correlates with a higher expression of CD138 (RFl= relative fluorescence index).

The observed sensitivity of, e.g., the breast carcinoma cell lines and pancreas carcinoma cell lines was substantially lower than that of that of the MM celi lines.

Nonetheless, as described in the experimental section in xenograft mouse models using cells from patients with breast cancer and pancreatic cancer, not only able, but significantly better results than in comparable xenograft models for MM were obtained. in both instances complete ion could eventually be obtained, while comparable MM models showed marked delay in tumor growth, but not complete remission.

While in pancreatic cancer there appears to be no difference in syndecan-1 mRNA expression between early and advanced tumors, in mammary carcinoma, it was ed that CD138 can be lost over time as reflected by weak or lacking lHC staining.

C0138 loss of expression had been ed and was often correlated with a shift of expression, i.e., de novo expression on surrounding stroma (Loussouarn, 2008). As a result, fewer targets for CD138 targeting agents can be expected over time.

Other cancers that have been shown to be positive for CD138 expression are many n adenocarcinomas, tional cell bladder carcinomas, kidney ciear cell carcinomas, squamous cell lung carcinomas; and uterine cancers (see, for example, Davies et al., 2004; Barbareschi et al., 2003; Mennerich et al., 2004; Anttonen et al., 2001; Wijdenes, 2002).

The treatment of active (symptomatic) multiple myeloma and related plasmaproliferative disorders shall serve as an example of diseases that can be treated via immunoconjugates of the present invention.

Plasmaproliferative disorder as used herein means plasma cell and/or hematoiogic disorders such as MGUS, SMM, active (symptomatic) MM, Waldenstrém's Macrogiobulinemia, solitary piasmacytoma, systemic AL amyloidosis and POEMS syndrome.

Multiple myeloma (MM) refers to a malignant proliferation of plasma cells that typically originates in bone marrow, involves chiefly the skeleton of a patient, and ts clinical features attributable to the particular sites of involvement and abnormalities in formation of plasma ns. The ion is usually characterized by numerous diffuse foci or nodular accumuiations of abnormal or malignant plasma cells in the marrow of s bones ially the skull), causing palpable sweilings of the bones, and onally in extraskeletal sites. Upon radiologicai exam, the bone lesions may have a characteristic "punched out" appearance. The ceils involved in the myeloma typically e abnormal proteins and/or abnormal protein levels in the serum and urine. The disease typically develops from monoclonal gammopathy of undetermined significance (MGUS) to smoldering multiple myeloma (SMM) to active le myeloma (MM). ms of these conditions vary, but may include hypercalcemia, renal insufficiency, fatigue, anemia, bone pain, spontaneous fractures, increased frequency or duration of infection, or abnormal urine color or odor. When the present ion refers to Muitipie Myeloma it refers to (MGUS), smoldering le myeloma (SMM) and active multiple myeloma (MM) as weil as other malignant proliferation of plasma cells that may eventually develop into active MM.

MGUS, a clinically benign precursor condition of MM is more common than MM, occurring in 1% of the population over age 50 and 3% of those over age 70 (Greipp and Lust, 1995). it is important to distinguish patients with MGUS from those with MM, as MGUS patients may be safely observed without resort to y.

However, during long-term follow-up, of 241 patients with MGUS, 59 ts (24.5%) went on to develop MM or a related disorder (See Kyle et al., 1993).

The term gammopathy refers to a primary bance in immunoglobulin synthesis of a patient.

Monoclonal athy refers to any of a group of disorders that are typically associated with the proliferation of a single clone of id or plasma cells lly visible on serum protein electrophoresis (SPEP) as a single peak) and characterized by the presence of monoclonal immunoglobulin in the serum or urine of a patient.

Smoldering MM (SWIM) has been reported to precede the onset of symptomatic multiple a in the elderly. Smoldering le myeloma is often considered as an advanced phase of MGUS; even at the time of progression, smoldering multiple myeloma usually lacks osteolytic lesions or other cardinal features of symptomatic multiple myeloma.

Clinical symptoms of MM include anemia, hypercalcemia, renal insufficiency, and lytic bone lesions. Distinctions in the course and the ty of the disease as it develops from monoclonal gammopathy of undetermined significance (MGUS) to smoldering multiple myeloma (SMM) to multiple a (MM) are provided in Table 3 below. The table also summarizes methods of detection, diagnosis, and monitoring of these conditions. Such symptoms and techniques are familiar to those of skill in the art.

TABLE 3 Serum M--rotein >=3 -/dl. >=3 uidL <3 nrdL Jones >=1-/24 h <1 -/24 h <1 -{24h -rotein in urine Yes Yes Anemia usuall oresent mAbsent H ercalcemia. renal insufficient: ma be uresenl m L icbone lesions usuall uresent M MM = multi le m eloma SMM = smolderin multiple myeloma ‘ MGUS = onal : ammoath of undetermined si-nificance Classif ‘na stages b severit and clinical features of multlle m atoma Stage l active MM Relatively few cancer cells have spread throughout the body. The number of red blood cells and the amount of calcium in the blood are normal. No tumors (plasmacytomas) are found in the bone. The amount of M—vrotein in the blood or urine is ve low. There ma be no 5 toms of disease. seen mmm _ A moderate number of cancer cells have sread throuhout the bod seem acme MM - A vely large number of cancer cells have spread throughout the body. There may be one or more of the following: A decrease in the number of red blood cells. g anemia.

The amount of calcium in the blood is very high, because the bones are being damaged.

More than three bone tumors (plasmacytomas) are found.

Hi-h levels of M—roteln are found in the blood or urine.

Clinical features of MM SPEP serum urotein electro-horesis —36- SPlEP serum rotein immunoelectro-horesis Urine urotein immunoelectro-horesis Bence- Jones n == _--- m>10%lasma cells In marrow or - re-ates on bIo-s ora olasmac oma Monoclonaspmem. ——_ Serum in >3 old! or —-- -—-— Active multiple a (MM) is typically recognized clinically by the proliferation of malignant plasma cells in the bone marrow of a patient. These neoplastic plasma cells produce immunoglobulins and evolve from B-lymphocytes. The immunoglobulins that are produced by the plasma cells may be ed in the blood serum and/or urine of a patient by electrophoresis testing.

As ted in Table 3, the measurement of serum M-protein is an important tool for ing MM at different stages.

"M-protein" refers to a monoclonal protein that is typically visualized as a narrow band on electrophoretic gel, or an abnormal arc in immunoelectrophoresis. it represents a proliferation of homogenous immunoglobulin produced by clone cells originating from a single common cell, e.g., a monoclonal immunoglobulin characterized by a heavy chain of a single class and ss, and light chain of a single type (also referred to as a M- spike and more broadly as a paraprotein).

"Serum protein electrophoresis" (SPE or SPEP) and "immunofixation electrophoresis" (IFE) can detect monoclonal globulin, which is produced in several plasma cell proliferative disorders ing multiple myeloma (MM). Population- wide, up to 61% of these findings are not associated with clinical symptoms, allowing for a diagnosis of monogammopathy of undetermined significance (MGUS). SPE and lFE do not, however. detect all monoclonal immunoglobulins, particularly when only light chains are secreted.

Those "free light chain molecules" (FLCs) include A and K light chains. Plasma cells produce one of the five heavy chain types together with either K or A molecules. There is normally approximately 40% excess free light chain production over heavy chain WO 2013083817 synthesis. Plasma cells secrete free light chains (FLC, kappa or lambda) in addition to intact immunoglobulin molecules, and serum light chain levels are determined by the relative rates of synthesis (K>)\) and renal excretion (K>A). In the presence of a monoclonal globulin. KzA ratios may be either higher or lowerthan the normal range. depending on the class of the involved FLC. The serum half—life of FLCs is 2-6 hours, compared with 5 days for 19A, 6 days for lgM and 21 days for lgG. Thus, measurement of serum FLC levels allows a far more rapid evaluation of tumor response to therapy than measurement of intact immunoglobulin. Likewise, serum FLC measurements allow earlier detection of relapse.

Non-plasmaproliferative diseases also are ated with CD138 expression.

Pancreatic oma The majority of cases se exocrine type. The majority of these exocrine cancers represent ductal adenocarcinoma (further more rare subtypes comprise cystic tumors, tumors of acinar cells and sarcoma). Endocrine cancer of the pancreas represents a e producing tumor.

Carcinoma in situ refers to the early stage of , when it is confined to the layer of cells where it began. in breast cancer, in situ means that the cancer cells remain confined to ducts (ductal carcinoma in situ) or lobules (lobular carcinoma in situ). They have not grown into deeper tissues in the breast or spread to other organs in the body, and are sometimes referred to as non-invasive or pre-invasive breast cancers.

Invasive (infiltrating) carcinoma.

The exocrine cells and endocrine cells of the as form completely ent types of tumors.

Exocrine tumors These are by far the most common type of pancreas cancer and most atic exocrine tumors are malignant. About 95% of cancers of the exocrine pancreas are adenocarcinomas (an adenocarcinoma is a cancer that starts in gland cells). These s usually begin in the ducts of the pancreas, but they sometimes develop from the cells that make the pancreatic enzymes (acinar cell carcinomas). 2012/074867 Less common types of ductal cancers of the exocrine pancreas include adenosquamous carcinomas, squamous cell carcinomas, and giant cell carcinomas.

Endocrine tumors Tumors of the endocrine pancreas are uncommon. As a group, they are known as pancreatic neuroendocrine tumors (NETs), or sometimes as islet cell tumors. There are several subtypes of islet cell tumors. Each is named according to the type of hormone— making cell it starts in: The main system used to describe the stages of cancers of the exocrine pancreas is the American Joint Committee on Cancer (AJCC) TNM system as provided by the American Cancer Society (A08). The TNM system for staging ns 3 key pieces of information: T describes the size of the primary tumor(s), measured in centimeters (cm), and whether the cancer has spread within the pancreas or to nearby organs. Distinctions are made between TX, T0, T1, T2, T3 and T4, wherein a higher number indicates advancement of the disease.

N describes the spread to nearby (regional) lymph nodes. N categories include, NX, NO and N1.

M indicates whetherthe cancer has metastasized (spread) to other organs of the body.

(The most common sites of pancreatic cancer spread are the liver, lungs. and the peritoneum - the space around the digestive organs.) M ries include: MX, M0 and M1.

After the T, N, and M categories have been determined, this information is combined to assign a stage, a process called stage grouping.

Stage 0 (Tie, N0, M0): The tumor is d to the top layers of atic duct cells and has not invaded deeper tissues. it has not spread outside of the pancreas. These tumors are sometimes ed to as atic oma in situ or pancreatic intraepithelial neoplasia ill (Panin ill).

Stage IA (T1, N0, M0): The tumor is confined to the as and is less than 2 cm in size. it has not spread to nearby lymph nodes or distant sites.

Stage lB (T2, N0, M0): The tumor is confined to the pancreas and is larger than 2 cm in size. it has not spread to nearby lymph nodes or distant sites.

Stage "A (T3, N0, M0): The tumor is growing outside the pancreas but not into large blood vessels. It has not spread to nearby lymph nodes or distant sites.

Stage "3 (T1-3, N1, M0): The tumor is either confined to the pancreas or growing outside the pancreas but not into nearby large blood vessels or major nerves. It has spread to nearby lymph nodes but not distant sites.

Stage ill (T4, Any N, M0): The tumor is g e the pancreas into nearby large blood vessels or major nerves. it may or may not have spread to nearby lymph nodes. it has not spread to distant sites.

Stage lV (Any T, Any N, M1): The cancer has spread to t sites.

Although not ly part of the TNM system, other factors are also important in determining prognosis (outlook). The grade of the cancer (how abnormal the cells look under the microscope) is sometimes listed on a scale from G1 to G4, with G1 cancers looking the most like normal cells and having the best outlook.

For patients who have y, another ant factor is the extent of the resection -- whether or not the entire tumor is removed. This is sometimes listed on a scale from R0 (where all visible and microscopic tumor was removed) to R2 (where some visible tumor could not be removed).

From a practical standpoint, how far the cancer has spread often can't be determined accurately without surgery. That's why doctors often use a simpler staging system, which divides cancers into groups based on whether or not it is likely they can be removed ally. These groups are called resectable, locally advanced (unresectable), and metastatic. These terms can be used to describe both exocrine and endocrine pancreatic cancers.

W0 2013l083817 ? Resectable: if the cancer is only in the pancreas (or has spread just beyond it) and the surgeon can remove the entire tumor, it is called resectable.

Locally advanced (unresectable): if the cancer has not yet spread to distant organs but it still can't be completely removed with surgery, it is called locally advanced. Often the reason the cancer can't be removed is because too much of it is present in nearby blood vessels. atic: when the cancer has spread to distant organs, it is called atic.

Surgery may still be done, but the goal would be to relieve symptoms, not to cure the cancer. atic neuroendocrine cancers are not staged like cancers of the exocrine pancreas. instead the statistics are broken down into different stages: localized (only in the pancreas), regional (spread to nearby lymph nodes or s). and distant d to distant sites, such as the liver).

Bladder tumors are grouped by the way the cancer cells look under a cope.

Transitional cell carcinoma (also called urothelial carcinoma) is by far the most common type of bladder . Within this group are also subtypes. They are named depending on the shape of the cells and whether they tend to spread and invade other organs. (if they are likely to grow deeper into the bladder wall they are called invasive. if not likely they are non-invasive.) These tumors are divided into grades based on how the cells look under the microscope. If the cells look more like normal cells, the cancer is called a low-grade cancer. When the cells look very abnormal, the cancer is high-grade. Lower- grade cancers tend to grow more slowly and have a better e than higher—grade cancers.

Also included in the definition, are squamous cell carcinoma (uncommon; usually invasive); adenocarcinoma (uncommon; almost all are invasive); small cell (rare).

Other rare bladder cancers are also included in this definition.

Bladder cancer is also staged: Stage 0a (Ta, N0, M0): The cancer is a noninvasive papillary carcinoma. it has grown toward the hollow center of the bladder but has not grown into the muscle or connective tissue of the bladder wall. it has not spread to lymph nodes or distant sites.

Stage Ois (Tis, N0, M0): The cancer is a flat, noninvasive carcinoma, also known as flat carcinoma in situ (ClS).

The cancer is growing in the lining layer of the bladder only. lt has neither grown inward toward the hollow part of the bladder nor has it invaded the muscle or connective tissue of the bladder wall. It has not spread to lymph nodes or distant sites.

Stage I (T1, N0, M0): The cancer has grown into the layer of tive tissue under the lining layer of the bladder without growing into the thick layer of muscle in the bladder wall. The cancer has not spread to lymph nodes or to distant sites.

Stage ll (T2, N0, M0): The cancer has grown into the thick muscle layer of the bladder wall but, it has not passed completely through the muscle to reach the layer of fatty tissue that surrounds the bladder. The cancer has not spread to lymph nodes or to distant sites.

Stage ill (T3 or T4a, N0, M0): The cancer has grown completely through the bladder into the layer of fatty tissue that surrounds the bladder (T3). It may have spread into the prostate, uterus, or vagina (T4a). It is not growing into the pelvic or abdominal wall. The cancer has not spread to lymph nodes or to t sites.

Stage lV (Tdb, N0, M0) or (any T, N 1 to 3, M0) or (any T, any N, M1): The cancer has spread through the bladder wall to the pelvic or abdominal wall (T4b) and/or has spread to lymph nodes (N1-3) and/or to distant sites such as bones, liver, or lungs (M1).

Types of gall bladder oma More than 9 out of 10 gallbladder cancers are adenocarcinomas. An adenocarcinoma is a cancer that starts in the cells with gland-like properties that line many internal and external surfaces of the body ding the inside the digestive system).

A type of gallbladder adenocarcinoma that deserves l mention is called ary adenocarcinoma orjust papillary . These are gallbladder cancers whose cells are arranged in finger-like projections when viewed under a microscope. in general, papillary cancers are not as likely to grow into the liver or nearby lymph nodes. They tend to have a better prognosis (outlook) than most other kinds of gallbladder adenocarcinomas. About 6% of all adder cancers are papillary arcinomas.

There are other types of cancer that can develop in the gallbladder, such as adenosquamous carcinomas, squamous cell carcinomas, and small cell carcinomas, but these are uncommon.

Following stages of gall bladder carcinomas are distinguished based on the TNM system of the AJCC: Stage 0: Tie, N0, M0: There is a small cancer only in the epithelial layer of the adder. lt has not spread outside of the gallbladder.

Stage EA: T1 (a or b), N0, M0: The tumor grows into the lamina propria (T1 a) or the muscle layer (T1 b). it has not spread outside of the gallbladder.

Stage l8: T2, N0, M0: The tumor grows into the perimuscular fibrous tissue. it has not spread outside of the gallbladder.

Stage "A: T3, N0, M0: The tumor extends h the serosa layer and/or directly grows into the liver and/or one other nearby structure. lt has not spread to lymph nodes or to tissues or organs far away from the gallbladder.

Stage "3: T1 to T3, N1, M0: in addition to any growth in the gallbladder. the tumor has spread to nearby lymph nodes (N1). it has not spread to tissues or organs far away from the gallbladder.

Stage ill: T4, any N, M0: Tumor invades the main blood vessels leading into the liver or has d more than one nearby organ other than the liver. it may or may not have spread to lymph nodes. lt has not spread to tissues or organs far away from the gallbladder.

Stage lV: Any T, any N, M1: The tumor has spread to tissues or organs far away from the gallbladder.

Mammary carcinoma An adenocarcinoma refers generally to a type of carcinoma that starts in glandular tissue (tissue that makes and es a substance). in the context of breast cancer, he ducts and lobules of the breast are glandular , so cancers starting in these areas are often called adenocarcinomas. There are several types of breast cancer, although some of them are quite rare. In some cases a single breast tumor can have a combination of these types or have a e of invasive and in situ cancer.

Ductal carcinoma in situ (DClS; also known as intraductal carcinoma) is the most common type of non—invasive breast cancer. invasive (or infiltrating) ductal carcinoma (lDC) is the most common type of breast cancer. Invasive (or infiltrating) ductal carcinoma (lDC) starts in a milk passage (duct) of the breast, breaks through the wall of the duct, and grows into the fatty tissue of the breast. At this point, it may be able to spread (metastasize) to other parts of the body through the lymphatic system and tream. About 8 of 10 invasive breast cancers are infiltrating ductal carcinomas. lDC patients revealed expression of 00138 (Loussouarn et al., 2008).

Triple-negative breast cancer describe breast cancers (usually ve ductal carcinomas) whose cells lack estrogen receptors and progesterone receptors, and do not have an excess of the HERZ n on their surfaces. Triple-negative breast cancers tend to grow and spread more quickly than most other types of breast cancer.

Because the tumor cells lack these certain ors, r hormone therapy nor drugs that target HERZ are effective against these cancers (although chemotherapy can still be useful if ).

Some other breast cancers that fall under the term "mammary oma" are inflammatory breast cancer, medullary carcinoma, metaplastic carcinoma, mucinous carcinoma, tubular carcinoma, papillary carcinoma, adenoid cystic carcinoma (adenocystic carcinoma), phyllodes tumor.

Surgery, radiation or herapy constitutes standard cancer therapies. Hormone therapy is sometimes employed. Hormone therapy is a form of systemic therapy. it is most often used as an adjuvant therapy to help reduce the risk of cancer recurrence after surgery. although it can be used as neoadjuvant treatment, as well. it is also used to treat cancer that has come back after treatment or has spread. en promotes the growth of about 2 out of 3 of breast cancers -— those containing estrogen receptors (ER—positive cancers) and/or progesterone receptors (PR-positive cancers). Because of this, several approaches to blocking the effect of estrogen or lowering estrogen levels are used to treat ER-positive and PR-positive breast cancers. However, hormone therapy is ineffective for patients lacking ERs or PR5. y carcinoma also follows such a g system: Stage 0: Atypical cells have not spread outside of the ducts or lobules, the milk producing organs, into the surrounding breast tissue. Referred to as carcinoma in situ, it is classified in two types: "Ductal Carcinoma in Situ" (DClS), which is very early cancer that is highly ble and able and "Lobular Carcinoma in Situ" (LClS), which is not a cancer but an tor that identifies a woman as having an increased risk of developing breast cancer.

Stage I: The cancer is no larger than two centimeters (approximately an inch) and has not spread to nding lymph nodes or outside the breast.

Stage II: This stage is divided into two categories according to the size of the tumor and whether or not it has spread to the lymph nodes: Stage ll A Breast —the tumor is less than two eters and has spread up to three auxiliary underarm lymph nodes. Or, the tumor has grown bigger than two centimeters, but no larger than five centimeters and has not spread to surrounding lymph nodes.

Stage ll B Breast Cancer— the tumor has grown to between two and five centimeters and has spread to up to three auxiliary rm lymph nodes. Or, the tumor is larger than five centimeters, but has not spread to the surrounding lymph nodes.

Stage lll: This stage is also divided into two categories: Stage iii: A Breast Cancer—~the tumor is larger than two centimeters but smaller than five centimeters and has spread to up to nine auxiliary underarm lymph nodes.

Stage ill 8 Breast Cancer— the cancer has spread to tissues near the breast including the skin, chest wall, ribs. muscles, or lymph nodes in the chest wall or above the collarbone.

Stage IV: Here, the cancer has spread to other organs or tissues, such as the liver, lungs, brain, skeletal system, or lymph nodes near the collarbone.

Lung cancer There are 4 types of neuroendocrine lung tumors, namely, large cell neuroenclocrine carcinoma, atypical carcinoid tumor, typical carcinoid tumor and small cell lung cancer.

Carcinoid tumors are tumors that start from cells of the diffuse neuroendocrine system.

Typical and atypical carcinoid tumors look different under the microscope. l carcinoids grow slowly and only rarely spread beyond the lungs and about 9 out of 10 lung oids are typical carcinoids.

For treatment purposes two main types of lung cancer, which are very differently treated, are distinguished, namely, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). if the cancer has features of both types, it is called mixed small cellflarge cell cancer.

About 10% t015% of all lung cancers are the small cell type. Other names for SCLC are oat cell carcinoma and small cell undifferentiated carcinoma.

This cancer often starts in the i near the center of the chest. Although the cancer cells are small, they can divide y. form large tumors, and spread to lymph nodes and other organs throughout the body. Surgery is rarely an option and never the only treatment given. Treatment includes cytotoxic agents, such as drugs to kill the widespread disease.

There are 3 sub-types of NSCLC, namely squamous cell carcinoma; adenocarcinoma; large-cell (undifferentiated) carcinoma.

Staging of non-small cell lung cancer The system used to stage non-small cell lung cancer is the AJCC (American Joint tee on Cancer) system. Stages are bed using Roman numerals from 0 to IV (0 to 4). Some stages are further divided into A and B. As a rule, the lower the number, the less the cancer has spread. A higher , such as stage IV (4), means a more advanced cancer.

A respective staging system, including Stages l to N, was also developed for squamous cell carcinoma (head and neck ). Stage l cancers are small, localized and usually curable, stage ii and Ill cancers typically are y advanced and/or have spread to local lymph nodes and Stage lV cancers usually are metastatic (have spread to distant parts of the body) and generally are considered inoperable.

Treatment in the context of the present invention includes preventing or slowing the progression, izing the disease state, remitting the e or ameliorating one or more ms of a disorder ated with cells sing CD138. Treatment thus includes preventing or slowing down the increase of severity or the remission of the disorder. ln the case of MM generally only patients with stage it or III active MM receive primary therapy (stage l patients or patients with SMM are lly only observed in 3 to 6 month intervals), a treatment according to the present invention does not only include the treatment of, e.g., any active stage of MM, but also includes the treatment of forms of disease states that precede the traditionally treated disease state. Treatment in ular also includes preventing the progression from one disease state to the next: in the case of MM, this would, e.g., be the progression from MGUS to SMM or from SMM to active MM stage i or another stage of MM. In case of cancers of the exocrine pancreas, e.g., a progression from Stage l to Stage ll, including any worsening as reflected by the ries established by the AJCC within the stages, eg. from lA to lB. However, the term also includes maintaining the status quo, such as to maintain stable disease and, as discussed below, eliciting certain responses in the patient treated. A patient is also successfully ed" it the patient shows observable and/or measurable reduction in or absence of, inter alia, one or more of the following: reduction in the number of cancer cells or e of the cancer cells; reduction in the tumor size; inhibition (i.e., slow to some extent and preferably stop) of cancer cell infiltration into peripheral organs including the spread of cancer into soft tissue and bone; inhibition (i.e., slow to some extent and ably stop) of tumor metastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent, one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues. in general, an effect of a certain treatment on the e status of a patient can be monitored, in the case of MM, by ing the M- protein levels in the patient's serum and/or urine and/or the FLC levels in the patient’s serum and/or urine. in the case of other disorders associated with cells expressing CD138, other parameters are ed to assess the effect of a treatment according to the present invention. CRP ctive protein) is an unspecific inflammation parameter for clinical cancer monitoring. To name just a few. for pancreatic cancer. relevant parameters that may be measured are CA 19-9 (carbohydrate antigen 19.9, a tumor marker often elevated in pancreatic cancer), bilirubin, or CRP. In addition imaging such as sonography, CT. MRT are used. in head and neck cancer, biomarkers which depend on the tumor type are used (e.g., NSE (neuron-specific enolase) for Merkel cell or CEA (carcinoembryonic antigen); in breast carcinoma, CA 15-3Her2 expression and Cadherin expression may be used as markers, while the treatment is monitored by serum markers such as NSE.

The bladder tumor antigen (BTA) and the NMP22 tests can be used along with copy (using a thin, lighted tube to look in the bladder) in sing the condition in matic subjects. These tests are also being used to follow some patients after treatment, though cystoscopy and urine cytology (using a microscope to look for cancer celis in the urine) are still recommended as the standard tests for diagnosis and follow- up. ETA and NMP22 tests are often used n cystoscopies. Normal values may allow cystoscopy to be done less often. However, these test tests cannot replace urine cytology and copy.

For advanced bladder cancer, some of the markers used for other cancers such as CEA, CA 125, CA 19-9, and TPA (tissue polypeptide antigen) may be elevated and can be used to follow patients during and after treatment. For lung cancer, no established marker exists, but CEA pr NSE might be elevated.

Tumor cells such as myeloma cells or mammary carcinoma cells are known to shed CD138. The toss of surface CD138 is correlated with poor prognosis in myeioma. High levels of soluble CD138 have been also detected in other oncologic indications such as head and neck or lung cancer (Anttonen et al. 1999). The loss of surface Syndecan-1 is correlated with EMT (epithelial mesenchymal transition) this s describes the transformation of a malignant cell into a less or poorly differentiated cell associated with invasiveness and metastatic stage. This is e.g. ed for metastatic breast cancer (Loussouarn et al., 2008).

An effective amount of an agent, in particular, an immunoconjugate or a pharmaceutical composition comprising an immunoconjugate according to the present invention refers to an amount required to "treat" a disease or disorder in a subject, in ular a human t (patient). ln the case of cancer such as MM, the effective amount of the agent may reduce the number of cancer celis; reduce the tumor size; t (i.e., slow to some extent and preferably stop) cancer cell infiltration into W0 2013f083817 eral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent, one or more of the symptoms associated with the cancer. See the definition herein of "treatment".

"A pharmacokinetic equivalent" of, e.g., 200 mg/m2 refers to the amount of immunoconjugate that results in equal pharmacokinetics observed at dosages of 200 mg/m2 when the immunoconjugate is administered in combination, including co- administered with an agent for treating actual including potential adverse side effects ily on non-target cells that also express CD138. Those equivalents might be at less than 200 or somewhat more than 200, depending on the other agent.

Included are, e.g., ive amounts of less than 160, less than 170, less than 180, less than 190 and less than 210, less than 220, less than 230 and less than 240 mg/mz. For example, the person skilled in the art would expect that co—administration with corticosteroids or with antibiotics would allow slightly higher doses of the immunoconjugate even in cases of side effects on skin, which, can, however, be readily ascertained by the person skilled in the art.

To evaluate the success of the administration of a drug, here an immunoconjugate (i.e., its ability to e a functional response, i.e., its efficacy), different nses" to an administration are distinguished.

Responses are often evaluated by measuring efficacy blood parameters. Typical efficacy blood parameters are M- n level, FLC level or other markers that correlate to the disease in question to the efficacy of the immunoconjugate (disease specific marker), in ular the cancer in question. The efficacy tes the capacity for beneficial change of a given treatment. in the context of MM and other plasmaproliferative diseases, responses are distinguished as follows: the term complete response (CR) refers to the negative fixation of serum and urine and disappearance of any soft tissue plasmacytomas and <5% plasma cells in bone marrow; the term stringent complete response (sCR) refers to OR as defined above, plus normal FLC ratio and absence of clonal cells in bone marrow by immunohistochemistry or immunofluorescence; the term very good l response (VGPR) refers to serum and urine M-component detectable by immunofixatlon, but not on electrophoresis or _>_ 90% or greater reduction in serum M-component plus urine M—component <100 mg per 24h; the term partial response (PR) refers to 350% reduction of serum M protein and reduction in 24—h urinary M protein by 390% or to <200 mg per 24 h, if the serum and urine M protein are immeasurable, a _>_50% decrease in the difference between involved and uninvolved FLC levels is required in place of the M protein ia, if serum and urine M protein are immeasurable, and serum free light assay is also immeasurable, _>_50% ion in bone marrow plasma cells is required in place of M protein, provided baseline percentage was ~>_30%, in addition to the above criteria, if present at baseline, _>_50% reduction in the size of soft tissue plasmacytomas is also required (Durie et al., 2006).

The term minor response (MR) in relation to patients with relapsed] refractory myeloma refers in the context of the present invention to 325% but <49% reduction of serum M protein and reduction in 24h urine M protein by 50-89%, which still exceeds 200 mg per 24h, in on to the above criteria, if present at baseline, 25—49% reduction in the size of soft tissue plasmacytomas is also required, no increase in size or number of lytic bone lesions (development of compression fracture does not exclude se). r, a response, though not formally classified, also includes an at least 30%, preferably at least 40% or 50% reduction in serum FLC levels. This is in particular of cance in cases where M—protein cannot be measured.

The term stable disease (SD) refers, in the context of the proliferative es of the present invention, to the not meeting of the criteria for CR, VGPR, PR or ssive disease, while the term progressive disease (PD) refers to the increase of % from lowest response value in any one or more of the ing: - Serum M-component (absolute increase must be _>_ 0.59/100 ml) and/or - Urine M-component (absolute increase must be 3 200 mg per 24h) and/or W0 20131083817 — Only in patients without able serum and urine M-protein : the difference between involved and uninvolved FLC levels ute increase must be > 100 mg/i) - Bone marrow plasma cell percentage (absolute % must be 310%) ~ Definite development of new bone lesions or soft tissue plasmacytomas or definite increase in the size of existing bone lesions or soft tissue plasmacytomas - Development of hypercaicemia (corrected serum calcium >11.5mgl100 mi) that can be attributed solely to the plasma cell proliferative disorder.

The term relapsed myeloma refers herein to a form of active MM in a t, wherein said subject underwent at least one prior treatment regime, and which does not meet the criteria for relapsed] refractory myeloma.

The term refractory myeloma generally refers to a state of the disease when the number of plasma celis continues to increase even though treatment is give, that is the disease has, at the time of assessment, been proven irrespective to the treatment regime administered.

The term reiapsed/ refractory myeioma refers herein to the relapse of disease while on salvage therapy, or progression within 60 days of most recent therapy.

The term refractory phenotype es any type of refractory myeloma, that is, refractory and relapsed/refractory myeloma.

The term relapsed or refractory a covers relapsed, refractory and ed/refractory a.

A tumor or a CD138 target cell is said to be refractory to, e.g., a therapy/treatment if the CD 138 target cell continues dividing and/or the tumor continues growing at the same rate during such a therapy/therapy as without such therapy/treatment.

Tumor growth delay refers to a tumor growth that is delayed relative to regular tumor growth without treatment.

WO 83817 2012/074867 Tumor stasis refers to a state at which there is no further growth in tumor size.

Remission refers to a decrease in tumor size (partial remission), including the complete eradication of the tumor and absence of regrowth (complete remission).

Hormone therapy includes a therapy with a hormone. Cancer hormone therapy is employed to fight target cells. A hormone therapy is used, e.g., in the context of mammary carcinoma or te cancer and include the administration of estrogen and progesterone or derivatives thereof.

Chemotherapy is the treatment of cancerous cells with an antineoplastic drug such as taxane or with a combination of such drugs in a standardized ent regime.

Maintenance therapy is a therapy that follows a prior treatment. and aims at maintaining the status obtained when completing said primary treatment. For example, if the prior treatment resulted in a partial response, the maintenance therapy is designed to maintain partial response.

In the clinical study discussed in more detail below, the ts had been treated with at least one immunomodulator and a proteosome inhibitor therapy. which have failed, prior to entering the study. Disease was considered treatment tory if the subject experienced progressive disease (PD) on his or her previous regimen.

The term "progression to"! e.g., "active MM" in relation to patients with SMM refers in the context of the present invention to evidence of progression based on the lMWG (international Myeloma g Group) criteria for progressive e in MM and any one or more of the following felt d to the underlying clonal plasma cell proliferative disorder, development of new soft tissue plasmacytomas or bone s, hypercalcemia (>11mg/100 ml), se in hemoglobin of 22g/100 ml, and serum creatinine level 22mg/100 ml. (Kyle & Rajkumar. 2009).

Progression free survival is the duration from start of a treatment to disease progression or death (regardless of cause of , whichever comes first. When a reference is made to "progression free survival" without a reference to time period. lack of progression of more than 3 months is implied.

The pathogenesis of multiple myeloma involves binding of myeloma cells, via oell~ surface adhesion molecules, to bone marrow stroma cells (BMSCs) as well as the ellular matrix (ECM). This binding triggers, and thus can be made ultimately responsible, for multiple myeloma cell growth, drug resistance, and migration of MM cells in the bone marrow milieu (Munshi et al. 2008). in particular, the adhesion of le a cells to ECM via syndecan-‘l (CD138) to type l en induces the expression of matrix metalloproteinase 1, thus promoting bone tion and tumor invasion (Hideshima et al. 2007). interactions between multiple myeloma cells and the bone marrow microenvironment results in activation of a pleiotrcpic proliferative and anti—apoptotic cascade.

For multiple myeloma patients, but also for patients suffering from other diseases that are associated with bone pains, a number of supportive treatments exist to treat this and other symptoms. Appropriate medications include bisphosphonates (eg. pamidronate, zoledronic acid) which can slow the bone damage. It has been demonstrated that these agents are able to reduce osteolytic bone lesions and prevent fractures (Ludwig et al., 2007). They are mostly given through a vein to se the risk of bone complications like fractures and to lower abnormally high blood calcium levels (Hypercalcemia). Data suggests that bisphosphonates reduce bone pain associated with MM. Patients may also have surgery if their bones are weak or break. in one embodiment, the immunoconjugates reduce; in particular reduce to an acceptable level, bone pains and/or bone complications, such as osteonecrosis. A reduction to an acceptable level involves in particular the ability to discontinue the administration of a medication that alleviates these pains or is aimed at reducing said bone complications. sphonates, such as pamidronate, zoledronic acid and clodronate, are commonly stered to alleviate bone complications, such as ecrosis in MM patients and thereby to alleviate bone pains associated with said compiications. Common bisphosphonates include, for oral administration, FOSOMAX, BONIVA, ACTONEL, DlDRONEL and SKELlD, for intravenous administration, S, AREDlA and ZOMETA.

Following the homing of multiple myeloma cells to the bone marrow stromai compartment, adhesion between multiple myeloma cells and BMSCs upregulates many W0 2013/08381 7 cytokines like interleukin-6 (IL-6) and insulin like growth factor 1 (lGF-1) which have angiogenic and tumor growth promoting activities (Hideshima et al. 2007). The signalling cascades initiated by these cytokines eventually result in MM cell ance to conventional therapeutics (Anderson et al. 2000; Hideshima et al. 2006).

In the normal human hematopoietic compartment, CD138 expression is restricted to plasma cells (Wijdenes, 1996; i, 1999) and CD138 is not expressed on peripheral blood cytes, monocytes, granulocytes, and red blood cells. In particular, CD34+ stem and progenitor cells do not express CD138, and D138 mAbs do not affect the number of colony forming units in hematopoietic stem cell es (Wijdenes, 1996). In non-hematopoietic compartments, CD138 is mainly expressed on simple and stratified lia within the lung, liver, skin, kidney and gut. Only a weak staining was seen on endothelial cells eld, 1992; Vooijs, 1996). it has been reported that CD138 exists in polymorphic forms in human lymphoma cells (Gattei, 1999). CD138 epithelial tissue of the gastrointestinal tract, skin, and eye are the non—target tissues that are most prone to be targeted by immunoconjugates of the present invention resulting in toxicities.

Monoclonal antibodies B-B4, BC/B—B4, B-BZ, DL-101, 1 D4, Ml15, 1.BB.210, 201484, 5F7, 104—9, 281 -2 in particular 8-84 have been reported to be specific to CD138, Of those B-B4, 1 D4 and M15 recognized both the intact molecule and the core protein of CD138 and were shown to recognize either the same or closely related epitopes i, 1999). us studies reported that B-B4 did not recognize soluble CD138, but only CD138 in membrane bound form (Wijdenes, 2002).

The initial anti-CD138 antibody was developed by Diaclone SAS (Besancon, France) as the murine parental Mab B-B4 generated by immunization with the human multiple myeloma cell line U266, using standard hybridoma technology nt,1995; Wijdenes, 1996). B-B4 binds to a linear epitope between residues 90-93 of the core protein on human syndecan-1 (CD138) (Wijdenes, 1996; Dore, 1998)- Consistent with the expression pattern of CD138, B-B4 was shown to strongly react with plasma cell line 26, but not to react with endothelial cells. Also consistent with the expression n of CD138, B-B4 also reacted with epithelial cells lines A431 (keratinocyte derived) and HepGZ (hepatocyte derived). An immunotoxin B-B4wsaporin was also highly toxic towards the plasma cell line RPMl8226, in fact considerably more toxic than free saporin. However, from the two epithelial cell lines tested, B-B4—saporin showed only ty towards cell line A431, although in a clonogenic assay B—B4— saporin showed no inhibitory effect on the outgrowth of A431 cells (Vooijs, 1996).

Other researchers reported lack of specificity of MM—associated ns against tumors (Couturier, 1999). 8—84 covalently linked to the maytansinoid DM1 showed selective cytotoxicity on multiple myeloma cell lines and cells, as well as anticancer activity in human le myeloma xenograft models in SCID mice (Tassone, 2004).

The present invention uses the term tumor cell to include cancer cells as well as pre- ous cells which may or may not form part of a solid tumor. Preferred tumor cells to be treated are cells of poietic malignancies.

A solid tumor according to the present invention is an abnormal mass of tissue that usually does not contain cysts or liquid areas. A solid tumor according to the present ion ses target tumor cells expressing 00138 and thus is a malignant solid tumor. Different types of solid tumors are named for the type of cells that form them. es of solid tumors are sarcomas, carcinomas, and lymphomas. Hematopoietic malignancies generally do not form solid tumors. Mammary carcinoma and prostate carcinoma are two examples of malignant solid tumors.

A targeting agent according to the present invention is able to associate with a molecule expressed by a target cell and includes peptides and non-peptides. In ular, targeting agents ing to the present invention include targeting antibodies and non—immunoglobulin targeting les, which may be based on non- immunogiobulin proteins, including, but not limited to, N® molecules, ANTlCALlNS® and AFFIBODIES®. Non—immunoglobulin targeting molecules also include non-peptidic targeting molecules such as targeting DNA and RNA oligonucleotides (aptamers), but also physiological ligands, in particular ligands of the antigen in question, such as CD138.

A targeting antibody according to the present invention is or is based on a natural antibody or is produced synthetically or by genetic engineering and binds to an antigen WO 83817 2012/074867 on a cell or cells (target cell(s)) of interest. A targeting antibody according to the present invention includes a monoclonal antibody, a polyclonal antibody, a multispecific antibody (for example, a bispecific dy), or an dy fragment. The targeting antibody may be engineered to, for example, improve its affinity to the target cells (Ross, 2003) or diminish its immunogenicity. The targeting antibody may be attached to a liposomal formulation ing effector molecules (Carter, 2001). An antibody fragment comprises a portion of an intact antibody, preferably the antigen binding or le region of the intact dy. Examples of dy fragments according to the present invention include Fab, Fab’, F(ab')2, and Fv nts, but also ies; domain dies (dAb) (Ward, 1989; United States Patent 079); linear antibodies; single-chain antibody molecules; and pecific antibodies formed from antibody fragments. ln a single chain variable fragment antibody (scFv) the heavy and light chains (VH and VL) can be linked by a short amino acid linker having, for example, the sequence (glycine4serine)n, which has sufficient flexibility to allow the two domains to assemble a functional antigen binding pocket. Addition of various signal sequences may allow for more precise ing of the targeting antibody Addition of the light chain constant region (CL) may allow dimerization via disulphide bonds, giving increased ity and avidity. Variable regions for constructing the scFv can, if a mAb against a target of interest is ble, be obtained by RT-PCR which clones out the variable regions from mRNA extracted from the parent hybridoma. Alternatively, the scFv can be generated de novo by phage display technology (Smith. 2001). As used herein. the term "functional fragment", when used in reference to a targeting antibody, is intended to refer to a portion of the targeting antibody which is capable of specifically binding an antigen that is cally bound by the antibody reference is made to. A bispecific antibody according to the present invention may, for example, have at least one arm that is reactive against a target tissue and one arm that is reactive against a linker moiety (United States Patent Publication 20020006379). A bispecific antibody according to the present invention may also bind to more than one antigen on a target cell r. 2001). An antibody according to the present invention may be modified by, for example, introducing cystein residues to introduce thiol groups (Olafsen, 2004). in accordance with the present invention, the targeting antibody may be derived from any source and may be, but is not limited to, a camel antibody, a murine antibody, a chimeric human/mouse antibody or a chimeric human/monkey antibody, in particular, a chimeric human/mouse antibody such as nBT062.

Humanized antibodies are antibodies that contain sequences derived from a human-antibody and from a man antibody and are also within the scope of the present invention. Suitable methods for humanizing dies include COR-grafting (complementarity determining region ng) (EP 0 239 400; WO 91/09967; United States Patents 5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padian, 1991; Studnicka et al., 1994; a et al., 1994), chain shuffling (United States Patent 5,565,332) and DelmmuncsatlonTM (Biovation, LTD). in CDR— ng, the mouse complementarity-detennining regions (CDRs) from, for example, mAb 8-84 are grafted into human variable frameworks, which are then joined to human constant regions, to create a human B—B4 antibody (hB-B4). Several antibodies humanized by CDR-grai’ting are now in clinical use, inciuding MYLOTARG -rs et al., 2001) and HECEPTIN (Pegram et al, 1998).

The resurfacing logy uses a combination of molecular modeiing, statistical analysis and mutagenesis to alter the non-CDR surfaces of dy variable regions to resemble the surfaces of known antibodies of the target host. Strategies and methods for the resurfacing of antibodies, and other methods for ng immunogenicity of antibodies within a ent host, are disclosed, for example, in United States Patent 5,639,641. Human antibodies can be made by a variety of methods known in the art including phage display methods. See also United States Patents 887, 4,716,111, 5,545,806, and 5,814,318; and international patent application publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96133735, and WO 91/10741.

Targeting antibodies that have undergone any tural modification such as chimeric human/mouse antibodies or a chimeric human/monkey antibodies, humanized antibodies or antibodies that were engineered to, for example, improve their affinity to the target cells or diminish their genicity but also antibody fragments, in particular functional fragments of such targeting antibodies that have undergone any non-natural modification, diabodies; domain antibodies; linear antibodies; single-chain antibody les; and multispecific antibodies are referred to herein as engineered targeting antibodies.

W0 2013.1083817 Chimerized antibodies, maintain the dy g region (ABR or Fab region) of the non-human antibody, e.g., the murine antibody they are based on, while any constant regions may be provided for by, e.g., a human dy. Generally, chimerization and/or the exchange of constant regions of an antibody will not affect the affinity of an antibody because the regions of the antibody which contribute to antigen binding are not affected by this exchange. in a preferred embodiment of the present invention, the engineered, in particular chimerized, antibody of the present invention, may have a higher binding affinity (as expressed by K0 values) than the respective non- human antibody it is based on. in particular, the nBT062 antibody and antibodies based thereon may have higher antibody affinity than the murine B—B4. ln another preferred ment of the present invention, immunoconjugates comprising those engineered/chimerized antibodies also display this higher antibody affinity. These immunoconjugates may also y in certain embodiments other advantageous properties, such as a higher reduction of tumor load than their B-B4 containing counterparts. in a preferred embodiment, the engineered, in particular ized targeting antibodies display binding affinities that are characterized by dissociation constants KD (nM) of less than 1.6, less than 1.5 or about or less than 1.4, while their murine counterparts are terized by dissociation constants Kg (nM) of about or more than 1.6. conjugates sing targeting agents such as targeting antibodies may be characterized by iation constants of K0 (nM) of less than 2.6, less than 2.5, less than 2.4, less than 2.3, less than 2.2, less than 2.1, less than 2.0, less than or about 1.9 are preferred, while immunoconjugates comprising the murine counterpart antibodies may be terized by dissociation constants KB (nM) of about or more than 2.6 (compare Table 12 Materials and Methods).

The basic antibody molecule is a tional structure wherein the variable regions bind antigen while the remaining constant regions may elicit antigen independent responses. The major classes of antibodies, lgA, lgD, lgE, lgG and lgM, are determined by the constant regions. These classes may be further divided into subclasses (isotypes). For example. the lgG class has four isotypes, namely, lth, lgGZ, lgGS, and lgG4 which are determined by the constant regions. Of the various human antibody classes, only human ith, lgG2, lgGS and lgM are known to effectively activate the compiement . While the constant regions do not form the antigen binding sites, the arrangement of the nt regions and hinge region may confer segmental flexibility on the le which allows it to bind with the antigen.

Different lgG isotypes can bind to Fc receptors on cells such as monocytes, B cells and NK cells, thereby activating the cells to release cytokines. ent isotypes may also activate complement, resulting in local or systemic inflammation. In particular, the different lgG isotypes may bind FcyR to different degrees. FcyRs are a group of surface glycoproteins belonging to the lg superfamily and expressed mostly on leucocytes. The chR glycoproteins are divided into three classes ated chRl (CD64), Fclel (C032) and chRlll (CD16). While lgG1, lgGZ and lgG3 bind ly to a variety of these classes of chR glycoproteins, lgG4 ys much weaker binding. in particular, 1964 is an intermediate binder of chRl, which results in relatively low or even no ADCC (antibody dependent cellular cytotoxicity), and does not bind to chRlllA or FclelA. lgG4 is also a weak binder of chRllB, which is an inhibitory receptor.

Furthermore, lgG4 mediates only weak or no complement fixation and weak or no complement dependent cytotoxicity (CDC). in the context of the present ion, lgG4 may be specifically employed to prevent Fc-mediated targeting of hepatic FcR as it displays no interaction with Fchll on LSECs (liver sinusoidal endothelial cells), no or weak interaction with FcRyl-lll on Kupffer cells (macrophages) and no interaction with FcRylil on hepatic NK cells. Certain mutations that further reduce any CDC are also part of the present invention. For example lgG4 residues at positions 327, 330 and 331 were shown to reduce ADCC (antibody dependent ar cytotoxicity) and CDC (Amour, 1999; Shields. 2001). One of more mutations that stabilize the antibody is also part of the present invention (also referred to herein as "stabilizing mutations"). Those mutations e in particular, e-to-glutamic acid mutations in the CH2 region of lgG4 and serine-to-proline exchanges in the lgG4 hinge core. These mutations decrease, in certain embodiments of the invention, the amount of half-molecules to less than 10%. less than 5% and preferably less than 2% or 1%. Moreover, the in vivo half life of so ized antibodies might be increased several days, including 1, 2, 3, 4 or more than 5 days (Schuurman, 1999).

When the present invention refers to an immunoconjugate comprising an engineered targeting antibody conferring lgG4 e properties, this means that the engineered targeting antibody shows significantly reduced affinity to Fe receptor sing cells as compared to the affinity of antibodies of [961 isotype. These properties are preferably red by a further antibody region, which is distinct from the ABR, wherein said further antibody region is in whole or part of a human antibody.

The result is a significantly reduced (more than 90% relative to its lgG1 isotype counterpart) or the total lack of a potential to induce CDC or ADCC as compared to the potential to induce CDC or ADCC usually observed with lgG1 isotype antibodies. This property can be ed in cell based assays by using the engineered ing antibody in its unconjugated form. CDC and ADCC can be measured via different methods such as the one disclosed in Cancer lmmunol. lmmunother., 36, 373 (1993) or the GUAVA Cell Toxicity Assay. The overall benefit of immunoconjugates comprising at least part of an engineered ing antibody conferring lgG4 isotype properties is an improvement of g specificity and a reduced toxicity. Also the resulting d affinity to Fc receptors improves antigen-specific targeting of tumor cells g to reduced toxicity against CD138 negative cells.

Targeting agents, including targeting antibodies disclosed herein may also be described or specified in terms of their binding affinity to an antigen, in particular to CD138. Preferred binding ies of targeting agents such as targeting antibodies are characterized by iation constants KD (nM) of less than 1.6, less than 1.5 or about or less than 1.4. For immunoconjugates comprising said targeting agents such as targeting antibodies dissociation constants Kn (nM) of less than 1.6, less than 1.5 or less than 2.5, less than 2.4, less than 2.3. less than 2.2. less than 2.1, less than 2.0, less than or about 1.9 are preferred.

An antigen binding region (ABR) according to the present ion will vary based on the type of targeting antibody or engineered targeting antibody employed. in a naturally occurring antibody and in most chimeric and humanized antibodies, the antigen binding region is made up of a light chain and the first two domains of a heavy chain. However, in a heavy chain antibody devoid of light chains, the antigen binding region will be made up of, e.g., the first two domains of the heavy chain only, while in single chain antibodies , which combine in a single polypeptide chain the light and heavy chain variable domains of an dy le, the ABR is provided by only one polypeptide molecule. FAB fragments are usually obtained by papain digestion and have one light chain and part of a heavy chain and thus comprise an ABR with only one antigen combining site. On the other hand. diabodies are small dy fragments with two antigen-binding regions. In the context of the present invention, however, an antigen binding region of a targeting dy or on engineered targeting antibody is any region that primarily determines the binding specificity of the targeting antibody or the engineered targeting dy.

If an ABR or another targeting antibody region is said to be "of a certain antibody", e.g., a human or non-human antibody, this means in the t of the present invention that the ABR is either cal to a ponding naturally occurring ABR or is based thereon. An ABR is based on a naturally occurring ABR if it has the binding specificity of the naturally occurring ABR. However, such an AER may comprise, e.g., point mutations, ons, deletions or posttranslational modification such as glycosyiation.

Such an ABR may in particular have more than 70%, more than 80%, more than 90%, ably more than 95%, more than 98% or more than 99% sequence identity with the sequence of the lly occurring ABR. nBT062 (see also is a murine human chimeric lgG4 mAb, namely a chimerized version of 8-84. This chimerized n of B~B4 was created to reduce the HAMA (Human Anti-Mouse Antibody) response, while maintaining the functionality of the antibody binding region of the 8—84 for CD138. Surprisingly, the results obtained using an immunoconjugate comprising this ered targeting antibody were much more homogenous (the variance in the results was reduced). The protocol for producing nBT062 is specified below. Chinese hamster ovary cells expressing nBT062 have been deposited with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1, D-38124 Braunschweig on December 11, 2007. The identification number is DSM ACCZ875. A CD138 specific chimeric antibody based on 8-84 is generically referred to herein as c—B-B4.

The amino acid sequence for both the heavy and the light chains has been predicted from the translation of the nucleotide sequence for nBT062. The amino acid sequences predicted for the heavy chain and light chain are presented in Table 4. Predicted variable regions are bolded, predicted CDRs are underlined. -61..

Tabie 4. Predicted Amino Acid Sequence for nBT062 - nBT082 heavy chain predicted sequence (SEQ ID NO:1): QVQLQQSGSE LMMPGASVKI SCKATGYTFS NYWIEWVKQR PGHGLEWIGE 51 ILPGTGR‘I‘IY NEKFKGKATF TADISSNTVQ MQLSSLTSED SAVYYCARBE 101 YYGNFYYAbflJ YWGQGTSVTV SSASTKGPSV FPLAPCSRST SESTAALGCL 151 VKDYFPEPVT VSWNSGALTS GVHTE‘PAVLQ SSGLYSLSSV VTVPSSSLGT 201 KTYTCNVDHK DKRV ESKYGPPCPS ‘LGGP SVFLFPPKPK 251 DTLMISRTPE VTCVVVDVSQ FNWY VDGVEVHNAK TKPREEQFNS 301 TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSS IEKTISK AKGQPREPQV 351 YTLPPSQEEM TKNQVSLTCL SDIA VEWESNGQPE NNYKTTPPVL 401 DSDGSFE‘LYS RLTVDKSRWQ EGNVFSCSVM HYTQKSLSLSLG (K) The C—terminal lysine is prone to clipping and might be present due to incomplete clipping to a certain extent. The (K) in parenthesis is not part of SEQ lD N021. — nBT062 light chain predicted sequence (SEQ ID NO:2): ‘QSTSS LSASLGDRV‘L‘ ISCSASQGIN NYLNWYQQKP DGTVELLIY: 51 TSTLQSGVPS RFSGSGSG'I'D YSLTISNLEP EDIGTYYCQQ. YSKLPRTFGG 101 GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV 151 GNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK THQG 201 LSSPVTKSFN RGEC Table 5. shows a comparision of the general CDR definitions of Krabat and Chothia and the predicted CDRs for nBT062 Kabat CDR definition nBT062 Light chain CDR1: residues 24-34 CDR1: es 24-34 CDRZ: residues 50-56 CDRZ: residues 50-56 CDR3: residues 89-97 CDRB: residues 89-97 Heavy chain CDR1: residues 31-35 CDR1: residues 31-35 CDRZ: residues 50-56 CDRZ: residues 51-68 CDRB: residues 95-102 CDRB; residues 99-111 W0 2013,!083817 Chothia CDR definition nBT062 Light chain CDRi: residues 28-32 CDR1: residues 24-34 CDRZ: residues 50-52 CDRZ: residues 50-56 CDRB: residues 91-96 CDRS: residues 89-97 Heavy chain 00th residues 26-32 CDRt: residues 31-35 CDRZ: es 52-56 CDR2: es 51-68 CDRS: residues 96-101 CDR3: residues 99—1 11 Fully human antibodies may also be used. Those antibodies can be selected by the phage display ch, where (30138 or an antigenic determinant thereof is used to selectively bind phage expressing, for example, B~B4 variable s (see, Krebs, 2001). This approach is advantageously coupled with an affinity tion technique to improve the affinity of the antibody. All antibodies referred to herein are isolated antibodies (See US Patent Publication 20090175863).

In one embodiment, the targeting antibody is, in its unconjugated form, moderately or poorly internalized. Moderate internalization constitutes about 30% to about 75% internalization of total antibody, poor internalization constitutes about 0.01% to up to about 30% internalization after 3 hours incubation at 37°C. in another preferred embodiment the targeting antibody binds to CD138, for example, antibodies B-B4, BC/B-B4, B—BZ, DL-iOi, 1 D4, M115, 1.88.210, , 5F7, 104-9, 281-2 in particular B—B4. oma cells, which were generated by izing SP02/0 myeloma cells with spleen cells of Balb/c mice have been deposited with the DSMZ- Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, roder Weg 1, D-38124 Braunschweig on December 11, 2007. The identification number of these 8- B4 expressing hybridoma cells is DSM AC02874. in another embodiment, the targeting antibody does not ntially bind non—cell~surface expressed C01 38.

When, in the context of the present invention, the name of a specific dy is combined with the term "targeting antibody" such as "nBT062 targeting antibody," this means that this targeting antibody has the binding specificity of the antibody nBT062. if a targeting antibody is said to be "based on" a specified antibody, this means that this targeting antibody has the binding specificity of this antibody. but might take any form consistent with the above description of a targeting antibody. When. in the context of the present invention, the name of a specific antigen is combined with the term "targeting antibody' such as "CD138 targeting antibody," this means that this targeting antibody has binding city for CD138. it, in the context of the present invention. for example, a targeting antibody is said to do something "selectively" such as "selectively targeting cell-surface expressed CD138" or, to be "selective" for something, this means that there is a significant selectivity (Le. a higher affinity towards CD138- positive cells compared with CD188-negative cells) for, in the case of the example provided. cell-surface expressed 001 38, compared to any other cell-surface expressed antigen. Adverse side effects in a given environment may be substantially reduced or even avoided due to this selectivity.

"Non-immunoglobulin targeting les" according to the present invention include targeting molecules derived from non-immunoglobulin proteins as well as non- peptidic targeting molecules. Small munoglobulin proteins which are included in this definition are designed to have specific affinities towards; in particular, surface expressed CD138. These small non-immuncglobulin proteins include scaffold based engineered molecules such as AFFlLlN molecules that have a relatively low molecular weight such as n 10 kDa and 20 kDa. Appropriate scaffolds include, for example, gamma crystalline. Those les have, in their natural state, no specific binding activity towards the target molecules. By engineering the protein surfaces through locally defined randomization of t exposed amino acids. tely new binding sites are created. Former non-binding proteins are y transformed into specific binding proteins. Such molecules can be specifically designed to bind a target, such as 00138, and allow for specific delivery of one or more effector molecules (see, scil Proteins GmbH at wvvwscilproteinscom, 2004). Another kind of non- globulin ing molecules are derived from lipocalins. and e, for example LlNS, which resemble in structure somewhat immunoglobulins.

However, Iipocalins are composed of a single polypeptide chain with 180 to 180 amino acid residues. The binding pocket of lins can be reshaped to recognize a molecule of interest with high affinity and specificity (see, for example, Beste et al., 1999). Artificial bacterial receptors such as those marketed under the trademark W0 2013I083817 Affibody® (Affibody AB) are also within the scope of the present invention. These artificiai bacterial receptor molecules are small. simple proteins and may be composed of a helix bundle based on the scaffold of one of the lgG-binding domains of Protein A (Staphylococcus aureus). These molecules have binding properties r to many globulins, but are substantially smaller, having a molecular weight often not exceeding 10kDa and are also comparatively stable. Suitable artificial ial receptor molecules are, for example, described in United States Patents 5.831 ,012; 6,534,628 and 6,740,734.

Other mmunoglobulin targeting molecules" are physiological ligands of the antigen in question. logical ligands of CD138 include for e, but not limited to, 4(aggrecanase-1), antithrombin—3, bFGF, cathepsin G, CCL5 S), CCL7, CCL11, CCL17, CD44, collagens (collagen type 1, collagen type 2. collagen type 3, collagen type 4, collagen type 5, collagen type 6), CXCL1, elastase, gp120 HGF [hepatocyte growth factor}, laminin—i, laminin-Z, laminin-S, midkine, MMP-7, neutrophil elastase, and pleiotrophin (HBNF, HBGF-B). Non—peptidic targeting molecules include, but are not limited to, to DNA and RNA oligonucleotides that bind to CD138 (aptamers).

An "effector molecule" according to the present invention is a molecule or a derivative, or an analogue thereof which is attached to a ing agent, in particular a targeting antibody and/or an ered targeting antibody, and that exerts a desired effect, e.g., apoptosis, or r type of cell death, or a continuous cell cycle arrest on the target cell or cells. Effector molecules according to the present invention include molecules that can exert desired effects in a target cell and include, but are not limited to, cytotoxic drugs, including low molecular weight cytotoxic drugs (Molecular mass of less than 1500 Da, preferably less than 1400, less than 1200, less than 1000, less than 800, less than 700, less than 600, less than 500, less than 300 but generally more than 120 Da). These cytotoxic drugs are, according to the present invention, generally non- proteinaceous biological cytotoxic drugs and contain or induce, upon administration, the production of another cytotoxic drug of at least 5 C atoms, 10 C atoms, preferably more than 12 C atoms, often more than 20 C atoms and sometimes more than 30, 40 or 50 C atoms and generally at least one ring structure, such as a benzene ring, which is often substituted. r, often interconnecting ring structures are part of these molecules.

These non-proteinaceous biological cytotoxic drugs may intercalate into DNA (DNA intercalators) or alkylate DNA, inhibit microtubule ion, are inhibitors of mitosis, 2012/074867 inhibitors of enzymes involved in the structural integrity of DNA, such as histone deacetylate or inhibitors of enzymes that are otherwise vital to a cell and cause disruption of cell metabolism. Effectors can also be rized as radionuclides, biological response modifiers, pore—forming agents, ribonucleases, proteins of apoptotic signaling cascades with apoptosis-inducing ties, nse oligonucleotides, anti~ metastatic agents, anti-oxidative substances, antibodies or cytokines as well as functional derivatives or ues/fragments thereof. in a preferred embodiment, the effector molecule increases internal effector ry of the immunoconjugate, in particular when the natural form of the antibody on which the targeting dy of the immunoconjugate is based is poorly intemalizable.

In another preferred embodiment the effector is, in its native form, non-selective. ln certain embodiments the effector has high lective toxicity, including systemic toxicity. when in its native form. The "native form" of an effector molecule of the present invention is an effector molecule before being attached to the targeting agent to form an immunoconjugate. In another preferred embodiment, the non-selective toxicity of the effector molecule is substantially eliminated upon conjugation to the targeting agent. In another preferred embodiment, the effector molecule causes, upon reaching the target cell, death or cell cycle arrest, including uous cell cycle arrest, in the target cell.

An effector molecule according to the present invention includes, but is not limited to, antineoplastic agents, in ular intracellular chemotherapeutic , which are defined below.

Moiecular mass 2012/074867 Sylimarin 482 com uonents Aminolevulinic 132 acid Epigallocatechin 459 - allate EGCG Psoralene 186 Melphalan 304 Table 6 es examples of low molecular weight cytotoxic drugs that may serve as effector molecules.

Low molecular weight cytotoxic drugs (see above for molecular weights) may ably be antimitotics, more particular, tubulin affecting agents, which include inhibitors of tubulin polymerization such as maytansinoids, dolastatins (and derivatives such as auristatin) and crytophycin and potent taxoid (taxane) drugs (Payne, 2003).

Further included in the definition of small highly cytotoxic drug are other tubulin interfering agents such as epothilones (e.g. ixabepilone) and colchicine derivatives (tubulin interfering agents are further discussed .

An effector molecule that is a maytansinoid includes maytansinoids of any origin. including, but not limited to synthetic sinol and maytansinol analogue and derivative.

Maytansine is a natural product originally derived from the Ethiopian shrub Maytenus serrate (Remillard, 1975; United States Patent 3,896,111). This drug inhibits tubulin rization, resulting in mitotic block and cell death (Remillard, 1975; Bhattacharyya, 1977; Kupchan, 1978). The cytotoxicity of maytansine is 200fold higher than that of anti-cancer drugs in clinical use that affect tubulin polymerization, such as Vinca alkaloids or taxol. However, clinical trials of maytansine indicated that it lacked a therapeutic window due to its high systemic toxicity. Maytansine and sinoids are highly cytotoxic but their clinical use in cancer y has been greatly limited by their severe systemic side-effects ily attributed to their poor selectivity for tumors. Clinical trials with maytansine showed serious adverse effects on the central s system and gastrointestinal system. ~67— WO 83817 2012/074867 Maytansinoids have also been isolated from other plants including seed tissue of Trewia nudifiora d States Patent 4,418,084) Certain microbes also produce maytansinoids, such as sinol and C-3 maytansinol esters (United States Patent 042).

The present invention is directed to maytansinoids of any origin, including synthetic maytansinol and maytansinoi analogues which are sed, for example, in United States Patents 4,137,230; 870; 4,256,746; 4,260,608; 4,265,814; 4294J57;v4307016; 4308268; 4308269; 4309A28; 4313348;4431&92& 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,371,533; 4,424,219 and 4,151,042. in a preferred embodiment, the maytansinoid is a thiol-containing maytansinoid and is more preferably produced according to the processes disclosed in United States Patent 6,333,410 to Cheri et al or in Chari et al. (Chari, 1992).

DM—1 acetyl—NZ-(B-mercapto-i~oxopropyl)—maytansine) is a preferred or molecule in the context of the present invention. DM1 is 3- to d more cytotoxic than maytansine, and has been converted into a pro-drug by linking it via disulfide bond(s) to a monoclonal antibody directed towards a tumor-associated antigen. Certain of these conjugates imes called "tumor activated prodrugs" (TAPs)) are not cytotoxic in the blood compartment, since they are activated upon associating with a target cells and internalized, thereby releasing the drug (Blattler, 2001). Several antibody-DM1 conjugates have been ped (Payne, 2003), and been evaluated in clinical trials. For example, hu0242-DM1 treatment in colorectal cancer patients was well tolerated, did not induce any detectable immune response, and had a long circulation time (Tolcher, 2003).

Other particularly preferred maytansinoids comprise a side chain that contains a sterically hindered thiol bond such as, but not limited to, maytansinoids NZ—deacetyl— N2'-(4-mercapto-1—oxopentyl)-maytansine, also referred to as "DM3," and Nzldeacetyh N2'-(4-methyl-4—mercapicoxopentyl)—maytansine, also referred to as "DM4." The synthesis of DM4 is shown in FIGS. 3 and 4 and is described elsewhere herein. 0M4 s from DM1 and DM3 in that it bears methyl groups at its 00. This results in a stericai hindrance when DM4 is attached via a linker in particular, but not limited to, a linker comprising a de bond, to a targeting agent such as nBT062. A wide y of maytansinoids bearing a sterically hindered thiol group (possessing one or two substituents, in particular alkyls substituents, such as the methyl substituents of DM4) are disclosed US. Patent Publication 200470235840, published Nov. 25, 2004, which is incorporated herein in its entirety by reference. The steric hindrance conferred by alkyl groups such as the methyl groups on the carbon adjacent to the sulfur atom of DM3 and DM4 may affect the rate of intracellular cleavage of the immunoconjugate. The variable alkyl unit may therefore affect potency, efficacy, and safety/toxicity in vitro and In vrvo.

As reported by Goidmakher et al. in U.S. Patent Publication 233814, such a hindrance induces tion (e.g., methylation) of the free drug once the drug is released at its target. The aikylation may increase the stability of the drug allowing for the so-called bystander effect. However, as the person d in the art will appreciate, other effector molecules comprising substituents such as alkyi groups at ons that result in a sterlcai hindrance when the effector is attached to a targeting agent via a linker are part of the present invention (US. Patent Publication 2004/0235840). Preferably this nce induces a chemical modification such as alkyiation of the free drug to increase its overall stability, which allows the drug to not only induce cell death or continuous cell cycle arrest in CD138 expressing tumor cells but, optionally, also to affect auxiliary cells that, e.g., support or protect the tumor from drugs, in particular cells of the tumor stroma and the tumor vasculature and which generally do not express CD138 to sh or lose their supporting or protecting function.

Maytansine was evaluated in Phase l and Phase ll clinical trials sponsored by the National Cancer institute (NCl) under IND #511,857 (submitted to FDA on September 19, 1975). Both complete and partial responses were seen in patients with hematologicai ancies and partial responses in patients with a broad spectrum of solid tumors (Blum and Kahiert., 1978, lssell and Crooke, 1978, r et al., 1978, Eagan et al., 1978, Cabanilias et ai., 1978). However, significant ties, including , vomiting, diarrhea, elevations of liver function tests, lethargy, and peripheral neuropathy were noted (see Maytansine iND #11857, Annual , February, 1984; Bium and Kahiert., 1978, lssell and Crooke, 1978, Chabner et al., 1978). Toxic effects precluded further development. -89— WO 83817 in another embodiment effector molecules might represent Taxanes. Taxanes are a class of tubulin interfering agents (Payne 2003). s are mitotic spindle poisons that inhibit the depolymerization of n, resulting in an increase in the rate of microtubuie assembly and cell death. Taxanes that are within the scope of the present invention are, for example, disclosed in United States Patents 6,436,931; 6,340,701; 6,706,708 and United States Patent Publications 20040087649; 20040024049 and 04210. Other taxanes are disclosed, for example, in United States Patent 6,002,023, United States Patent 5,998,656. United States Patent 5,892,063, United States Patent 5,763,477, United States Patent 5,705,508, United States Patent ,703,247 and United States Patent 5,367,086. A red embodiment of the present ion might be highly potent Taxanes that contain thiol or disulfide . As the person skilled in the art will appreciate, PEGylated taxanes such as the ones described in United States Patent 6,596,757 are also within the scope of the present ion.

The present invention inciudes further DNA affecting effector molecules, in more particular, alating agents such as anthracyclines and derivatives (daunorubicin, valrubicin, doxorubicin, aclarubicin, epirubicin, idarubicin. amrubicin, pirarubicin, zorubicin) and anthracenediones, such as Streptomyces derived substances (actinomycin, mitomycin, bieomycin, aactinomycin) or amsacrine.

A effector molecule might represent more particular DNA alkylating agents like, and more particular, Nitrogen mustard and analogues (e.g. Cyclophosphamide, Meiphalan, Estramustin), Aikyisuifonates, Nitrosoureas, Aziridines, Hydrazines, Ethylene lmines, and other substances such as Trenimon and Mitobronitoi (a mannitoi analogue). In ular, preferred DNA aikylating agents are CC-1065 anaiogues or derivatives (United States Patents 5,475,092; 5,585,499; 6,716,821) and duocarmycin.

CC~1065 represents a potent antitumor—antibiotic isolated from cultures of Streptomyces zelensis and has been shown to be exceptionaily cytotoxic in vitro d States Patent 4,169,888). Within the scope of the present invention are, for example, the 00-1065 ues or derivatives bed in United States Patents ,475,092, 5,585,499 and 5,739,350. As the person skilled in the art will readiiy appreciate, modified 5 analogues or derivatives as described in United States Patent 5,846,545 and gs of CC-1065 analogues or derivatives as described, for example, in United States Patent 6,756,397 are also within the scope of the present invention. in certain ments of the invention, CC-‘i 065 analogues or derivatives may, for example, be sized as described in United States Patent 6,534,660.

Other DNA alkylating or molecules such as platinum based substances are further included (e.g. e.g. carboplatin, nedaplatin, oxaliplatin, triplatin, satraplatin).

Among the DNA ing effector molecules, also Topoisomerase l and ii inhibitors are ed, such as Camptotheca derived substances (belotecan, can) and yllotoxin and derivatives (etoposide, teniposide).

Further subclass of DNA affecting effector molecules include antimetabolites such as folic acid analogues (methotrexate, known as a dihydrofolate reductase inhibitors) or Aminopterin. Also included are metabolites interfering with purine or pyrimidine metabolism, in particular adenosine deaminase inhibitor (pentostatin), or halogenated/ribonucleotide reductase inhibitors (cladribine, clofarabine), thiopurine and tiazofurine. Further antimetabolites include DNA polymerase inhibitor (cytarabine), ribonucleotide reductase inhibitor (gemcitabine), and hypomethylating agents (azacitidine, decitabine) and ribonucleotide reductase inhibitors. More general included are also DNA crosslinking substances such as tin.

Effector molecules according to the present invention may be antitumor antibiotics, defined as DNA modifying or damaging effector molecules including enediyne antibiotics such as callcheamicin which include, e.g., gamma 1 l, N-acetyl calicheamicin and other tives of calicheamicin. Calicheamicin binds in a sequence-specific mannerto the minor groove of DNA, undergoes rearrangement and exposes free radicals, g to breakage of doubie-stranded DNA, resulting in cell apoptosis and death. One example of a eamicin or molecule that can be used in the context of the present invention is described in United States Patent ,053,394.This compound is used in immunoconjugates with the monoclonal antibodies published as gemtuzumab icin and inotuzumab ozogamicin.

A subgroup of enediyne comprises the chromoproteins esperamycln and neocarzinostatin. in particular, trabectedin, which is also categorized as a DNA damaging agent (anti—tumor antibiotics)? tedin causes DNA backbone cleavage and can be isolated from a sea squirt (also known as ascidin 743 or ET-743) is sold by ZELiTA and JOHNSON & JOHNSON under the brand name YONDELIS.

Another group of preferred effector molecules are substances such as, but not limited to, toxins affecting cell metabolism. in particular enzyme tors such as but not only, olaprib, or more preferred proteasome (e.g. bortezomib) and protein kinase inhibitors, or lipoxygenase inhibitors such as masoprocol are part of the present invention. Also included are receptor antagonists such as, but not limited to, endothelin A receptor antagonist (eg. atrasentan), or sex steroids such as testoiactone, interfering with estrone metabolism. r included are estrogen receptor interacting substances such as plant d polyphenols, for e but not only vonoids, stilbenes, silymarin, phenylpropanoid glycosides.

Also suitable as effector les are substances affecting cell metabolism, such as substances used for photodynamic or radiation therapy, including, but not limited to, porphyrin derivatives e.g. flaminolevulinic acid. Efaproxiral represents a radiosensitizer, which increases oxygen levels by decreasing hemoglobin—oxygen affinity. Further included are retinoids (first, second and third generation), in particular tretinoine, all trans retinoic acid (ATRA), which is used to treat acute promyelocytic leukemia (APML) sold for this indication by ROCHE under the brand name VESANOID.

Retinoids are a class of chemical compounds that are related chemically to vitamin A, ng diverse functions as for example activation of tumor suppressor genes. At present they are used to treat skin cancer and inflammatory skin disorders. in another preferred embodiment, effector molecules might affect signaling pathways, such as but not limited to, calcium signaling. Examples are arsenic trioxide or trimethyltin chloride, the latter of which is a highly toxic organotin compound.

The present ion also includes effector molecules that are affecting drug resistance mechanisms which might include, for example, anti-multidrug ance activity (via P—glycoprotein inhibition). ic heteroaromatic nds and derivatives might severe as non—limiting examples.

Another effector molecule class might include substances, or more particular ns interfering with apoptotic signaling pathways, including, but not limited to, antisense ucleotides, more particular, oligodeoxynucleotides such as oblimersen (INN, trade name genasense; also known as augmerosen and bcl-2 antisense oligodeoxynucleotide G3139) which is an antisense oligodeoxyribonucleotide actually d as a possible treatment for several types of cancer, including chronic Iymphocytic leukemia, B-cell lymphoma, and breast . It has been proposed that this compound may kill cancer cells by blocking the production of Bcl-2 and by rendering them more sensitive to herapy. Further apoptosis inducing classes of nces that may serve as effector molecules comprise plant polyphenols such as, PCTI’EP2012/074867 WO 83817 cell cycle regulators and but not d to, rins, which are able to interfere with proteins involved in apoptosis Effector molecules might also be proteins, such as those of apoptotic signaling limited to, Granzyme cascades with apoptosis-inducing activities, including, but are not truncated Bid (tBid), B, me A, Caspase-B, Caspase-7, Caspase-S, Caspase-9, Bax and Bak. limited to, Other effector molecules might include enzymes such as but not asparaginase or other enzymes with antineoplastic activities. also be an A drug~effector molecule according to the t invention may antiprotozoal drug such as miltefosine. in another embodiment effector molecules might ent plant polyphenoles, such as, but not limited to, ens and their y metabolites.

Plant polyphenoles such as flavonoids, tannins (proanthocyanidins), stilbenoids, activities (eg. curcuminoids and ligands having one of the above mentioned antitumor radical apoptosis inducing, cell cycle arrest) or additional activity such as free antioxidant, ging, metal chelating activity, estrogen receptor interfering activity, molecules. More interfering with drug metabolizing enzymes are also possible effector intercalate into specifically, psoralens and their hydroxy metabolites which are able to DNA acting as metal chelators having antioxidant and cytoprotective properties are preferred effector molecules. Particularly preferred are reservatol and polyhydroxylated derivatives and flavonoids, such as catechins and epicatechins, more specifically epigallocatechin 3-0 gallate, which may act as antioxidants. r embodiment of effector molecules might represent Toxins. Toxins may Exotoxin A, plant include ial toxins, such as, but not limited to, Diphtheria toxin or , such as but not limited to, Ricin other alkaloids and polyphenols, mycotoxins, Toxins might not such as alpha amanitin or more specially Amatoxins and phallotoxins. all of only be of bacterial origin, but also fungal, plant, vertebrate and invertebrate , also be which can be genetically or chemically modified. Moreover toxins might environmental toxins such as, but not limited to, mercury. Toxins may also Dolabella dolastatins 10 and 15 are small peptides ed from the marine sea hare auricularia that have been shown to interact with tubuiin is also A broad classification of effector molecules according to their mechanism possible: WO 2013083817 PCTIEP2012/074867 Antineopiastic agents and modulating agents (According to ATC code L01) in particular "intracellular chemotherapeutic agents" ATC: Anatomical Therapeutical Chemical classification system (WHO) 1) Antimitotics, or molecules affecting microtubules in binding agents) such as vinca alkaloids and analogues (Vinca alkaloids (Vinblastine, Vincristine, Vinflunine, Vindesine, Vinorelblne) and taxanes (Paclitaxel, Larotaxel.

Docetaxel) dolastatins (and derivatives e.g. auristatin) and crytophycin, maytansine and colchicine derivatives, epothilones (e.g., ixabepilone) 2) affecting DNA replication 8) Intercalating agents such as anthracyciines (Daunorubicin, Valrubicin, Doxorubicin, Aclarubicin, Epirubicin, ldarubicin, Amrubicin, pirarubicin, Zombicin) and cenediones, such as Streptomyces derived substances (Actinomycin, cin. Bleomycin, Dactinomycin) or Amsacrine b) Alkylating agents such as Nitrogen mustards, Nitrosoureas, Alkylsultonates.

Aziridines, ines rbazine), Triazenes, Epoxides, Ethylene lmines, Altretamine, onitol, duocarmycin and analogues/stereoisomers, Trenimon, Estramustine, 00-1065 Alkylating-like agents such as um (e.g. Carboplatin Nedaplatin, Oxaliplatin, tin Tetranitrate, Satraplatin) Topoisomerase l c inhibitors such as theca (Belotecan, Topotecan) Topoisomerase ll c inhibitors such as Podophyllotoxin and derivatives (Etoposide, Teniposide) Antimetabolites affecting DNA/RNA synthesis by interfering with - folic acid such as Dihydrofolate reductase inhibitors (e.g. Aminopterin, Methotrexate), thymidilate synthase inhibitor ~ purine such as adenosine deaminase inhibitor statin), haiogenated/ribonucieotide reductase inhibitor (Ciadribine. Ciofarabine), Thiopurine, Tiazofurine - Pyrimidine such as DNA Polymerase inhibitor (Cytarabine), ribonucleotide reductase inhibitor (Gemcitabine), hypomethylating agent (Azacitidine, Decitabine) 2012/074867 - deoxyribonucleotide such as ribonucleotide reductase tor Hydroxycarbamid 9) other DNA crosslinking agents such as platinum based compounds (e.g. tin) 3) Other DNA ering substances e.g. "antitumor/cytotoxic antibiotics" such as elsamicin A, further antibiotics such as 00-1065, and subclasses of antibiotics such as bacteria derived enediyne chalicheamin or chromoprotein enediyne esperamicin (extremely toxic DNA splicing agent) or neocarzinostatin (other members of the neocarzinostatin group of antibiotics are macromomycin, actinoxanthin, kedarcidin and maduropeptin.) or Trabectedin (DNA backbone cleavage) 4) toxins affecting cell metabolism e.g. HSPQO inhibitors, Lonidamide (inhibits both ation and glycolysis leading to a decrease in cellular ATP) a) Enzyme inhibitors e.g. Olaprib (PARP inhibitor), CDK inhibitors (Alvocidib), Proteasome (Bortezomib), n kinase inhibitors, Masoprocol (Lipoxyenase inhibitor) b) Receptor antagonists such as tutin (Glycin receptor antagonist (plant toxin), Atrasentan, retinoid X receptor (Bexarotene), sex steroids such as testolactone, estrogen receptor interfering substances 0) Photosensitizers or other compounds used for photodynamic therapy (Porfirmer Sodium),Porphyrin derivatives e.g. fi-Aminolevulinic acid) d) Radiosensitizer such as Efaproxiral which ses oxygen levels by sing hemoglobin—oxygen y e) Substances affecting signaling pathways e.g. Ca2+ signaling such as arsenic trioxide and trimethyltin chloride f) Other substances ering with metabolism such as retinoids and derivatives Tretincine (ATRA) ) Affecting epigenetic processes such as HDAC inhibitors (e.g. Panobinostat, Vorinostat, Valporic acid, MGCD0103 (Mocetinostat), which are at present in clinical pment for cutaneous T—cell lymphoma, acute myeloid leukemia, Hodgkin lymphoma or follicular lymphoma) 6) Affecting drug resistance isms such as bicyclic heteroaromatic nds, which inhibit P-glycoprotein WO 83817 7) Substances inducing apoptotic signaling/mechanisms include proteins but also antisense oligodeoxynucleotides such as Oblimersen (trade name Genasense) 8) Enzymes such as Asparaginase 9) Antiprotozoal drugs such as Miltefosine ) Plant polyphenoles such as Flavonoids, Tannins (Proanthocyanidins), Stilbenoids, curcuminoids and lignans having one of the above ned antitumor activities (eg. apoptosis inducing, cell cycle arrest) or additional activity such as free l scavenging , metai cheiating activity, estrogen receptor interfering activity, antioxidant. ering with drug metabolizing enzymes). More specifically psoralens and their hydroxy metabolites. reservatoi and pclyhydroxylated derivatives, Fiavonoids, such as Catechins and Epicatechins, more specifically epigailocatechin 3‘0 gailate 11) Further natural substances and derivatives such as exotoxin A, diphtheria toxin, and derivatives thereof, wherein the derivatives can be ally or genetically modified.

Effector moiecuies can also be categorized according to the substance class they belong to such as anorganic nds, aromatic compounds, metal based compounds, proteins related to cell metabolism, enzymes, peptides, oiigonucleotides, such as nse nucleotides, bacterial toxins, plant derived toxins and polyphenols such as tannins, fiavonoids and coumarins as well as terpenoids, ids, anti-tumor antibiotics (e.g. enediyne antibiotics), mycotoxins, toxins from invertebrates as well as vertebrates, nmental .

An immunoconjugate according to the present invention comprises at least one targeting agent, in particular targeting antibody and one effector molecule. The immunoconjugate might comprise further molecules for example for stabilization. For immunoconjugates, the term "conjugate" is generally used to define the operative association of the targeting agent with one or more effector les and is not intended to refer solely to any type of operative association, and is particularly not limited to chemical gation". So long as the targeting agent is able to bind to the target site and the attached effector functions sufficiently as ed, particuiariy when delivered to the target site, any mode of attachment will be le. The conjugation methods according to the present invention include, but are not limited to, direct attachment of the effector molecule to the targeting antibody, with or without prior modification of the effector molecule and/or the targeting antibody or ment via linkers. Linkers can be categorized functionally into, for e, acid , photolabile linkers, enzyme cleavable linkers, such as linkers that can be cleaved by peptidases. Cleavable linkers are preferred in many ments of the invention.

Such cleavable linkers can be cleaved under conditions present in the cellular nment, in particular, an intracellular environment with no detrimental effect on the drug released upon cleavage. Low pHs such as pH of 4 to 5, as they exist in certain intracellular departments, will cleave acid labile linkers, while photolabile linkers can be cleaved by, e.g., infrared light. However, linkers that are cleaved by/under physiological conditions present in the majority of cells are preferred and are referred to herein as physiologically cleavable linkers. Accordingly, disulfide linkers are preferred in many ments of the invention. These linkers are cleavable through disulfide exchange, which can occur under physiological conditions. Preferred heterobifunctional disulfide s include, but are not limited to, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (see, e.g., Carlsson et al. (1978)), N—succinimidyi 4-(2~pyridyldithio)butanoate (SPDB) (see, e.g., US. Pat. No. 4,563,304), N—succinimidyl 4-(2— pyridyldithio)pentanoate (SPP) (see, e.g., CAS Registry number 341498—08—6), N- succinimidyl 4-(N-maleimldomethyl)cyclohexanecarboxylate (SMCC) (see, e.g., Yoshitake et al., (1979)), and N-succinimidyl 4-methyl—4—[2—(5-nitro—pyridyl)— ]pentanoate (SMNP) (see, e.g., U.S. Pat. No. 4,563,304). The most preferred linker les for use in the ive composition are SPP, SMCC, and SPDB.

Other suitable linkers may include "non-cleavable" bonds, such as, but not limited to Sulfosuccinimidyl maleimidomethyl cyclohexane carboxylate (SMCC), which is a heterobifunctional linker capable of linking compounds with SH—containing nds. Bifunctional and heterobifunctional linker molecules, such as carbohydrate-directed bifunctional linker molecules, such as S—(2—thiopyridyl)—L— ne hydrazide (TPCH), are also within the scope of the present ion (Vogel, 2004). The effector molecule, such as a maytansinoid, may be conjugated to the targeting antibody via a two reaction step process. This includes as a first step the modification of the targeting antibody with a cross-linking reagent such as N- succinimidyl pyridyldithiopropionate (SPDP) to introduce dithiopyridyl groups into the targeting dy. in a second step, a reactive maytansinoid having a thiol group, such as DM1, may be added to the modified antibody, resulting in the displacement of the thiopyridyi groups in the modified antibody, and the production of disulfide-linked xic maytansinoid/antibody conjugate (United States Patent 5,208,020). However, one-step conjugation processes such as the one disclosed in United States Patent Publication 20030055226 to Chari et at are also within the scope of the t invention. in one embodiment of the present invention multiple effector molecules of the same or different kind are ed to a targeting antibody. As sed elsewhere herein, the nature of the s employed may influence der killing (Kovtun et al., 2006). See also Pat. Nos. 030; 064; 6,333,410; 6,441,163; 6,716,821; 6,913,748; 7,276,497 and US Application No. 200510169933 for method for preparing immunoconjugates. 00—1065 analogues or derivatives may be conjugated to the ing agent via, for example, PEG linking groups as described in United States Patent 6,716,821.

Calicheamicins may be conjugated to the targeting antibodies via linkers (United States Patent 5,877,296 and United States Patent 5,773,001) or according to the conjugation methods disclosed in United States Patent 5,712,374 and United States Patent ,714,586. Another red method for preparing calicheamicin conjugates is disclosed in Unites States Patent Publication 20040082764. The immunoconjugates of the present invention may take the form of recombinant fusion proteins.

Operations! association in form of an attachment with or t a linker is referred to herein as "functional attachment." One milligram (mg) of immunoconjugate BT062 comprises approx. 3.5 DM4 molecules (1 DM4 has an approximate molecuiar weight of 800 Da), thus 1 mg immunoconjugate comprises 2800 Da of DM4.

The molecular weight of BT062 is about 150000 Be. Thus, 1 mg immunoconjugate comprises about 1/53 mg DM4 molecules. Thus 4 mg/mi of antibody corresponds to about 4/53 DM4 molecules, which is 75 pg/mi. 160 mg/m2 of immunoconjugate corresponds to about 2.5 to 3.5, in particular to about 3 mg/m2 of DM4.

According to the present invention more than 2, 2.5, 3, 3.5 or even 4 mg/m2 Bit/i4 can be administered to a subject in either repeated single doses or multiple doses, including repeated multiple doses, without DLTs.

An immunoconjugate consisting essentially of certain components means in the context of the present invention that the antibody/immuncconjugate consists of the specified components and any onal materials or ents that do not ally affect the basic characteristics of the antibody.

Some of the immunoconjugates of the present invention have an effector molecule that is sterically hindered and contains a cleavable linker (HICL red immunoconjugate, cleavable linker). An unhindered counterpart (Ul: unhindered immunoconjugate) of an immunoconjugate comprising an engineered targeting dy against CD138 attached to an effector molecule via a cleavabie linker (CL) and is described herein as UlCL. The UlCL is an immunoconjugate equivalent to the HlCL comprising an engineered targeting antibody in which the effector molecule is, r, not sterically ed. Examples of a pair of HiCL/UICL are BTOSZ and nBT062-SPP-0M1. An unhindered counterpart of such an immunoconjugate comprising a avable linker (UiNCL) refers to the equivalent immunoconjugate comprising an engineered targeting antibody in which the effector molecule is not sterically hindered and comprises a noncleavable linker. For BT062 (nBTOGZ—SPDB—DM4), nBTOBZ- SMCC—DM1 would tute an example of such an ered counterpart comprising a non-cleavable linker (UNlCL).

A growth of a tumor ting activity (=tumor growth inhibiting activity) of an immunoconjugate is a relative measure. it describes the tumor growth inhibiting activity of a conjugate relative to the activity of the highest performing immunoconjugate whose activity is set as 100%. For example, if the activity of the highest ming immunoconjugate. say, BTOSZ, which causes a tumor growth delay (TGD) of 32 days, is set as 100%, the activity of, e.g., nBT062-DM1, which displays a tumor growth delay (TGD) of 18 days is calculated as follows: Tumor Growth inhibiting Activity= 100x (TG DnBTDGZ-DMt/TGDBT062)a more cally: Tumor Growth inhibiting Activity= 100x (TGDSamplerrGDReferenCE)' TGD" da 5 % Activit ** PBS 0 0 nBTDGZ-SMCC-Dllfl‘l 18 56 BT062 nBT062~SPP-DM1 Table 7: Tumor growth delay (TGD) and "/0 Activity of —DMX against MOLP-S tumor xenografts in SCID mice based on treatment groups receiving a 450 ug/kg dose.

(*) Tumor growth delay in days (TGD) as mean time in days for ent group to reach a predetermined size (160 mm ) minus the mean time for the control group to reach this predetermined size.

(**)Tumor Growth inhibiting Activity =100x(TGDSEmp.e/TGD37052). The activity of BT062 is defined to be 100%. in the example provided in Table 7, BT062 provides a growth of a tumor inhibiting activity that exceeds that of its unhindered counterpart (nBTOGZ—SPP—DMi) by 60%, and a growth of a tumor inhibiting activity that exceeds that of its unhindered counterpart immunoconjugate comprising a non-cleavable linker (nBTOBZ—SMCC~DM1) by 44%.

As discussed above, certain drugs such as maytansinoids, while effective, are highly toxic, destroying in their native, i.e., unccnjugated form, cells non-selectively. Linking the cytotoxic maytansinoid to an antibody can keep the drug inactive until it reaches the target cell (Lambert 2005). l antibody—maytansinoid conjugates have undergone clinical development.

Phase I and it studies with lMGNQO‘l (huNQO‘l -DM‘l, 88—1 0901) for treating C056- positive solid tumors (small cell lung cancer and neuroendocrine cancers) were performed. In these studies 1 was administered on 4 consecutive weeks every 6 weeks and was generally well tolerated (Fossella et al., 2005, Lorigan et al., 2006, McCanri et al., 2007, Carter and , 2008, Johnson et al. 2008). The antibody n of the immunoconjugate, , shows significant CDC or ADCC activity. The same immunoconjugate is investigated for treatment of CD56-positive le myeloma. ln a phase l study administration of lMGNQOi on 2 consecutive weeks every 2012/074867 3 weeks to patients with CD56-positive multiple myeloma who have failed established multiple myeloma treatments has shown preliminary evidence of safety as well as clinical activity. Eighteen ts were ed to have received lMGNQO'l (3 patients each at 40, 60, 75. 90, 112. and 140 mg/mzlweek). Preliminary pharmacokinetic (PK) results were reported to te an approximately linear relationship between dosing and observed maximal serum concentration. interesting clinical activity has been observed with a tolerable safety profile. A confirmed minor response (MR) was documented in 3 heavily pretreated ts (1 patient each at 60. 90, and 112 mg/mzlweek) using the European Bone Marrow Transplant criteria. e stable disease was reported at doses of 60, 90, 112. and 140 mg/mzlweek (Chanan-Khan et al., 2007. Chanan-Khan et al., 2008). lMGN901 at a dose of 75 mg/mzlweek will be taken forward for further investigation in the expansion phase of the trial. At higher doses. peripheral neuropathy was reported with the treatment combination with lenalidomide and dexamethasone, the standard ent regimen for multiple myeloma.

MLN2704 (huJ591-DM1) is investigated for treating castration-resistant prostate cancer (Miiowsky et al., 2006, Brand and Tolcher 2006). A Phase l trial of 4 in patients with progressive metastatic castration-resistant prostate cancer investigated the safety profile, pharmacokinetics, immunogenicity, and antitumor activity of 4 when administered once every four weeks. Results demonstrated that therapeutic doses of MLN2704 can be administered safely on a repetitive basis (Gaisky et al., 2008).

Parallel trials were performed with another DM1-immunoconjugate, namely bivatuzumab mertansine which targets CD44V6, which is expressed on head and neck carcinomas and other solid tumors. in the clinical trial with the most condensed stration schedule (weekly administration) binding to CD44v6 on skin keratinocytes mediated serious skin toxicity with a fatal outcome in one t, which led to the termination of the development program of bivatuzumab mertansine (Tijink et al., 2006, Sauter et al., 2007, Rupp et al., 2007, Riechelmann et al., 2008).

CD44v6 is not only expressed on various cancer cells, but also in normal skin tissue and resembles in this respect CD138 which is also expressed not only on cancer cells but in normal skin tissue. singly, it was found that BT062 shows ai efficacy without intolerable side effects like skin toxicity as found in bivatuzumab mertansine.

See Fig. 28, which shows that repeated single doses BT062 of up to 160 mg/m2 led to _31- at least stable disease with manageable side effects over extended periods of time.

The figure in particular shows a minor se defined by serum M-protein (M-protein levels were reduced by .>. 25%). Only after a hold period (days 400 to 421) did the M- protein levels increase, but could be stabilized after the next dose was ed. In sum, there was progression free al for about 22 months, with a duration of a minor response for 19 months. it was also usly shown that 10 repeated single doses of 20 mg/m2 (treatment over more than 6 months). 5 repeated single doses of 40 mg/m2, 5 repeated single doses of 80 mg/m2. 6 repeated single doses of 160 mg/mz, and 1 single doses of 200 mg/m2 followed by 6 repeated single doses of 160 mg/m2 (ergo, a total dose of 1160 mglmz) were well tolerated (See also US Patent Publication 20110123554).

CD138 is also expressed on normal blood cells and other cells whose destruction would lead to intolerable side effects, ergo severe adverse events (SAEs) discussed later herein. irrespective of this. no dose limiting toxicity towards non-cancer/non-tumor cells expressing CD138 of any sort were found in the repeated single dose treatment regimens up to 120 mg/mz. An aggregate dose of 360mg/m2 resulted in 3 weeks (21 days) when 120 mg/m2 was administered ion day 1, 8, and 15 and a resting period of 1 week. Thus, while the aggregate maximum ble dose (AMTD) in the context of this once a week treatment regime is higher than the maximum tolerable dose (MTD) which, in the case of BT062, has usly been determined to be 160 mglm2 when the immunoconjugate was only stered as a single dose, here on day one in a 21 day cycle. in fact, the AMTD is , including more than 50%, 60%, 70%, 80%, 90%, 100% higher than the usly determined dose limiting toxicity (DLT), in the case of BT062, 200 mg/m2 for administration of the immunoconiugate as a single dose, e.g., once, e.g. on day 1, in a three week (21 days) active ent cycle. This constitutes a significant difference to other immunoconjugates, where no difference in the BLT or MTD could be found between an administration of the immunoconjugate as a single dose (including repeated single dose), e.g., a one time administration within three weeks and in multiple dose regimen, e.g., a three time administration once a week for three weeks (21 days).

The effects aggregate maximum tolerable dose (AMTD) are identical to the effects of an MTD defined elsewhere herein. However, the term "aggregate" conveys that the -32_ 2012/074867 administration is not performed as a single dose or repeated single dose within a certain time period, e.g. an active treatment cycle of, e.g., three weeks (e.g., 21 days), but that, within said certain time period, the immunoconjugate is administered in intervals, e.g., weekly als such as on day 1, 8 and 15 of a 21 day period.

Preferably, equal doses are stered, e.g., in 7 day intervals (e.g., day 1. 8 and 15). 3 day intervals (e.g., day 1, 4, 7, 10, 13 and 16), 4 day intervals, 5 day intervals or 6 day intervals. However, slight variation in the administrations such as an initial booster administration described elsewhere herein are also within the scope of the present invention. The stration intervals may be increased or decreased after each cycie (see also maintenance y discussed elsewhere ). For example, the first and optional second cycle might involve administration every 3rd day, while in the following cycies the intervals may be, e.g., progressively, increased to 4, 5, 6 or 7 days. A fraction of a of the AMTD includes e.g. about 95% of the AMTD, about 90% of the AMTD, about 85%, about 80%, about 75% about 70%, about 65%, about 60%, about 55%, about 50%, about 45% of the AMTD. Assuming, e.g., that the AMTD of a theoretical immunoconjugate is 100mg/m2, a 95% fraction wouid be, e.g., 95mgim2.

Adverse events (AEs) can be evaluated according to the NCi-CTCAE version 4.0 (Cancer Therapy Evaluation Program, Common Terminology ia for Adverse Events, Version 3.0. DCTD. NCl, NiH, DHHS March 31, 2003), Nationai Cancer institute, US National institutes of Health, Publishing Date: August 9, 2006). For AEs not listed in the CTCAE v4.03, severity will be assessed by the investigator by the following criteria: Only grade 1 and grade 2 AEs are able, whereby Grade 1 (Mild) requires minimal or no treatment and does not interfere with the ts daily ties and Grade 2 (Moderate) results in a low level of inconvenience or concern with the therapeutic measures. te events may cause some interference with the subject’s functioning.

AEs of Grade 3 (Severe) and Grade 4 (Life threatening) are considered not acceptable their occurrence defines the BLT (dose limiting toxicity), if not otherwise defined by study specific DLT criteria (see below). —83- W0 2013/0838]? AEs of Grade 3 and 4 are also referred to as severe adverse events (SAE) and include lymphopenia, enia, thrombopenia, neutropenia, cardiac arrest. atrial ation, pulmonary embolism and deep vein thrombosis. Other study c criteria may be employed (see below).

Dose limiting toxicities (DLT) are determined using the grading according to NCl CTCAE v4.0 nced to above. Generally, all toxicities of at least grade 3 are defined as DLT. Further study specific DLT criteria which can be employed are listed below: Nonhematological: - Alopecia, of any grade, is not considered a DLT ~ Grade 3-4 nausea and vomiting lasting longer than 3 days despite optimal antiemetic medication.a . Grade 3-4 diarrhea lasting longer than 3 days despite optimal antidiarrheal medication.a a. Optimal arrheal and antiemetic treatment were determined by each investigator.

Hematologic: - Grade 4 neutropenia lasting longer than 5 days.

° Grade 3 or higher neutropenia with temperature r than or equal 101 °F, for 2 consecutive determinations spaced 4 hours apart.

° Grade 4 thrombocytopenia - Grade 3 or higher thrombocytopenia with bleeding and requiring the use of platelet transfusion.

- Grade 3 neutropenia, grade 3 thrombocytopenia were NOT ered DLTs.

The maximum tolerated dose (MTD) is defined as the dose at which any subject to whom a single dose or a repeated single dose has been administered experiences dose limiting ties (DLTs). As is readily apparent, a MTD can be readily determined for a wide variety of immunoconjugates according to the present invention. These DLTs may occur in a first or a uent treatment cycle. In particular, 1 out of 6 subjects to whom a single dose or a repeated single dose has been administered experience DLTs. Preferably DLTs in the first cycle are considered.

W0 2013i/083817 During dose escalations, preferably only DLTs in the first cycle are considered.

Study specific adverse event (AE) Any rabie or unintended sign, symptom, or disease that appears or worsens in a t or clinical investigation subject during the period of observation in a clinical study The AE may be any of the following: o a new illness a an exacerbation of a sign or symptom or the underlying condition under treatment or of a concomitant s, 9 unrelated to participation in the clinical study or an effect of the study medication or comparator drug, a a combination of one or more of the above factors.

Generaiiy, no causal relationship with the study medication is implied by the use of the term "adverse event". s adverse event (SAE) An SAE is any rd l occurrence or effect that at any dose: 0 results in death, — death is an outcome of an AE and not an AE in itself. All deaths, regardless of cause or relationship must be reported for patients on study a is life—threatening, — life-threatening means that the patient was at ate risk of death from the event as it occurred. This does not include an event that might have led to death if it had been more severe . results in tent or significant disability or incapacity, . is a congenital anomaly or birth defect, or . is another medically important condition — An important medical event that is not immediately life—threatening or will result in death or hospitalization, but which may jeopardize the patient/subject or may require medical intervention to prevent one of the outcomes listed above, should be reported as "serious" as well WO 83817 Causality of adverse event Refers to the relationship of the AE to investigational product. Causality will be categorized according to the following criteria: Not related AEs for which a reasonable explanation for an alternative cause is considered plausible, e.g., non investigational t taken, plausible clinical alternative like accidental injury, ed progression of underlying or concomitant disease, pharrnacologically incompatible temporal relationship, intercurrent illness Related AEs for which a reasonably le clinical and I or phannacologicai relationship to investigational product cannot be excluded, e.g. lacking plausible alternatives.

Phase l studies with the immunoconjugated form of trastuzumab (T-DM1 ) for ent of HER2 over-expressing metastatic breast cancer are performed to investigate safety and pharmacokinetics of T~DM1 administered weekly or once every 3 weeks. in both studies AEs of grade 2 2 related to T-DM‘i have been infrequent and manageable.

Objective tumor responses have been observed at doses at or below the MTD (Burris et. al., 2006, Krop et al., 2007, Beeram et al., 2008, Holden et al., 2008). A phase ll study investigating T-DMl in HERZ—positive metastatic breast cancer when administered once every 3 weeks has been initiated (Beeram et al., 2008, Carter and Senter, 2008, Holden et al., 2008). A Phase ill clinical trial evaluating T-DM1 for second —-line HERZ-positive metastatic breast cancer and Phase ll al trials evaluating T- DMl for first-, second- and third-line HERZ—positive metastatic breast cancer are ongoing. A Phase lb clinical trial in combination with pertuzumab for HERZ-positive metastatic breast cancer ts who have been progressed on tin—basecl treatment is planned. Three phase 1 clinical trials have been completed with umab sine, a njugate of the huC242 antibody that targets an antigen found on colorectal cancers and other CZ42-expressing cancers. Treatment with huC242~DM1 administered on a weekly basis as well as once every 3 weeks was found to be safe and tolerated (Rowinsky et al., 2002, Tolcher et al., 2003, Helft et al., 2004) Four studies are investigating conjugates using the thiol-containing DM4 maytansinoid. which is also a component of BT062: An anaiog of cantuzumab mertansine, lMGN242 (huCZ42-DM4), was investigated in a phase I study in subjects with CanAg-expressing cancer (Tolcher et al., 2006). Subjects received a single iv infusion of lMGN242 once every 3 weeks with a dose ranging from 18 to 297 mg/mz. Dose-limiting toxicity was experienced by 2 of 6 subjects treated at the 223 mg/m2 dose level during their second cycle of treatment.

The drug was well tolerated at the 168 mgfm2 level and did not induce any detectable antibody se (Mita et al., 2007). Based on first safety results from the Phase i study, a Phase ll study was initiated to evaluate IMGN242 for treating CanAg- expressing gastric cancer at the dose of 168 mg/m2 (Sankhala et al., 2007). five ts have been treated with lMGN242 in two clinical trials. Based on the safety and thorough clinical phannacokinetic (PK)/pharmacodynamic (PD) analyses, the Phase ll study was amended to treat patients with low plasma CanAg ievels at the dose of 126 mg/m2 and patients with high plasma CanAg levels at 168 mg/m2 (Qin et al. 2008).

A phase 1 study with huMy9-6 antibody conjugated to DM4 (AVE9633) was also performed for the treatment of subjects with CD33-positive Acute Myeioid Leukemia (AML). The treatment regimen consisted of 1V infusions once every 3 week using a dose range of 15 to 260 mg/mz. Neither associated myelosuppression nor responses have been noted in a single-dose study (Giles et al., 2006). A second phase 1 study investigating AVE9633 with a treatment regimen consisting of N infusions on day1 and day 8 of a 28-day cycle also shows that AVE9633 was well tolerated; it also provides evidence of antileukemia activity including 1 subject with complete response quate et se, transfusion dependent) lasting for at least 4 months (Legrand et al., 2007). Two further DM¢immunoconjugates (SAR3419 and BllBO15) have entered into clinical trials. 9 (huB4-DM4) is an antibody-drug ate composed of a humanized lgG1 onal antibody, huB4, which specifically targets the CD19 antigen, conjugated h a disulfide link to the maytansinoid derivative 0M4. sion of the CD19 molecule is found on all B lymphocytes, including pro-B cells, but is lost during maturation to plasma cells. The CD19 antigen is also sed on the membrane of follicular dendritic cells and on most stabilized B cell lines. After binding to the CD19 antigen, SAR3419 undergoes internalization and intracellular release of DM4.

In a phase 1/" study SAR3419 was administered by intravenous infusion, weekly with 8 to 12 doses, to patients with relapsed/refractory B-cell NHL expressing CD19. Forty— four patients were enrolled at 7 dose levels from 10 to 70 mg/m". Main histologies were follicular (18; 41%) and diffuse large B-cell (17; 39%). The median number of prior regimens was 3 (1-8) and 19 patients had ed priortransplantation. Twenty-eight patients were enrolled in the dose escalation part. Of 6 patients at 70 mg/m2, 1 patient had a ol defined dose limiting toxicity (DLT) of penia and 2 patients had grade 2 significant toxicities with late onset: blurred vision associated with corneal deposits and left bundle branch block. The maximum tolerated dose (MTD) was defined at 55 mg/m2, while the MTD in a regimen involving a single administration every three weeks was m2. Of 22 ts at the MTD of 55mg/m2, 4 patients had related reversible grade 3-4 toxicities after 6-8 closes: optic neuropathy, paraesthesia, penia and thrombocytopenia. Of 38 patients at doses of 20 mg/m2 or higher. 12 (32%) patients achieved an objective response including 6 CR/CRu (complete response/complete response unconfirmed), with no obvious dose effect. Of 22 patients at the MTD (55 mg/mz). 8 (36%) had a response, including 3 CR/CRu. Of 9 ts evaluable for se duration (RD), 4 patients had a RD g from 6 to at least 12 months. in sum it can be said that the aggregate maximum tolerated dose (AMTD) in a three weeks (21 days) dosing regimen involving 3 doses did not exceed the MTD in a three weeks (21 days) dosing regimen involving a single dose (e.g., on day one). l—W’" T Once weekly regimens ‘ Corresponds to totalconcentration of (assuming 1’0 kg and 1.9 in2 body surface L area) BTO62 MTD 140 mg/m SGN-35 Up to 1,2 mg/kg 84 mg (Batlett et al., 2008) SAR3419 MTD 55 mg/m 110mg (Coiffer et ai., 2011) T-DM1 MTD at 2.4 mg/kg 168 mg (Holden et al., 2008) SEEN-75 CD70; MMAF) Study 0.3 to 0.6 mg/kg (SEATTLE GENETICS) (MTD has not been Table 8: Comparison 0? lmmunoconjugates administered in repeated multiple dose ns (once weekly).

As can be seen from the table above, BT062 can be administered at higher doses once weekly (at least in total an amount of 266 mg). In contrast to the other immunoconjugates listed, BTOBZ displayed characteristic pharmacokinetics. In particular BT062 shows a characteristic discrepancy between observed and theoretical Cmax values BT062 described elsewhere herein.

Also. it is known from other immunoconjugates, such as Mylotarg which is ing C033, that the activity of the immunoconjugate may not be sufficient to treat patients at low doses. This problem has been alleviated by, e.g., stration of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to ize 0033 expressing target cells (Fianchi et al., Annals of Oncology 2008 19(1):128-134).

The above studies demonstrate that the responses to different immunoconjugates, in particular maytansinoid (such as DM1 or DM4) containing immunoconjugates, vary widely. The BTOGZ trials in human subjects showed not only tolerable toxicity against non-cancer cells expressing CD138 at different stable disease doses, especially at doses up to 160 mg/mz, but also fast plasma clearance at dosages up to about 50 mg/m2 in a weekly administration scheme.

The immunoconjugate described herein can be stered in ation with cytotoxic agents. These combinations are also referred to herein as anticancer combinations.

Selection of drug combination partners A set of guidelines for designing combination chemotherapy regimens has been developed oto, 2006). Abiding to these guidelines will generally increase the chances that a particular combination es at least one of the three most important theoretical ages of combination chemotherapy over single-agent therapy: 1.) ze cell kill while minimizing host toxicities by using agents with noninteriering dose-limiting toxicities; W0 2013/08381 7 2.) Increasing the range of drug activity against tumor cells with endogenous resistance to specific types of therapy; and 3.) Preventing or slowing the pment of newly resistant tumor cells.

Recommended principles to consider for selecting agents for use in combination chemotherapy regimens se: a) selecting drugs known to induce complete remission as single agents, b) selecting drugs with different mode of actions and with ve or synergistic cytotoxic effects, 0) selecting drugs with different dose limiting toxicities, d) selecting drugs with different patterns of resistance to minimize cross resistance.

Also, drugs should be administered at their optimal dose and le (e), and the administration should be performed at consistent intervals, whereas the treatment free period should be as short as possible to allow for recovery of the normal tissue (f) (Takimoto et al, 2009).

Synergistic effects orjust additive s can be counteracted by a variety of factors: For example, the components of an anticancer combination might inactivate each other, e.g., by g each other. in addition. one component of an ncer combination might interfere with the mode of action of another component. For e: Lenalidomide downregulates cell adhesion receptors such as CD138, which is the target of the immunoconjugate of present invention (Quach et al., 2010. Udi et al, 2010). The proteasome inhibitor bortezomib causes GZ/M cell cycle arrest (Wang et al., 2009) which is also ed by anti-mitotic agents. Thus, if the effector molecule of the immunoconjugate is a maytansinoid, it will share a target for action with bortezomib. which is considered disadvantageous.

Dosages, routes of administration and recommended usage of the cytotoxic agents according of the t invention which have been widely used in cancer therapy are known in the art and have been described in such literature as the Physician's Desk Reference (PDR). The PDR discloses dosages of the agents that have been used in 2012/074867 treatment of various cancers. The dosing regimen and dosages of these cytotoxic agents that are ive will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be ined by the physician. The 2006 edition of the Physician's Desk Reference (PDR) discloses the mechanism of action and preferred doses of treatment and dosing schedules for omide (p 979-983), VELCADE (p 2102-2106) and melphalan (p 976- 979). One of skill in the art can review the PDR, using one or more of the ing parameters. to determine dosing n and dosages of the chemotherapeutic agents and conjugates that can be used in accordance with the teachings of this invention.

These parameters include: 1. Comprehensive index according to a) Manufacturer b) ts (by company's or trademarked drug name) c) Category index (for example, "proteasome inhibitors", "DNA alkylating agents, I! llmelphalan" etc.) d) Generic/chemical index (non-trademark common drug . 2. Color images of medications 3. Product information, consistent with FDA labeling including a) Chemical information b) Function/action c) indications & Contraindications d) Trial research, side s, warnings. in the present context, one goal of ing combinations are a reduction in the effective doses of the immunoconjugate of the t invention, lowering their side effects and opening new therapeutic windows with acceptable side effects. Another goal is to reduce the effective dose of previously employed cytotoxic agents such as VELCADE or lenalidomlde and preferably reducing the side effects of these agents.

Similarly, the dosages Positive consequences include, but are not limited to, prolongation of treatment, higher dosages, other application schedules, better and more sustained response to treatment.

Patients displaying a refractory phenotype towards drugs such as lenalidomide, melphalan (study ongoing) might be rendered sensitive again by the use of immunoconiugates according to the present invention.

The term "cytotoxic agents" comprises "cytotoxic/cancer drugs" including chemotherapeutic agents, in particular chemotherapeutic agents that are generally used in rapidly ng cells, namely: - Aikylating agents such as nitrogen mustards (e.g. melphalan, cyclophosphamide, mechiorethamine, uramustine, chlorambucil, ifosfarnide) or nitrosureas (e.g. carmustine, lomustine. streptozocin) or alkylsuifonates; - Alkylating like agents such as cisplatin, carboplatin, nedaplatin, oxaliplatin; or non classical alkylating agents such as tetrazines, dacarbizine, procarbazine, amine — Anthracyclines such as doxorubicin and iiposomal doxorubicin (DOXlL) - Alkaloids such as vincristine The term oxic agents" also comprises immunomodulatory drugs ) such as thalidomide (or analogs), lenalidomide (CC—5013), pomalidomide, actimid, which are used for myeloma therapy in View of their pleitropic immunomodulatory ties. They commonly display nflammatory activity by inhibition of TNF alpha production, but display also anti-angiogenic activity and immunomodulatory properties such as T-cell co stimulation and ce on regulatory T-cells (Quach et al., 2010).

The term "cytotoxic agent" also comprises steroids, such as, but not limited to, dexamethasone and prednisone as well as proteasomal inhibitors such as bortezomib (VELCADE) or carfilzomib which induces the activation of programmed cell death in neoplastic cells dependent upon suppression of pro-apoptotic ys.

Further potent cytotoxic agents, include etoposide, which inhibits the enzyme topoisomerse ll, cytarabine, which, upon conversion damages DNA when a cell cycle holds in the S phase (synthesis of DNA) and thus in particular affects rapidly dividing cells such as cancer cells. in addition, ubule inhibitory agents such as vinca ids, taxanes (as described above in the context of effector molecules) can also serve as cytotoxic agents ing to the present invention.

Also ed in the definition are kinase inhibitors such as sorafenib or HDAC ne deacetylase), inhibitors such as romidepsin as well as growth inhibitory agents, anti- hormonal agents, anti-angiogenic agents, cardioprotectants, immunostimulatory agents, W0 2013f083817 2012/074867 immunosuppressive agents, angiogenesis inhibitors and protein tyrosine kinase (PTK) inhibitors. r included in this definition are antibody based cytotoxic agents including immunoconjugates and antibodies that have an art recognized cytotoxic effect. Anti- CD40 is a preferred antibody. Other antibodies include, but are not limited to, e.g..

AVASTlN (bevacizumab) or MYELOMAClDE (milatuzumab).

Thalomide (o~(N-phthalimido) glutarimide; thalidomide), is an immunomodulatory agent. The empirical formula for thalidomide is 013H10N204 and the gram molecular weight is 258.2. The CAS number of thalidomide is 501. it appears to have multiple actions, including the ability to inhibit the growth and survival of myeloma cells in various ways and to inhibit the growth of new blood vessels.

Lenalidomide (REVLlMlD) is a derivative of thalidomide representing the second generation of immunomodulatory compounds (lmiDs) which were initially developed as inhibitors of TNF alpha. Effects of lenalidomide e growth arrest or sis, abrogation of myeloma cell adhesion to bone marrow stromal cells and modulation of cytokines promoting cell growth, survival and drug resistance of myeloma cells n et al., 2006). Lenalidomide is effective in patients refractory to thalidomide. in on to effects on immune cells, lmiDs such as domide were suggested to cause cell cycle arrest in GO/G1 phase. In addition, it is d that lmiDs downregulate cell adhesion receptors (VLA—4, VLA-5, CD138) (Quach et al., 2010; Udi et al, 2010).

A downregulation of CD138 would be expected to cause a reduced g of any CD138 targeting agent, such as BT062, to target cells.

Proteasomal inhibitors can be divided into further subgroups: a) naturally occurring peptide derivatives which have a inal epoxy ketone ure, beta-lactone derivatives. aclacinomycin A, lactacystin, clastoiactacystin; and b) tic inhibitors (comprising modified peptide ds. alpha, beta epoxyketon structures, vinyl sulfones, boric acid residues, pinacolesters. A preferred proteasomal inhibitor of the present invention is bortezomib (PS 341; VELCADE, see discussion below). One of the proposed mechanisms suggests that somal inhibition may prevent degradation of pro-apoptotic factors, permitting activation of programmed cell death in neoplastic cells dependent upon suppression of pro-apoptotic pathways. in addition, bortezomib causes GZiM cell cycle arrest (Wang et al., 2009).

Thus, bortezomib might interfere with anti-mitotic agents which are part of the immunoconjugate of the present invention, e.g., with the effect of maytansinoid 0M4, which acts also at this cell cycle phase. Furthermore, PARP (Poiy(ADP-ribose) Polymerase) cleavage, which takes part in sls, is also affected by both DM4 and bortezomib. Accordingly, the combination of an immunoconjugate comprising an anti~ mitotic agent and a proteasomal inhibitor displaying the features of bortezomib do not conform to the l guidelines set forth previously to obtain synergistic effects oto et al, 2009).

VELCADE (bortezomib) is a proteasome inhibitor used to treat le myeloma. it is believed that VELCADE acts on myeloma celis to cause celi death, and/or acts indirectly to inhibit myeloma cell growth and survival by acting on the bone microenvironment. Without being limited to a specific theory or mode of action, VELCADE thus disrupts normal cellular processes, resulting in proteasome inhibition that es apoptosis.

Dexamethasone is a synthetic glucocorticoid steroid hormone that acts as an anti- inflammatory and immunosuppressant. When stered to cancer patients, dexamethasone can counteract side effects of cancer therapy. Dexamethasone can also be given alone or together with other anticancer agents, including thalidomide, lenalidomide, bortezomib, ycin or vincristine.

Substances for treatment, which may be used in combination with BTOBZ also include immunomodulatory agents (eg. thalidomide, and lenalidomide, and pomalidomide), proteasome inhibitors (e.g. omib and omib), steroids (e.g. dexamethasone), alkylating agents and ose chemotherapy, combinations (e.g. Melphalan and Prednisone (MP), Vincristine, bicin (Adriamycin), and dexamethasone (VAD)), and bisphosphonates.

Currently, many combinations of in particular anti—myeloma drugs are investigated in clinical trials. The purpose of the use of a combination is generally either to enhance iveness, to overcome a refractory ype, e.g., of myeloma cells, to reduce W0 2013f083817 2012/074867 side effects due to the use of lower concentrations of one of the ation partners or a combination thereof. Using a low dose, for example, of lenalidomide plus a low dose of dexamethasone was shown to reduce toxicity (Rajkumar et al., 2010). ally in patients with relapsed or refractory multiple myeloma several drug combination are and have been investigated.

A standard example for combined chemotherapeutics represents the triple combination of vincristine, dexamethasone, doxorubicin (VAD Regimen).

Proteasomal inhibitors such as bortezomib (VELCADE) have been combined with myeloma drugs such as melphalan and prednisone (VMP). This combination resulted in a complete response rate of 16% and an overall response rate of 89% (Mateos et al., 2006).

Bortezomib has been also approved for use in combination with mal doxorubicin for relapsed or refractory patients (Ning et al., 2007).

Bortezomib is investigated in several al s for use in ation with dexamethasone. melphalan. prednisone and/or thalidomide.

Bcrtezomib is also under investigation combined with liposomal doxorubicin. cyclophosphamide and thasone in multiple myeloma patients. Combinations with vorincstat are currently under investigation aiming at resensitizing patients to bortezcrnib which are refractory to this drug.

Thalidomide, which is administered orally, has been combined with melphalan/ prednisone (MPT) (Facon et al., 2006) or dexamethasone or bendamustine (Ponisch et al., 2008).

Moreover, lenalidomide (REVLlMlD), an immunomodulatory drug, used in combination with dexamethasone, resulted in a prolonged time to tumor progression and increased survival compared to dexamethasone alone (Weber et al., 2006).

Lenalidomide combined with thasone has been also studied in newly diagnosed patients (Rajkumar et al., 2005) as well as the combination with melphalan/prednisone (RMP) (Palumbo et al., 2006).

US Patent Publication 201010028346 to Lutz et al., bes synergistic effects of n conjugates with chemotherapeutic agents.

The term "in combination with" is not limited to the administration at exactly the same time. instead, the term encompassed administration of the immunoconjugate of the W0 20131083817 present invention and the other regime (e.g. radiotherapy) or agent. in particular the xic agents referred to above in a sequence and within a time interval such that they may act together to provide a benefit (e.g., sed activity, decreased side effects) that is increased compared to treatment with only either the immunoconjugate of the present invention or, e.g., the other agent or agents. it is preferred that the immunoconjugate and the other agent or agents act additively, and especially preferred that they act synergistically. Such molecules are suitably provided in amounts that are effective for the purpose intended. The d medical practitioner can determine empirically, or by ering the pharmacokinetics and modes of action of the agents, the appropriate dose or doses of each therapeutic agent, as well as the appropriate timings and methods of administration. As used in the t of the present invention "co-administration" refers to administration at the same time as the immunoconjugate, often in a combined dosage form.

Synergistic effects that are effects of two ents such as an immunoconjugate and a xic agent that exceeds a strictly additive effect. These synergistic effects might be counteracted by a number of factors further discussed below.

Synergism has been calculated as follows (Yu et al., 2001; Gunaratnam et at, 2009): RAM) (r) = ed FTV (combination)/observed FTV (combination) FTV: Fractional tumor volume = mean tumor volume (test)/mean tumor volume (control) A ratio > 1 is ed as istic, whereas r < 1 is less than additive.

The ratio (r) is, when above 1, also referred to herein as "SYNERGY RATlO." The ACTIViTY RATING is another measurement for the effects of a combination. This rating is based on the Logic cell kill Logm cetl kill = (T-C) I Td x 3.32 where (T-C) or tumor growth delay, is the median time in days required for the treatment group (T) and the control group (C) tumours, to reach a predetermined size (600 mm3). Td is the tumor doubling time, based on the median tumor volume in the control mice, and 3.32 is the number of cell doublings per log of cell growth (Bissery et al., 1991). A Logm cell kill of higher than 2.8 indicates that the combination is highly active, a logm cell kill of 2.0- 2.8 indicates that the combination is very active, a log", cell kill of 1.3- 1.9 indicates that the combination is active, at logw can kill of 0.7- 1.2 indicates that the combination is moderately active and a logm cell kill of less than 0.7 indicates that the ation is inactive.

As the person skilled in the art will appreciate, the amino acid sequence of the preferred engineered targeting antibody portion of an immunoconiugate, nBT062, can be varied without loss of the functionality of the antibody portion in targeting CD138. This is in particular true when the heavy chain variable region CDR3 comprising amino acid residues 99 to 111 of SEQ lD NO: 1, and light chain variable region CDR3 comprising amino acid residues 89 to 97 of SEQ lD NO: 2, respectively of the antigen binding region (ABR). Advantageously, the heavy chain variable region CDR1 and CDR2 sing amino acid residues 31 to 35 and 51 to 68 of SEQ 10 NO: 1, and/or (b) light chain le region CDR1 and CDR 2 comprising amino acid residues 24 to 34 and 50 to 56 of SEQ iD NO: 2, respectively of the antigen binding region (ABR) are also maintained.

The term "sequence identity’ refers to a measure of the identity of nucleotide ces or amino acid sequences. ln l, the sequences are aligned so that the highest order match is obtained. "identity", per as, has recognized meaning in the art and can be calculated using published techniques. (See, e.g.: Computational Molecular Biology, Lesk, A. M.. ed., Oxford University Press, New York, 1988; Biocomputing: atics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1998; Computer Analysis of Sequence Data, Part l, Griffin, A. M., and Griffin, H. G., eds, Humana Press, New , 1994; Sequence Analysis in Molecular Biology, von Heinje, 6., Academic Press, 1987; and Sequence Analysis Primer, ov. M. and Devereux, J., eds., M on Press, New York, 1991). While there exist a number of methods to e identity between two polynucleotide or polypeptide sequences, the ' term "identity" is well known to skilled artisans (Carillo, H. 8: , D., SIAM J Applied Math 4821073 (1988)).

Whether any particular nucleic acid molecule is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nBT062 nucleic acid sequence, or a part thereof, can be determined conventionally using known computer programs such as DNAsis software (Hitachi Software, San Bruno, Calif.) for initial sequence alignment followed by ESEE version 3.0 DNA/protein sequence software @trog.mbb.sfu.ca) for multiple sequence alignments.

Whether the amino acid sequence is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance SEQ lD N021 or SEQ 1D NO:2, or a part thereof, can be determined conventionally using known computer programs such the BESTFlT m (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). BESTFlT uses the local homology algorithm of Smith and Waterman. es in Applied Mathematics 2482-4893 (1981), to find the best t of homology between two sequences.

When using DNAsis, ESEE, BESTFlT or any other sequence alignment program to determine whether a ular ce is, for instance. 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleic acid or amino acid sequence and that gaps in gy of up to 5% of the total number of nucleotides in the nce sequence are allowed. if, in the context of the present invention. reference is made to a certain sequence identity with a combination of residues of a particular ce, this sequence identity relates to the sum of all the residues ed.

As discussed above, BT062 is an conjugate sing the CD138 targeting chimeric antibody nBT062 that is attached via a linker, here SPDB, to the cytostatic maytansinoid derivative DM4. A chemical representation of BT062 is provided in FlGS. 1 and 2. lmmunoconjugates comprising nBT062 and a maytansinoid effector molecule are often characterized in terms of their linker and maytansinoid effector, e.g., nBTOSZ- SMCC-DM1, is an immunoconjugate comprising nBT062, SMCC (a "noncleavable" linker ning a thioester bond) and mm as an or. More generically, immunoconjugate containing nBT062 and an effector molecule may also be described as nBTOGZ-iinker—effectcr orjust as nBTOBZ-efi’ector (nBT062N, wherein N is any effector described herein (see also US Patent ation 20090232810). in one example, BT062 binds to CD138-positive multiple myeloma cells. Once the target cell internalizes and/or releases the immunoconjugate, DM4 is released from the targeting molecule, y restoring its original cytotoxic potency of DM4. Thus, BT062 provides a targeted antibody payload (TAP), wherein the functional attachment of DM4 to nBTOfiZ keeps the cytotoxic drug inactive until it reaches/is internalized into the CD138 expressing target cell.

Data from nonclinical studies investigating cytotoxicity of BT062 in multiple myeloma cells and animal models discussed herein demonstrate that BT062 has highly significant antimyeloma activity at doses that are well tolerated in a murine model.

A phase i open-label, dose escalation, repeated single dose study in patients with relapsed or relapsed/refractory multiple myeloma has been conducted (US patent publication: 20110123554; lntemational publication: WC 2010 128087).

The immunoconjugates disclosed herein can be administered by any route. including intravenously, erally, orally, intramuscularly, intrathecally or as an aerosol. The mode of delivery will depend on the d effect. A skilled artisan will readily know the best route of stration for a particular treatment in accordance with the t invention. The appropriate dosage will depend on the route of administration and the ent indicated, and can readily be determined by a skilled artisan in view of current treatment protocols.

Pharmaceutical compositions containing the immunoconjugate of the present invention and/or any further xic agent as active ingredients can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Phamiaceutioal Sciences, 17th Ed. (1985, Mack hing 00., , Pa).

Typically, effective amounts of active ingredients will be admixed with a ceuticaliy acceptable carrier. The carrier may take a wide variety of forms depending on the form of preparation desired for administration. for e, intravenous, oral, parenteral, intrathecal, transden'nal, or by aerosol. -gg- The anticancer combinations of the present invention can preferably be either in the form of pharmaceutical compositions or in the form of kits comprising the components of the anticancer combination in different containers. The components of the kit are usually administered in combination with each other, often they are co—administered either in a combined dosage form or in separate dosage forms. Such kits can also include, for example, other components, a device for administering the components or combination, a device for combining the components and/or instructions how to use and administer the components.

For oral administration, the immunoconjugate and/or cytotoxic agent can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, s, suspensions or emulsions. in ing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, ls, flavoring agents, preservatives, coloring agents, ding agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, ating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for e, powders, capsules and tablets).

Because of their ease in administration, tablets and capsules ent the most ageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. if desired, tablets may be sugar-coated or enteric-ccated by standard techniques. The active agent must be stable to passage through the intestinal tract. If necessary, suitable agents for stable passage can be used. and may include phospholipids or lecithin derivatives described in the ture, as well as liposomes, articles (including pheres and macrospheres).

For parenteral administration, the immunoconjugate and/or cytotoxic agent may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. illustrative of suitable carriers are water, saline, phosphate buffer solution (PBS), se solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic . The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like. When the unconjugated targeting agent and/or immunoconjugate and/or cytotoxic agent are being —100- administered intracerebroventricularly or intrathecally, they may also be dissolved in cerebrospinai fluid.

Dosages administered to a subject may be specified as amount, per e area of the subject (which includes humans as well as man animals). The dose may be, in a (multiple) single dose regimen, generally lasting 21 days, administered to such a subject in amounts, preferably, but not ively from about 5 mg/m2 to about 300 mg/mz, including about 10 mg/m2, about 20 mg/m2, about 40 mg/m2, about 50 mg/m2, about 60 mg/m2, about 80 mg/m2, about 100 mglmz, about 120 mglmz, about 140 mg/mz, about 150 mg/mz, about 160 mg/m2 and about 200 mg/mz. ln a (repeated) multiple dose regimen, the aggregate dose may administered within one cycle, generally lasting 21 days, to such a subject may preferably, but not exclusively be from about 120 mg/m2 to about 840 mg/mz, including about 120 mg/mz, about 130 mg/mz, about 140 mg/mz, about 150 mg/mz, about 180 mg/mz, about 195 mg/mz, about 240 mg/mz, about 300 mg/m2, about 360 mg/mz, about 420 mg/mz, about 450 mg/mz. about 480 mgi’m2 , 600 mg/mz, 720 mg/m2 about 840 mg/mz. The aggregate dose is administered ably in at least three individual doses, wherein the dose administration can be isochronously. e.g., once every week. preferably on days 1, 8, 15 or anisochronously within, e.g., the period of 21 days. dual dosages administered may be about 3 x 40 mg/m2. about 3 x 50 mg/m2, about 3 x 60 mg/m2, about 3 x 65 mg/m2, about 3 X 80 mg/m2, about 3 x 100 mg/mz, about 3 x 120 mg/mz, about 3 x 140 mg/mz, about 3 x 150 mg/mz, about 3 x 160 mglm2 , 3 x 200 mg/mz, 3 x 240 mg/m2 about 3 x 280 mg/mz.

The immunoconjugates are suitably administered at one time or over a series of treatments. in a multiple dose regime these amounts may be administered once a day, once a week or once every two weeks. Loading doses with a single high dose or. alternatively, lower doses that are administered shortly after one another followed by dosages timed at longer intervals constitute a preferred ment of the t invention. E.g., in a le dose regimen, a g dose of anywhere between 100 to 160 mg/2 could be combined with one or two subsequent doses of 40 to 1OOmg/m2. In a preferred embodiment, the timing of the dosages are adjusted for a subject so that enough time has passed prior to a second and/or any subsequent treatment so that the previous dose has been metabolized substantially, but the amount of immunoconjugate ~101— present in the subject’s system still inhibits, delays and/or prevents the growth of a tumor. An exemplary "repeated multiple dose" regime comprises stering doses of immunoconjugate of about 10, 20,40, 50,60, 65. 80, 100, 120, 140, 160, 180, 200, 220 or 240 mgz’m2 once every week. Alternatively, a high initial dose of, e.g., 160 mglm2 may be followed by a one, two, or tri-weekly maintenance dose of, e.g., about 20 mg/m2. Other combinations can be readily ascertained by the person skilled in the art.

However, other dosage regimens may be useful. The progress of this y is easily monitored by known techniques and assays. Dosage may vary, amongst others, depending on whether they are administered for preventive or therapeutic purposes, the course of any us therapy, the patients clinical y, the patient‘s disease status, the patient's tumor load, the patient's genetic predisposition, the patient’s concomitant diseases. the disease stage upon first treatment and se to the targeting agent/immunoconjugate, the side effects experienced by the patient and the discretion of the attending ian.

When a dose X of an immunoconjugate is said to significantly exceed another dose Y, it means that total (e.g., aggregate) dose X exceeds total (e.g., single) dose Y by at least 10% (e.g., if dose X is 100mg/m2, a dose Y that significantly exceeds dose X is at least "Ding/mg), preferably about 20% more preferably about 30%, 40%, 50%, 60% or even more.

The term individual dose is in particular when used in the t of a multiple dose regime used to describe a defined dose stered in a single administration and can be contrasted to the aggregate dose administered, e.g., in an active treatment cycle, which is the sum of the individual doses stered in said treatment cycle. E.g., three individual doses in an active treatment cycle lasting, e.g., 21 days of n'i2 result in an aggregate dose of 300 mglmz.

The level of an conjugate in a patients body fluid such a, e.g., in the patient‘s plasma, serum or plasma is measured by methods well known in the art. Plasma levels can be assessed via different phamtacokinetic (PK) assay like the one described under Materials and Methods. The levels of the immunoconjugate in the serum or plasma or other blood derived body fluid is generally determined 2 to 4 hours after the start of the respective administration, respectively, wherein said administrations are preferably in 402- form of an infusion. This generally corresponds to 0-2 hours after completion of an administration, in particular an infusion.

The present invention is, in one ment, directed to a maintenance therapy.

As can be seen in Fig. 28 long-term therapy with up to 160 mg/m2 once every 21 days is sfully ing stable disease or even minor response, but at least progression free survival. The Figure also shows that plasma concentrations of BT062 sed over time indicating a decrease of tumor burden under treatment.

This type of therapy is well suited to follow a repeated multiple dose regimen of, 9.9., weekly administration. A typical nance therapy with dosing up to 160 mg/m2 once every three weeks may follow a repeated multiple dose regimen (e.g. once every week for three weeks). Depending on the tumor burden, lowering the doses may be employed, whereby, e.g., plasma levels of the immunoconjugate or other relevant parameters may serve to determine the appropriate closing for maintenance therapy.

Maintenance can be achieved by threshold levels of the immunoconjugate, which are permanently present/maintained in the subject, so that there is a constant amount of immunoconjugate available. in a preferred embodiment. a tumor in a subject/the target cells are permanently exposed to immunoconjugate, so that no new tumor cells can grow, or that they are quickly destroyed due to a constant ce of the immunoconjugate in the subject, which is reflected by a certain measurable level of conjugate in the subject’s. e.g., plasma.

The maintenance y preferably reduces administration frequency. However, other maintenance therapies resulting in particular in, e.g., reduced aggregate doses of immunoconjugate administered are also preferred. The particular design of a maintenance therapy will depend, among others, on tumor . The level immunoconjugate and/or other efficacy blood ters such as M-protein, FLC or a tumor/cancer specific marker can be determined in a body fluid, such as the plasma, serum or urine of the subject (patient) and the nance dose and frequency of the dose can be made dependent on the level or a change in the level of the efficacy blood parameter. A kit that may be employed in this as weli as other contexts of the present invention may include markers, in particular antibodies, preferably labeied dies, t the immunoconjugate, e.g., against the toxin portion of the immunoconjugate, which can be used to quantify the immunoconjugate in a body fluid of a subject. A -103~ 2012/074867 signal obtained from the binding of the, e.g. labeled antibody, can be correlated to the amount of immunoconjugate present in the body fluid of a subject. Suitable individual dose levels, both for repeated multiple doses as well as repeated single doses, for maintenance therapy are, e.g., 60-160 mg/mz.

Extended treatment free s may be beneficial for the patient. Surprisingly, it was found that after even an extended restng period (see days 400 to 421) stable disease could still be maintained (see Fig. 28).

The present invention is, in one embodiment, directed to an administration regimen, ably with rapid plasma clearance. The n provides generally less than about 280 mg/mz, less than 120 mg/mz. less than 100 mg/mz, less than 80 mg/m2, including no more than about 40 mg/mz, more preferably no more than about 20 mg/m2, even more ably no more than about 10 mg/m2 in a given week for at least three consecutive weeks which define an interval (cycle). The 10 mg/m2 to 280 mgim2 range translates to an average daily dose of about 1.43 mglm2 to 40 mg/m2 Thus, average daily doses of about .4 mg/m2 to about 17.14 mg/m2 , ing about 5.7 mg/m2 , about 7.1 mg/m2 , 8.58 mm2 , 9.28 mg/m2 , 11.4 mg/m2 . 14.28 mg/m2 , 17.1 mg/m2 , 22.85 mg/m2 , 25.7 mg/m2 (180 mg/mz), 28.58 mg/m2 342 mg/m2 , 40 mg/m2 are part of the present invention. Low dose administration schemes up to 1OOmg/m2 are associated with rapid plasma clearance at the in early ation phase. that is. any time during administration up to two hours after administration is completed. What distinguishes the low dose administration regime from other low dose regimens is the rapid plasma clearance, which is defined by a measured Cmax during that period that is preferably less than 55%, less than 50%, less than 40%, or less than 35% of the theoretical Cmax (Tables 11). stration regimens, are, at higher ievels, accompanied by less rapid plasma clearance, that is by plasma clearances that exceed 55%, often 60%, 70% 80% or 90% of the theoretical Cmax value, which are referred to herein as moderate (equal or > 55%, but < 80% of the theoretical Cmax value) or slow plasma clearance (equal or >80% of the theoretical Cmax value). At these clearances it was surprisingly found that, despite the relative high concentration of immunoconjugate in the , these administration regimens still did not result in DLTs. This is despite the fact that expression levels of 00138 on non target cells that express CD138, e.g., cells of vital ~104- organs, such as the lium which are not target of any treatment, are also ve high in 00138 (immunohistochemistry analyses with the CD138 antibody BB4 showed that the reactivity to this antibody to the epithelium matched that of MM patient plasma cells (US Patent Publication 20070183971». Expression levels of CD138 on target and non target cells that produce equal scores (e.g. plus three ((+++) as in the above example) in histochemistry analyses are ed to herein as comparable expression levels and are part of the present invention. in an alternative embodiment. the expression levels on target cells were actually consistently below that of the epithelium (e.g., plus one (+) or plus two (++) vs. plus three (+++) for the epithelium).

Some tumor target cells show mixed expression levels, such as, that some cells have an expression level of plus two and some an sion level of plus three. The mean of a representative number of cells (such as 100 randomly d cells) will determine r these tumor target cells in question fall under the definition of having expression levels comparable or below that of the epithelium. These treatment regimens are generally above 40 mg/m2, but below 180 or even 280 mgl’m2 given weekly at least for three consecutive weeks which define an active treatment cycle. which translates to a daily doses of about 5.71 mg/m2 to about 25.71 mg/m2 (180), 40 mg/m2 (280).

With respect to Patient 8 (see Figure 18 for numbering) it was noticeable. that this patient had. during the entire treatment of 168 days. while there was an increase in FLC until day 141, no disease progression (see also Figure 21), ing the efficacy of BTDBZ administration, while Patient 6 showed no disease progression for 6 cycles (Figure 20).

Figure 195 clearly demonstrates that a constant amount of approx. 20 pg/ml is lost, presumable during infusion. This has been calculated from the difference between the plasma levels (defined here as Cmax) determined in the samples and the theoretically achievable Cmax value. ln Table 110 the absolute values for the plasma level determined between 0 and 2 hours after the end of infusion are displayed and compared to the theoretically achievable Cmax plasma values ("Theoretical Cmax" as calculated by the formula below) ~105- tical Cmax was calculated according to the following assumed ters: Patients Body e Area 1.9 m2 Patients Weight 70 Kg Patient’s Plasma Volume 40 mlle istered dose x e area) / body weight Plasma Volume in certain embodiments, the invention is also directed to a treatment regimen, wherein the dose can be adapted according to the measured ievel of an efficacy blood parameter found in a body fluid such as plasma. This allows for a patient tailored treatment. For example, the dose of BTOBZ may be adapted according to plasma levels determined between 0 and 4 hours after tion of an administration, such as an infusion.

As can be seen in Fig. 29, the ter M-protein (decrease in its ievel) indicated in this t disease improvement. At the same time plasma levels (Cmax values) of BT062 increased. With increasing treatment cycles, the tumor load was d and concomitantly, plasma levels ("Cmax values") determined in the plasma after 0-4 hours after completition of the infusion with BT062 increased, so that a negative correlation between the M-protein level and the Cmax were observed. The increase in Cmax values can be explained by a decrease in tumor volume, which means that fewer tumor cells are present, which is reflected in the decrease in M—Protein level. Less CD138 as a target source would lead to less binding sites for BT062. As a consequence, more BT062 can be detected in the plasma. Thus, the Cmax values can be used to evaluate the response to treatment (increase in Cmax correlates with efficacy). if, in a given instance, the Cmax values increases, when compared to Cmax of a prior injection (or any injection, where no efficacy was seen) e.g. by 10% , 20% or more, this indicates that fewer binding sites on the tumor are present and that thus tumor size decrease. in this example, the dose can be ed to a lower dose in the next treatment cycle. As a result, a lower amount of drug needed and toxicities can be prevented.

A repeated single dose refers to a sequence of administrations, wherein the administration following an administration is regarded to be ndent of this preceding administration. Thus, in the present context, the level of immunoconjugate in a subject’s blood can be regarded as equal after each administration. Each time the conjugate is administered, it is ed that equal levels of immunoconjugate are lly present in the blood.

Administration intervals n the "single doses" of the repeated single doses are defined according to the theoreticaliy calculated half life of an isotype of an immunoconjugate, in the case of BT062, lgG4.

In general, the half life of therapeutic antibodies depends mainly on the dy characteristics/ its structural features (e.g. binding to Fc receptors) and the target. For example, the binding affinity of the Fc part to the neonatal receptor FcRn is affecting the half life. By binding to FcRn in endosomes, the antibody is salvaged from lysosomal degradation and recycled to the circulation, which prolongs the half life. For an lgG4 a half life of 15.6 (+/~ 4.5) days (Alyanakian et at, 2003; Saifeld et at, 2007) has been reported. In the study referenced herein, a "repeated single dose" has been chosen that has stration intervals of three weeks. However. about three weeks. about four weeks, but also about five or about six weeks are alternative intervals for repeated single doses. A reference to "about" refers in the t of three weeks to +/- 96 hours and in the context of four to six weeks to +/— 120 hours.

A multiple dose n or a multiple dose refers to a ce of administrations. wherein the administration following an administration is regarded to be dependent of the preceding administration. Thus. in the present context. the level of immunoconjugate in a subject’s blood is expected in a second and subsequent administration to be above the base level that existed prior to the initial administration.

At each stration following the initial administration of the immunoconjugate, a certain ievel of immunoconjugate is expected to be present in the blood.

Administration intervals between the individual "doses" of the muitipie doses are defined, as in the context of the repeated single doses. according to the theoretically ated half life of an isotype of an immunoconjugate, in the case of BT062, igG4.

For an igG4 a half life of 15.6 (+1— 4.5) days (Aiyanakian et at, 2003; Saifeld et at, 2007) has been reported. in the study referenced herein, a "multiple dose" has been chosen that has administration intervais of one week. However, even shorter administration intervals may be chosen such as 4 days or even 3 days. Aiternatively, a longer interval can be chosen. r, at a minimum a multiple dose implies at least 2 administrations in a 21 day period. A reference to "about" refers in the context of one week to +/- 32 hours, in the t of 4 days, +/- 18 hours and in the context of 3 days +/- 12 hours. A repeated multiple dose refers to multiple doses administered in subsequent treatment cycles, which may e intermittent resting period(s) or ent free period(s), including extended g period(s) ortreatment free period(s), that do not obliterate in whole the effects of the previously administered multiple ).

The actual level of immunoconiugate after the first and each subsequent administration, however, s on the de facto "clearance" of the immunoconjugate from the,_e.g., the plasma ("plasma clearance") immediately during/ after completion of the stration, in particular, 0—2 hours after completion of administration. At 40 mg/m2 median infusion time was 40 min within a range of 32 min to 1 hour 30 min. At the dose level of 120 mg/m2 median on time was 2 hours 2 min within a range of 1 hour 40 min to 2 hour 30 min. Accordingly, in an iv administration, about 1 mg/m2 may, in certain embodiments, be administered on e per minute, but administration times, of about 1 mg/m2 per 30 seconds to about 1 mg/m2 per 120 seconds are well within range. Surprisingly, it was found that BT062 cleared from the plasma considerably faster than either the theoretical expected values or the values encountered with similar immunoconjugates. This observation allowed for the design of new administration regimens for the immunoconjugate both alone in a monotherapy as well as in combination with other relevant agents, in particular cytotoxic agents to provide effective anticancer combinations.

Aggregate effective amount is the effective amount of immunoconjugate administered within a period of a dosing regimen, preferably in equal doses, e.g., once a week, for e.g. three weeks such as on days 1, 8, and 15 ofa 21 day dosing regimen or on days 1, 8, 15 of a 28 day dosing regimen wherein no dose is stered on day 22.

The progress of the therapy is easily monitored by known techniques and assays.

Dosage may vary, amongst others, depending on whether they are administered for preventative or therapeutic purposes, the course of any previous therapy, the patients clinical history, the patient’s disease status, the patient’s tumor load, the patient’s genetic predisposition, the patient's concomitant es, the disease stage upon first ent and response to the ing agent/immunoconjugate, the side effects experienced by the patient and the discretion of the attending physician. ~108- The advantages of a low dose regime are wide-ranging. However, the probably most significant advantage is minimizing the risk of adverse side s. While immunoconjugates generally permit ive discrimination between target and normal cells, resulting in fewer toxic side effects than most conventional chemotherapeutic drugs. many immunoconjugates are still not completely free of side s. e superior targeting, the antigen of interest is generaiiy also expressed on non-cancer cells whose ction during therapy can lead to adverse side effects. in the case of C0138, the antigen is in particular expressed on epithelial cells. Also, the immunoconjugate might undergo processing within the body that is unrelated to the procession in or at a target cell and a certain percentage of effector molecules might be released at ons remote from the target cells leading to toxic side effects. it was shown that the immunoconjugate of the present invention was effective at low doses, while displaying clinically acceptable toxicities (dosages up to 160 mg/m2 provided once every three . At doses up to at least 120 mg/m2 but in any event at doses of less than 160 mg/m2 provided once every three weeks (e.g.. on day 1). the tested immunoconjugate also showed rapid plasma clearance in human subjects.

Tables 9 and 10 show the clearance observed in repeated single dose regimens. plasma level of BTOGZ (pg/ml) human theoretical Cmax effective Cmax effective Cmax dosage BT062 (cycle 1) mean (cycle 4) mean mglmz (lowest; highest) (lowest; highest) —_7 1 4 2.9 7.06 1.66; 4.44 6.79; 7.34 40 27 4 31 2.51 (O 97; 9 86 1.02; 3.68 13.4; 23.6 7.4‘ 21 120 3 SD .1; 28.7 (73.7; 85.5) 68.0; 102.4 n.a. data not available Table 9: Plasma trations after end of infusion and effective Cmax mean values of BT062 from plasma obtained in patients having received a single doseirepeated single dose BT082 (first and fourth cycle). ed dose administration in cycles of 21 days. Cmax values were obtained between 0 and 2 hours post infusion. Administration cycles: cycle 1: day 1, cycle 2: day 22; cycle 3: day 43; cycle 4: day 64 etc. lasma level of BT062 theoretical effective percentage of ive percentage of Cmax Cmax theoretical theoretical (cycle 1} Cmax (n) Cmax (n) —__‘1l_m M34%3_4262°/20 120 214 -_l_. - 200 136 82.0 60%03 n..a n.a. data not available n: number of patients Table 10: Effective Cmax mean values of BT062 from plasma obtained in patients having received a single dose/repeated single dose BT062 (first and fourth cycle). Repeated dose administration in cycles of 21 days. Maximum values were obtained within the first 2 hours post ion. Cmax values were obtained between 0 and 2 hours post infusion. Effective Cmax is indicated in percentage of theoretically calculated Cmax. Administration cycles: cycle 1: day 1, cycle 2: day 22; cycle 3: day 43; cycle 4: day 64 etc.

The theoretical Cmax was calculates as described above.

Although the half life of BT062 in plasma of human subjects treated proved to be significantly lower than the plasma half life observed in cynomolgus monkeys (days) and in human plasma ex vivo (14 days), the immunoconjugate still showed efficacy in human subjects, even at administrations as low as 20 mg/m2 suggesting an accelerated tumor ing and tumor cell binding which results in an increased efficacy.

The accelerated tumor targeting could be confirmed by measurements of the receptor ) occupancy on le a cells in the bone marrow of a multiple myeloma patients. As can be seen in Table 1162 at different repeated multiple doses regimen, the receptor occupancy at the tumor site in the bone marrow came close to 100% within four hours after the end of the administration of the immunoconjugate, supporting an antibody mediated rated tumor targeting. Accordingly, the present invention is directed at immunoconjugates having an early, that is 0—12, 0-10, 0-8, 0-6 or 0-4 hours after completion of administration, target tissue receptor (CD138) occupancy of between 70-100%, preferably 80-100%, more ably SIG-100%, even more ably more than 94, 95, 96, 97 or 98% "receptor occupancy" (R0). The "receptor" is hereby CD138 and the R0 is measured according to the following formula: R0: (MFl Sample 1 — MFI Sample 3) I (MFI Sample 2 — MFI Sample 3) MF! = Mean Fluorescence intensity measured via flow cytometry s of a cells in bone marrow aspirates.

Sample 1: Bound immunoconjugate, here, BTOGZ was stained with anti-May (May: maytansinoid) antibodies.

Sample 2: Total CD138 was measured with anti-May antibodies after or saturation with the immunoconjugate.

Sample 3: unspecific binding measure by incubation with an lgGi isotope antibody.

As noted above. unusual rapid nce from plasma of treated MM patients was observed in the early elimination phase (observed already during infusion and about 0 to 2 hours post on. ergo completition of infusion) followed by generally normal terminal elimination phase at dose levels up to 120 mg/mz. whereas a more typical clearance profile was observed for all 4 patients at the 160 mg/m2 and 200 mg/m2 dose (3 patients), even though the clearance was still below the theoretical Cmax value. in addition, in the administration regimens that showed rapid plasma clearance at the early elimination phase, eg. 20, 40, 80 and 120 mg/mz) not only rapid plasma clearance at the early elimination phase was observed, but a response (decrease of urine M-protein) was observed, ing responses that manifested themselves in a decrease of urine M-protein by more than 50% after repeated single dosages (results not shown).

As discussed above, data supports that the rapid clearance from plasma of treated MM patients observed in the early elimination phase can be correlated to a high receptor occupancy at the target cells.

Surprisingly it was found that in a multiple dose regimen rapid plasma clearance ed at aggregate dosages that were well above the 1.2Omg/m2 and in fact close to the determined DLT of 'i 60 mg/m2 for a repeated single dose regime, which opened up the possibility for potent mono- or combination ies due to low the ties of the immunoconjugate in the multiple dose regime.

Table 11a shows the % of theoretical Cmax values following differently dosed weekly administration s lasting for 3 weeks (21 days). ergo multiple dose regimens.

The percent of tical Cmax in the 65 mg/m2 cohort is higher than in the lower dosed cohorts shown: 65 m/m BOm-Im 100m-im 120m-lm c1 01 43% 42% c1 D8 63% I 42% 61 015 72% i 44% 79% 02 01 52% 45% 94% 62% 02 DB 61% 50% 102% c2, D15 41% 35% 52% 43% 111% 0313152%39% 109% _ c3 08 71% 53% 121% _ CS D15 73% 41% 142% - —C4D? -24%—— 125% — C4 D8 123% - C4 D15 35% 42% 135% Mean % 33% 33% 60% 44% 108% Standard 0 a Table 11a: °/o of theoretical Cmax: CX refers to the number of cycle: C31 is cycle 1, wherein each cycle is 21 days long fotlowed by one treatment free week (or each cycle is ered 28 days long with no administration on day 22) DXlS the day within the cycle at which the conjugate is administered; D8Is day 8 of the cycle The "/0 theoretical Cmax was calculated as set forth above The high standard deviation for 100mg/m: and the relative lower percentiles of Cmax at ‘l20mg/m2 indicate that the high percentiles at 1OOmg/m2 are a deviation Cone. Missing to theor. Cmax [mglm ] 40 mg/m2 50 mg/m2 65 mg/m 80mg/m _00mglm 120mglm C_1D1 25.4 C1D8 _1._64 242 130 21,6 02D1 182249 312 CS, D1 19,4 23,8 21,3 33,0 -6,0 CS, DB 19.1 24,2 12,8 25,8 w14,4 C401_—=:_ c4, 08 188_15,8 Mean 18. 6 SD 1.5 22340 17.77 .0 13. 2 Table 11 b Mean conc. missing to theor. Cmax per Cycle 40 mg/m2 50 mg/m2 65 mg/m 80 mg/m 100 mg/m 120 mg/m C1 D1 C1-DS Table 1 1:: Table 11b and Ho: CX refers to the number of cycle. Ci is cycle 1, wherein each cycle is 21 days long followed by one treatment free week (or each cycle is considered 28 days long with no administration on day 22). DX is the day within the cycle at which the immunoconjugate is administered; DB is day S of the cycle. Shown in 1‘": are the trations (mg/m2) in absolute terms that, based on the actual Cmax, are missing at each dosage level to reach the theoretical Cmax. in 110 the actual numbers per administration are shown, on the right side the mean concentrations in each cycle are shown. it is also noticeable that the mean concentration (110) within one cycle and over the three cycles shown is comparable and constant. Apart from a deviation at 1OOmg/m2, the missing concentration also remains relatively constant.

Plasma Dose level/Mean m-Im Time da 8 Patient uiml SD n/ml Pre~dose 0 (day of stration).2 (hr after completion ofI! 7,78 2,23 4O administration 4 40 before next dose 0,33 40 I 7.2 4 10,67 4,93 mmmmmcceOOOOOOOO before next dose 0,55 14,2 11,61 5,50 before next dose 0,62 Predose l0,2 before next dose! 0,46 NM w 3,58 before next dose 3 0,45 50 14,2 3 10,43 3,47 before next dose 2 0,38 65 Predose 1 4 O 65 0,2 4 19,18 8,43 010010) 101 before next dose . 4 \l N 4: 27,83 3.95 !beforenextdose 14,2I 31,94 18.12 -i‘l 3.. 65 before next dose 4 80 Predose 80 before next dose 3 80 14,2 3 80 before next dose 3 3.77 0‘78 54.85 24.34 before next dose 3 4,13 1,82 ,70 29.81 120 7,2 before next dose before next dose Table 1 1d represents the mean values of plasma level (pg/ml) of conjugate at dose levels between 40 and 120 mg/m2 before and after the weekly administration. The mean plasma level before the next administration ("before next dose") starts to increase slightly. At 65 and 80 mg/m2, plasma levels before the next dose stay above 1 uglml. At 100 and 120 mg/m2 , the level before the next administration is between . 2 and 4 pg/ml, thus the plasma levels of subsequent treatments, are somewhat higher than those in the first cycle, indicating some accumulation prior to the next injection. l i , Week! Patient ID. y Dose Level i No. Cycles at i Receptor occupancy f , i (mg/m2) 3’ Term. 1 (R0) ____________________________2 "Wowcwmmmfiw1mm g m -__ __ 2 MW 12 80 3 .‘ i came 99 % .......w. ,,_ mime -,,,,,,,,,,,,,,,,,,, -. ,. m _ ML," wmimc MW -WA ow E 12 g 80 (313015" E 37 % c .fl l , - , W i i E c M W,,,,,,,,, N i i g g i 12 80 E c14015** i 51 % i " 2 2 i 22 140 § Cl o1* 86 % l i t 3 i_.___...W...2-w__._m_.-,2.w-w~._g ..........................._ J .......... .___ 2A} ..2 _,_~,w _ .z i 23 i 140 0101* 1 ound to high ,3- H. - missus u... ,,,,,,,,. "._-...._3,._2 .. ~_..»ul 2.- W"W ,_ ,V._ o "m2-4.. -"2a l ‘ i i 26 140 0101* 95 % l __., J i V c..__~_ .... _-m.,_. ....... m mm... ., ~_M...... -wa i i 23 i 140 g c1 os** I 58% i i l t 24 i 140 i 0101* i 94 % ‘"WWW«WMMWVWWJ am25 i 140 i c101* g 98 % _.............u W_.MJW. c Madam- "at l i . 30 160 l 01 01* I 98 % a E g 31 E 160 i J 0101* 76 % , macaw W W;-WWWWWWWwwwwmj. ..» u .u_w...mWWWWWW_ Table 12: Receptor Occupancy (R0) in Repeated le Dose Regimen: Bone marrow receptor occupancy was measured via flow cytometry. Myeloma cells in bone marrow aspirates were characterized by CD138 and CD38 staining (not shown). Bound BT062 was stained with anti-May antibodies (Sample 1). Total 00138 was measured with anti-May antibodies after receptor saturation with BT062 (Sample 2). incubation with an lth isotype antibody determined unspecific binding to the sample (Sample 3). The occupancy of 00138 was calculated with the following equation.

RO= (MFI Sample 1 - MFI Sample 3) I (MFI Sample 2 - MFI Sample 3) wherein, MFl = Mean Fiuorescence intensity Each cycle lasted 28 days with administration of the indicated dose on days 1, 8 and 15. 013015, for example, indicates the dose administered on day 15 in the 13‘h cycle. In three of the above, measurements are based on s that were taken just prior n 12 hours) to the next administration and are marked with double asterisks (**) and thus more than 6 days after the last administration. The remainder of the measurements were taken directly after an administration of BT062, here within 4 or 12 or 24 hours after completion of administration (*). As can be seen, the R0 was relatively low just prior to the next stration, while the R0 was high right after administration.

Figure 13 illustrates the rapid plasma clearance for single dose administrations ranging from 40 rag/m2 to 120 mg/mz, while higher doses as illustrated here by a dose of 160 mg/mz, showed plasma clearance closer to the theoretical value. Figure 17 clarifies that the rapid plasma clearance cannot be attributed to a buffering effect caused by soluble CD138. Figure 14 shows how the measured Cmax values of BT062 compared to the tical Cmax values.

Figure 19A and 19A as well as Table 11a illustrate the rapid plasma clearance in an administration scheme involving multiple doses. As can be seen in a scheme that involves individual doses that are administered on days 1, 8, and 15 and that add up within a cycle (e.g., 21 days) to nearly a dosage that corresponds to DLTs of a repeated single dose (3x50 mg/m2= 150 mg/mz', vs. 160 mg/mz) of the immunoconjugate, the actual Cmax values remain well h 50% of the theoretical Cmax value, while at —115- the BLT levels in a repeated single dose, the actual Cmax values are well over 50% of the theoretical Cmax value.

Table 11b shows that at concentrations at which the actual Cmax is on average already well above 50% of the theoretical Cmax, the concentration missing to the tical Cmax, remains on average similar, in the examples provided, namely around 20 ug/ml (see also Figure 198). This may point towards a , which "ab/adsorps" a certain portion of the lmmunoconjugate quickly, but becomes less noticeable as doses increase. In fact at 100 mg/m2 this effect appears only to occur during the first dual dose. However, it rebounded at 120 aklng it likely that that 100 mg/m2 are a deviation. However, the sink of 20ug/ml is observed here also at higher cycles.

Accordingly, the invention is also directed to a method of atment with an targeting agent, preferably an unconjugated antibody, that is fed into this sink instead of the immunoconjugate, which contain or molecules, which are not only toxic, but generally also costly. As the person skilled in the art will tand, the sink may include tumor target cells as well as CD138 expressing cells of other s. Thus, in one aspect of the invention, the constant amount of +/-20 ug/ml of immunoconjugate which is consistently missing to reach the theoretically Cmax value (Figure 198) promptly after or during infusion, ergo is considered quickly to be ad-labsorbed by/ bind to said sink (also referred to herein as "antigen sink"). Such a sink is filled in such an embodiment not by the immunoconjugate, but by another agent, preferably an agent that binds to CD138. ln this embodiment, rather than having the conjugate be ad-labsorbed during/after administration, an alternative ad-labsorbent, e.g., unconjugated antibody, is administered. ng that the immunoconjugate is lost in the sink, and thus potentially does not contribute to the therapeutic effect, a pretreatment can be used to a) minimize the toxicities which might be related to that "sink" and b) lower the required amount of immunoconjugate to obtain equivalent results.

This pretreatment may consist of administration of 20 ug/ml (+/-) of an unconjugated anti-CD138 dy or fragment thereof. preferably nBT062 and may fill this sink.

At repeated single doses of 160 mg/m2 which tute a low dose compared to administration schemes of other immunoconjugates. terminal clearance profiles were closer to normal, that is, closer to the theoretical Cmax values. However, a rapid reduction of FLC in the serum could be observed afterjust a single administration, which manifested itself in a partial response after the 2"", 3rd and 4‘h administration (Figure 26). ues and Derivatives One skilled in the art of therapeutic agents. such as xic , will readily understand that each of such agents described herein can be modified in such a manner that the resulting compound still retains the specificity and/or activity of the ng nd. The skilled artisan will also understand that many of these compounds can be used in place of the therapeutic agents described herein. Thus, the therapeutic agents of the present invention include analogues and derivatives of the compounds described .

For illustrative purposes of the uses of the immunoconjugates some non-limiting appiications will now be given and are illustrated.

Materials and Methods Chimeric Antibody uction (oB-B4z nBTOBZ) 8-84 Murine antibody 8-84 as previously characterized (Wijdenes et al., Br J Haematol., 94 (1996), 318) was used in these experiments.

Cloning and expression of 8—84 and 03-54 / nBT062 Standard recombinant DNA techniques were performed as described in detail in text books, for example in J. Sambrook; Molecular Cloning, A Laboratory Manual; 2nd Ed. (1989), Cold Spring Harbor tory Press, USA, or as recommended by the manufacturer's instruction in the cases when kits were used. FOR-cloning and modification of the mouse variable regions have been conducted using standard PCR methodology. Primers indicated in the respective results section have been used.

Expression of cB-B4 / nBT062 Exponentially growing COS cells, cultured in DMEM supplemented with 10% FCS, 580 pg/mi L-glutamine, 50 ml penicillin and 50 ug/mi streptomycin were harvested by trypsinisation and centrifugation and washed in PBS. Cells were ~117— 2012/074867 resuspended in PBS to a final concentration of 1x107 cells/ml. 700 ul of COS cell suspension was transferred to a Gene Pulser cuvette and mixed with heavy and kappa light chain expression vector DNA (10 pg each or 13 ug of ector). Cells were electroporated at 1900 V, 25 uF using a Bio-Rad Gene Pulser. Transformed cells were cultured in DMEM supplemented with 10% gamma—globulin free FBS, 580 ug/ml L- glutamine, 50 Units/ml penicillin and 50 ug/ml streptomycin for 72 h before antibody- containing cell culture supernatants were harvested.

Capture ELISA to measure expression levels of 08-84 / nBT062 96 well plates were coated with 100 pl aliquots of 0.4 ug/ml goat anti—human lgG antibody diluted in PBS (4°C, overnight). Plates were washed three times with 200 ul/well washing buffer (PBS+0.1% Tween-20). Wells were blocked with 0.2% BSA, 0.02% Tween-20 in PBS, before addition of 200 pl cell culture supernatants containing the secreted antibody (incubation at 37°C for one hour). The wells were washed six times with washing buffer. before detection of bound dy with goat anti-human kappa light chain peroxidase conjugate.

Purification of 08-84 / nBT062 from cell culture supernatants The cB-B4 antibody was purified from supernatants of transformed C08 7 cells using the n A lmmunoPure Plus kit (Pierce, rd. lL), according to the manufacturer‘s recommendation. cB-B4 binding and competition assay Analysis of binding activity of 8-84 and cB-B4 to CD138 was med using the Dlaclone (Besancon. France) 8001 38 kit according to the manufacturer's recommendation, considering the changes described in the results section.

RNA preparation and cDNA sis Hybridoma B-B4 cells were grown and processed using the QlAGEN Midi kit (Hilden, Germany) to isolate RNA following the manufacturer‘s protocol. About 5 pg of 8-84 RNA was subjected to reverse transcription to produce B-B4 cDNA using the am Biosciences (Piscataway, NJ) ist strand synthesis kit ing the manufacturer‘s protocol. -'l18- Cloning of 3-84 immunoglobulin cDNA lmmunoglobulin heavy chain (lgH) cDNA was amplified by PCR using the lgH primer MHV?’ (5‘-ATGGGCATCAAGATGGAGTCACAGACCCAGG-S') [SEQ lD N023] and the [961 constant region primer MHCGi (5'-CAGTGGATAGACAGATGGGGG-B‘) [SEQ ID NO:4]. Similarly, immunoglobulin light chain (lgL) was amplified using the three ent ng primers MKVZ (5‘-ATGGAGACAGACACACTCCTGCTATGGGTG-3') [SEQ lD N025}, MKV4 (5‘—ATGAGGGCCCCTGCTCAGTTTTTTGGCTTCTTG-B‘) [SEQ ID NO:6] and MKVQ (5'-ATGGTATCCACACCTCAGTTCCTI‘G—B‘) [SEQ ID N027]. each in combination with primer MKC (5'-ACTGGATGGTGGGAAGATGG-S') [SEQ lD NO:8]. All amplification products were ly ligated with the pCR2.1-TOPO vector using the TOPO~TA cloning kit (lnvitrogen, Carlsbad, CA) according to the manufacturer's instruction.

E. coli TOPiO bacteria (lnvitrogen) transformed with the ligated pCR2.1 vector constructs were selected on LB-ampicillin—Xgal agar plates. Small scale cultures were inoculated with single white colonies, grown overnight and plasmids were isolated using the QlAprep Spin ep kit according to the manufacturer's instruction. cDNA sequence determination Plasmids were sequenced using the BigDye Termination v3.0 Cycle Sequencing Ready Reaction Kit (ABl, Foster City, CA). Each selected plasmid was sequenced in both directions using the 1210 and 1233 s cycled on a GeneAmeBOO PCR machine. The ophoretic sequence analysis was done on an ABI capillary sequencen The complete cycle of RT-PCR, cloning and DNA sequence analysis was repeated to obtain three completely independent sets of sequence ation for each immunoglobulin chain.

B—B4 VK DNA sequence ist strand synthesis was performed in three independent reactions. The PCR products ted by using primers MKC and MKV2 nces given above) were ligated into pCR2.1-TOPO vectors according to the manufacturer's instruction. Clones from each ndent set of RT—PCR reactions were sequenced in both ions.

MKVZ-primed product sequence was highly similar to sterile kappa transcripts originating from the a fusion partner such as MOPC-21, SP2 and A98 (Carroll et ~119- at, Mol Immunol., 25 (1988), 991; Cabilly et at, Gene, 40 (1985); 157) and was ore disregarded.

The PCR products using MKC with MKV4 and MKVQ primers were similar to each other and differed only at the wobble positions within the leader sequence primer.

B-B4 VH DNA sequence 1st strand synthesis was performed in three independent reactions and PCR ts were cloned and sequenced from each ist strand product. Five clones were ced from each 1st .

Construction of chimeric cB~B4 expression vectors The uction of the ic expression vectors entails adding a suitable leader sequence to VH and VK, preceded by a BamHI restriction site and a Kozak ce. The Kozak consensus ce is l for the nt translation of a variable region sequence. it defines the correct AUG codon from which a ribosome can commence translation, and the single most critical base is the adenine (or less preferably, a guanine) at position —3, upstream of the AUG start. The leader sequence is selected as the most similar sequence in the Kabat database (Kabat et a/., NIH National Technical Information Service, 1991). These additions are encoded within the forward (For) primers (both having the sequence 5'-AGAGA&TTGCCGCCACCAT.

GATTGCCTCTGCTCAGTTCCTTGGTCTCC-B' [SEQ lD NO:9]; restriction site is underlined; Kozak sequence is in bold type). Furthermore, the construction of the chimeric expression vectors entails introducing a 5' fragment of the human gammai constant region, up to a natural Apal restriction site, uous with the 3' end of the J region of 8-84 and, for the light chain, adding a splice donor site and Hindlli site. The splice donor sequence is important for the t in-frame attachment of the variable region to its appropriate constant region, thus splicing out the V:C intron. The kappa intrcn + CK are encoded in the expression construct downstream of the 8-84 VK sequence. Similarly, the gamma—4 CH is encoded in the expression construct downstream of the 8-34 VH sequence.

The 8-84 VH and VK genes were first carefully analyzed to identify any unwanted splice donor sites, splice acceptor sites, Kozak sequences and for the presence of any extra sub-cioning restriction sites which would later interfere with the subcloning and/or expression of functional whole antibody. An unwanted Hindlll site was found in the VK ~120— W0 20131083817 ce which necessarily was removed by site-directed mutagenesis via PCR without changing the amino acid sequence. For this reactions, oligonucleotide primers BT03 ACAGTATAGTAAGCTCCCTCGGACG'ITCGGTGG-B') [SEQ lD NO:10] and BT04 (5’-CCACCGAACGTCCGAGGGAGCTTACTATACTGTTG—S') [SEQ ID NO:1 1] were used and mutagenesis was med according to the Stratagene (La Jolla, CA) Quickchange Mutagenesis Kit protocol.

Kappa chain chimerization s The non-ambiguous B—B4 VK leader sequence, independent of the PCR primer sequence, was aligned with murine leader sequences in the Kabat database. The t match for the 3-84 VH leader was VK—1 0 ARS-A (Sanz at al., PNAS, 84 (1987'), 1085). This leader sequence is predicted to be out correctly by the SignalP algorithm (Nielsen et 231., Protein Eng, 10 (1997); 1). Primers CBB4Kfor (see above) and 92258 (5'-CGCGGGATCCACTCACGTTTGATTTCCAGCTTGGTGCCTCC-S‘ [SEQ ID NO:12]; Restriction site is underlined) were designed to generate a PCR product containing this complete leader, the 8-84 VK region, and Hindlll and BamHl terminal restriction sites, for cloning into the pKN100 expression vector. The forward primer, CBB4K introduces a Hindill ction site, a Kozak translation initiation site and the VK~10 ARS-A leader sequence. The reverse primer g2258 introduces a splice donor site and a BamHl restriction site. The resulting fragment was cloned into the Hindlll/Baml-ll restriction sites oprN100.

Heavy chain chimerization s The non—ambiguous B-B4 VH leader sequence, independent of the PCR primer ce, was aligned with murine leader sequences in the Kabat database. The nearest match for the 8-84 VK leader was VH17-1A (Sun et al., PNAS, 84 (1987), 214).

This leader sequence is predicted to be out correctly by the SignalP algorithm. Primers cBB4Hfor (see above) and 5322949 (5‘-CGATGGGCCCTTGGTGGAGGCTGAGGA— GACGGTGACTGAGGTTCC-S’ [SEQ lD NO:13]; Restriction site is underlined) were designed to generate a PCR product ning VH1 7-1A leader, the B~B4 VH region, and terminal Hindill and Apal restriction sites, for g into the pG4D200 expression vector. The forward primer cBBHFor introduces a Hindlll restriction site, a Kozak translation initiation site and the VH17-1A leader sequence. The reverse primer 922949 uces the 5' end of the gamma4 C region and a l Apal restriction site. The —121- WO 83817 2012/074867 resulting fragment was cloned into the Hindlll/Apal restriction sites of pG4DZOO, resulting in vector pG4D200cBB4.

Production of 0884 antibody One vial of COS 7 cells was thawed and grown in DMEM supplemented with % Fetal clone I serum with antibiotics. One week later, cells (0.7 ml at 107 cells/mi) were electroporated with pG4D200cBB4 plus pKN100cBB4 (10 119 DNA each) or no DNA. The cells were piated in 8 ml growth medium for 4 days. Electroporation was repeated seven times.

Detection of ic antibody A sandwich ELISA was used to measure antibody concentrations in COS 7 supernatants. Transientiy transformed COS 7 cells secreted about 6956 ng/ml antibody (data not shown).

Binding activity of 08-84 To assay the binding activity of cB-B4 in COS 7 e supernatants, the Diaclone sCD138 kit has been used, a solid phase sandwich ELlSA. A monoclonal antibody specific for sCD138 has been coated onto the wells of the microtiter strips provided. Dun‘ng the first incubation, sCD138 and biotinylated B-B4 (bio-BB4) antibody are simultaneously incubated together with a dilution series of unlabeled test antibody (8-84 or cB—B4).

The concentrations of bio-B—B4 in this assay have been reduced in order to obtain competition with low concentrations of unlabeled antibody (concentration of cB—B4 in COS 7 cell e supematante were ise too iow to obtain sufficient competition). Results from this assay reveal that both antibodies have the same specificity for CD138 (data not shown).

Purification of 03—84 Chimeric B-B4 was purified from COS 7 cell supematants using the Protein A lmmunoPure Plus kit (Pierce), ing to the manufacturer’s reoom mendation (data not shown).

Ku~determinationz ison nBTOGZI BB4 -122~ Purification of e CD138 e CD138 antigen from U-266 cell e supernatant was purified by FPLC using a 1 ml "HiTrap NHS-activated HP " column coupled with 8-84. Cell culture supernatant was loaded in PBS-Buffer pH 7.4 onto the column and later on CD138 antigen was eluted with 50 mM tri-ethylamine pH 11 in 2 ml fractions. Eluted CD138 was immediately lised with 375 pL 1 M Tris-HCl, pH 3 to prevent structural and/or functional damages.

Biotinyiation of CD 138 Sulfo-NHS-LC (Pierce) was used to label CD138. NHS-activated s react efficiently with primary amino groups like lysine residues in pH 7-9 buffers to form stable amide bonds.

For biotinylation of CD138, 50 ul of CD138 were desalted using protein desalting spin columns (Pierce). The biotinylation reagent (EZ-Link Sulfo NHS-LC-Biotin, Pierce) was dissolved in ice-cooled deionised H20 to a final concentration of 0.5 mgiml. Biotinylation reagent and capture reagent solution were mixed having a 12 times molar excess of biotinylation reagent compared to capture t (50 pmol CD138 to 600 pmol biotinylation t) and incubated ‘l h at room temperature while shaking the vial gently. The unbound biotinylation reagent was removed using protein desalting columns.

Immobilization of DC!) 1'38 The sensorchip (SENSOR CHlP SA, BlACORE AB) used in the E assay is designed to bind ylated molecules for ction analysis in BlACORE systems.

The surface consists of a carboxymethylated dextran matrix pre-immobilized with streptavidin and ready for high~affinity capture of biotinylated ligands. immobilization of b00138 was performed on SENSOR CHlP SA using a flow rate of 10 ul/min by manual injection. The chip surface was conditioned with three consecutive 1-minute injections of 1 M NaCi in 50 mM NaOH. Then biotinylated CD138 was injected for 1 minute.

KD-Determination of different antibodies using BIACORE ~123— The software of BIACORE C uses ore-defined masks. so called "Wizards" for different experiments where only certain settings can be d. As the BIACORE C was originaiiy developed to measure concentrations, there is no wizard designed to carry out affinity measurements. However, with the adequate gs, the wizard for "non- specific binding" could be used to measure affinity rate constants and was therefore used for KD-determination. With this wizard, two flow cells were measured and the dissociation phase was set to 90 s by performing the "Regeneration 1" with BIACORE running buffer. "Regeneration 2" which is equivalent to the real regeneration was performed with 10 mM Glycine-HG! pH 2.5. After this step, the ligand CD138 was in its binding competent state again. During the whole procedure HBS-EP was used as running and on butter. To determine binding of the ent antibodies (~150 kDa) to CD138. association and dissociation was analysed at different trations (100, 50, 25 12.5, 6.25 and 3.13 nM). The dissociation brium constants were determined by calculating the rate constants ka and kd. Afterwards, the KD-vaiues of the analytes were calculated by the quotient of kd and ka with the BIAevaluation software. The results are shown in Table 13. mean KD nM 1.4 1.4 +1. 0.06 1.7 1.6 +/- 0.06 f 1.9 neTosz-SPoB-om 1.9 1.9 +1- 0.00 . 1.9 ’T‘" 2.6 B—BILSPP-DM1 2.7 2.6 +/- 0.06 Tabie 13 Comparative analysis of KD values of nBT062 and 8-34. Standard deviations are given for mean KD values.

Discussion Mean KD vaiues for each antibody were calculated from three independent experiments. The results show that in all measurements nBT062 exhibits ly decreased K0 values ed to 8-84 (mean K0 values were 1.4 and 1.6 nM, respectively).

Preparation of Immunoconjugates nBT062-DM1 and hu0242-DM1 The thiol~containing maytansinoid DM1 was synthesized from the microbial fermentation product ansamitocin P~3, as previously described by Chari (Chari et al., Cancer Res. 1 (1992), 127). Preparation of humanized 0242 (hu0242) (Roguska et al., PNAS, 91 (1994), 969) has been previously described. Antibody-drug conjugates were prepared as previously described (Liu at al., PNAS, 93 (1996), 8618). An average of 3.5 DM1 molecules was linked per antibody molecule. nBT062-DM4 BT062 is an antibody—drug conjugate composed of the cytotoxic sinoid drug, DM4, linked via de bonds h a linker to the nBT062 chimerized monoclonal antibody. Maytansinoids are anti~mitotics that t tubulin polymerization and microtubule assembly (Remillard et al., e 189 (1977), 1002). Chemical and schematic representations of BT062 (nBT062-DM4) are shown in FIGS. 1 and .2.

FACS analysis and WST xicity assays FACS anaiysis OPM-2 cells are plasma cell leukaemia cell lines showing highly expressing CD138. OPM~2 cells were incubated with nBTOGZ, nBTOBZ-SPDB-DM4, nBT062-SPP- DM1 or nBTOBZ-SMCC—DM1 at different concentrations (indicated in . The cells were washed and CD138—bound antibody or conjugates were detected using a fluorescence—labelled secondary antibody in FACS analysis. The mean cence measured in these experiments was plotted t the antibody concentration.

Cell ity assay CD138+ MOLP—S cells were seeded in flat bottom plates at 3000 cells/well.

CD138 BJAB control cells were seeded at 1000 cells/well. The cells were treated with nBT082-SPDB-DM4, nBTOGZ-SPP-DM1 or -SMCC-DW at different concentrations (indicated in for five days. WST reagent (water-soluble tetrazolium salt, ROCHE) was added in order to measure cell viability according to the manufacturer's instruction ). The reagent was incubated for 7.5 h on MOLP-8 cells and for 2 h on BJAB cells. The fraction of surviving cells was calculated based on the optical densities measured in a microplate reader using standard procedures. 2012/074867 Discussion Binding of nBTOBZ-SPDB-DM4, nBTOBZ-SPP-DM‘l, nBTOBZ-SMCC—DM‘l or nBT062 was analyzed by FACS. CD138+ OPM-Z as target cells were incubated with nBTOBZ or immunoconjugates and cell-bound molecules were detected using a fluorescence~labeled secondary antibody. in the mean fluorescences as measure for the amount of cell bound antibody is plotted against different antibody or conjugate concentrations. The results show, that nBTOBZ-SPDB-DNM, -SF’P- DM1 and nBTOBZ—SMCC-DM’i show very similar binding characteristics. in addition, the results strongly suggest that the binding teristics of the ugated antibody is not affected by the conjugated toxins.

In cell viabiiity assays, the cytotoxic activity of the antibody t CD138+ MOLP-8 target cells and against CD138" BJAB hoblastoma control cells were analyzed. Both cell lines were seeded in flat-bottom plates and incubated with increasing concentrations of the immunoconjugates. Unconjugated antibody was used as a control. The xic activity was analyzed five days after addition of the immunoconjugates by using WST reagent in order to measure cell viability. in (A)-(C). the fraction of surviving cells relative to control cells treated with vehicle control is plotted against increasing immunoconjugate concentrations. The s show that cytotoxic activity of nBTOGZ—SPDB-DM4, nBTOGZ—SPP—DMi and nBTOGZ-SMCC—DMi against MOLP—S cells is very similar. As expected, CD138’ BJAB l cells were not killed by the conjugates, indicating that all immunoconjugates act via cell specific binding to 00138. in competition experiments, in which MOLP-B cells were ubated with a molar excess of unconjugated nBT062. Preincubation substantially blocked the oytotoxicity of nBTDSZ—SPDB—DlWi, providing further evidence that the immunoconjugates kill the cells via specific g to CD138 onto the cell surface (HQ. 7 (D)).

Indicator: Pancreas] Mammary and other Carcinoma -- Xenograft Models General Experimental Set-up In accordance with the CD138 expression analysis (lmmunohistochemistry analysis on tumor tissue microarrays) tumor candidates were selected from a primary tumor collection. that is, from patient derived tumors. These tumors display similar characteristics as the patient tumors, since they are passaged in mice at low numbers, -126~ WO 83817 to retain original characteristics. Following subcutaneous transplantation and establishment of tumors (induction time 30 days), the immunoconiugate BT062 was injected intravenously at 2 different concentrations of the maytansinoid 0M4, 450 pg/kg and 250 ug/kg (each based on the molecular weight of the linked DM4 (1 mg of DM4 is conjugated to 52 mg of antibody, equalling a total mass of 53 mg; 450 pg/kg DM4 = 23.850 pg) The immunoconjugate was administered once weekly for 10 weeks (in case of treatment of pancreatic tumor implanted mice) and 5 weeks (in case of mammary, lung and bladder tumor ted mice). A treatment free observation period followed to investigate a possible tumor regrowth.

Example 1: Pancreas Carcinoma Pancreatic tumor tissue (PAXF 736 (Kuesters et al., 2006) was implanted (bilateral) into NMRI mice. The implanted tumor originated from a patient’s y atic carcinoma (poorly differentiated, infiltrating adenocarcinoma (an exocrine carcinoma».

No side s were observed. The tumor of this patient was identified as a high CD138 expressing tissue by immunohistochemistry studies. However, CD138 is not expressed to a degree comparable to myelomatous plasma cells in multiple myeloma patients. as ed on tumorgenic cell lines by flow cytometric surface ng.

Treatment with BT062 was ted after tumors have reached a size of approx. 6—8 mm diameter (minimum 5 mm). Tumor diameters have been measured two times a week.

Tumor volumes were ated according to the formula aXbXb/Z where "a" is the longest axis and "b" the perpendicular axis thereto. lnhibition of tumor volume in the test groups relative to the vehicle l group was calculated as the ratio of the median ve tumor volumes (TIC).

Tumor inhibition for a particular day (TIC in %) was calculated from the ratio of the median RTV (relative tumor volume) values of test versus control groups multiplied by 100%.

Median relative tumor volume of the test group DayX TIC (Dayx) = --------—---—--—--—-------—--------~»--—~«—---~——-----—--- x 100% Median relative tumor volume of the control group Dayx Tumor volume could be significantly reduced by this weekly administration of BT062. As can be seen in Fig. 8. dose dependent partial and complete remission was observed.

The Figure shows that at a dose of 23.85 mg/kg, complete remission could be obtained 28 days after tumor implantation, while at a dose of 13.25 mg/kg, complete remission could be obtained 35 days after tumor implantation. Notably. after 52 days all mice in the 13.25 mg/kg administration ns were still alive (8/8), while the eight mice of the control group had been reduced to 1. A TIC value below 10% indicates complete remission (CR) (Bissery et al., 1991). According to this criteria, CR was achieved in both treatment groups, reflecting the complete remission that was achieved by BT062.

Remarkably in a treatment free observation phase, no tumor regrowth was detected, confirming the complete curance of in this model.

Relative Tumor BT062—DM4; 13.25 mg/kg BT062-DM4; Table 14: Tumor volumeiS atic cancer xenograft mouse model Example 2: Mammary Carcinoma NMRl (nude) mice were implanted (bilateral) with primary mammary tumor of a patient (determined via lHC analysis as strongly CD138 positive). A breast carcinoma skin metastasis was taken at stage M1. it was a tumor which did not respond to Herceptin (low Herz with an intermediate expression). The tumor was estrogen receptor ve and progesterone or ve and thus not responsive towards hormone y.

Tumors to be implanted were selected according to [H0 staining results (strong, homogenous expression of CD138 detected by BT062 (triple negative expression of hormone receptors estrogen and progesterone); Herz expression scored 2 or less (regarded as Herceptin non responsive).

Treatment with BT062 was initiated after tumors had reached a size of approx. 100 mm3. Tumor s were calculated according to the formula aXbXb/Z, with "a" being the t axis and "b" the perpendicular axis thereto. inhibition of tumor volume in the test groups relative to the e control group was calculated as the ratio of the median relative tumor volumes . BT062 was administered once weekly at a loading dose of 13.25 mg/kg (which was given on day 1) followed by doses of 4 mg/kg once weekly. in the other dose group a high dose of 23.85 mg/kg was administered.

Tumor volume could be significantly reduced by weekly administration of BT062. A dose dependent partial and te remission was observed. The conjugate was well tolerated, having no influence on body weight after each injection. A T/C value below 10% was obtained in both treatment groups. reflecting a complete remission achieved by the administration of BT062. As can be seen in Figure 9, the anti—tumor effect (i.e., complete remission) was achieved after 21 days, which can be considered a rapid response to BT062. As can been seen from Figures 35 and 36, lower dose regimens were also effective. As can been seen from Fig. 37, a mouse model that did not respond to Docetaxel treatment, also did not respond to BTOGZ treatment, while a model that did not respond to taxol ded well to BT062 treatment (Fig. 35).

Compared to the pancreatic model, duration of treatment could be cut short by half (5 weeks instead of 10 weeks) and the low dose of 13.25 mg/kg was reduced to 4mg/kg to achieve a similar , namely complete remission and no tumor regrowth. The shorter treatment period for mammary carcinoma was not expected, since on IHC analysis the level of CD138 expression was similar. Thus, no conclusions can be drawn from the level of CD138 expression to a general recommendation for the treatment duration. After 21 days all mice of both the treated groups as well as the control group were still alive. in a treatment free observation period (39 days after the last stration of the immunoconjugate) no tumor th was detected, confirming the complete curance.

Relative Tumor Mean Ran e Volume (%) (Day 21) 9 533 (3‘: 149.5) 339 -— 878 l (P83) BT062-DM4; 13.25 mg/kg/4 mg/kg 0 (i 0-02) 0 - 0-1 BT062-DM4; Table 15: Tumor volume is mammary carcinoma xenograft mouse model.

FFPE tissue samples Stainin- score membrane 0.25 - ml 0.05 - ml ~129- Breast, tumor 3 Homo Mets, 051909-13 Unknown, 9—12 Mets, 4161909439 Prime , 44 B'eaS"t"’"°’ Prime , 4119044 Normal Skin sam-le‘i 3 Home Normal Skin samnie 1 3 Homo Table 16: Expression of CD138 on mammary carcinoma cells vs. epithelium ceils Example 3: Bladder Carcinoma NMRl (nude) mice are implanted with a bladder tumor (determined via iHC analysis as CD138 strong positive), namely a transitional cell carcinoma.

Treatment with BT062 is initiated after tumors had reached a size of approx. 100 mm3.

Tumor volumes are calculated according to the formula aXbXbIZ, with "a" being the longest axis and "b" the perpendicular axis thereto. inhibition of tumor volume in a test groups relative to the vehicle control group is ated as the ratio of the median relative tumor volumes (TIC).

Tumor volume is sought to be significantly d by weekly administration of BT062.

Any dose dependent partial and te remission is tracked.

Example 4: Lung Carcinoma NMRI (nude) mice are implanted with a Lung carcinoma (determined via IHC analysis as 00138 strong positive).

Treatment with BTDSZ is initiated after tumors had reached a size of more than 5 mm.

Tumor diameters are measured two times a week. Tumor volumes are calculated according to the formula a*b*b12, with "a" being the iongest axis and "b" the perpendicular axis thereto. inhibition of tumor volume in a test groups relative to the e control group is calculated as the ratio of the median relative tumor volumes (TIC). te ion could be achieved in both dose groups (4 mg/kg and 23.85 mg/kg once per week). in a treatment free period, no re-growth was observed, confirming the te eradication of the tumors. ~130- Example 5: To investigate metastatic tumors, NMRI (nude) mice were implanted with a metastatic patient s derived from a bladder tumor (determined via lHC analysis as CD138 strong ve).

Complete remission could be also achieved in this model, in both dose groups (4 mg/kg and 23.85 mglkg once per week). in a treatment free period, no re-growth was observed. confirming the complete eradication of the tumors.

Example 6: To investigate efficacy of BT062 at lower doses and in comparison with a clinically used drugs taxol (Paclitaxel), NMRI (nude) mice were implanted with the mammary tumor of example 2. Lower doses of BT062 (0.5 mg/kg, 1 mg/kg, 2 mg/kg and 4 mg/kg) were administered once weekly (Fig. 35). At 4 and 2 mg/kg once weekly, a te remission was observed, without re-growth in a treatment free period. Taxol treated mice showed only a minor tumor growth delay at 10 mg/kg. DM4 was used in an amount corresponding to that in 4 mg BT062, but did not result in tumor response. At concentrations 1 mg/kg a "tumor stasis" could be achieved. i.e. the tumor neither grow but the volume did not se. This is also called the minimal effective dose, since in this group 2/7 mice had a partial ion and 3/7 mice had complete ion without tumor regrowth.

The minimal effective dose can also be somewhat lower than 1 mg/kg but higher than 0.5 mg/kg.

Examples 7 and 8: Here BT062 was investigated at lower doses and compared with the clinically used drug xel (10 mg/kg), as in Example 6. Lower doses of BT062 (1 mg/kg, 2 mg/kg, 4 mg/kg and 8 mg/kg) were administered once weekly. At 8 mg/kg once weekly, a complete remission was observed during treatment in mice that had tumors g a 2-3 scored CD138 lHC staining and that also response to docetaxel, while mice that had tumors showing a 1-2 scored lHC staining and did not respond to docetaxel, also did not respond to BT062 (Figs. 36 and 37).

Example 9: -13‘i— To igate efficacy of BTOSZ at lower doses and in comparison with a ally used used drug docetaxel (10 mg/kg) NMRl (nude) mice were implanted with a primary pancreatic tumor. The patient derived tumor had a high but heterogeneous CD138 staining determined by IHC is and scored with 3. Lower doses of BTOSZ (1 mglkg, 2 mgikg, 4 mglkg and 8 mg/kg) were administered once weekly (Fig. 38). At 4 and 8 mg/kg once , a complete remission was observed, but re-growth in a treatment free period occurred which could be an effect of the heterogeneity of the tumor. Docetaxel treated mice showed complete remission during the treatment period as well as the treatment free period.

Human Trials with BT062 ln the context of the present invention, human subjects responded well to a low dose regime. This was even the case in absence of any additional treatments that would compensate for potential variations in qualitative or quantitative expression of the 001 38 on the target cells (compare RG). While mouse models demonstrated that BT062 has highly cant antimyeloma activity at doses that are well tolerated in mice. effectiveness was considerably better at vely high doses (results not shown). posing the question how higher doses would be tolerated by human subjects that express CD138 on a wide variety of non-tumor celis.

Phase I research study This study was performed to test the effects (good and bad) and to determine the MTD (maximum tolerated dose) of BT062 in treating ts with relapsed or relapsed refractory multiple myeloma.

Up to now. 32 ts were recruited. At least 12 out of 32 patients experienced diminished disease progression as represented by receiving at least a forth treatment cycle. The trial is being performed at different sites, with groups of 3 and 4 patients being treated with different dose levels (10 mg/m2, 20 mgfmz, 40 mg/m2, 80 mgfmz. 120 mg/mz, 160 mg/mz, 200 mg/mz) for anywhere between 1 to 31 ent cycles (results not shown). As the person skilled in the art will appreciate a higher number of treatment cycles is possible and within the scope of the present inventions, such as 10 to 50, 10 to 100, 10 to 200 and more.

Disease progression diminished with relatively low dosage levels, namely 20 mg/m2, 40 mg/m2, 80 mg/m2 and 120 mg/mz, with one patient at the 2"d dosage level of 20 mg/m2 ~132— displaying no disease progression for 10 treatment cycles of 21 days. in some ts stable disease and responses, including minor and partial responses could be observed.

At these dose levels, as described above (see Tables 9 and 10), rapid clearance of BT062 from plasma was also observed. Some pharmacokinetic profiles of these low dose administration schemes are shown in Fig. 13.

Doses of 160 mg/m2 and 200 mg/m2 were also administered. A dose of 160 mg/m2 was identified as MTD and studies in this group were expanded. A dose of 200 mg/m2 was fied as MAD.

Repeated single doses regimens of 10 mg/m2. 20 mg/m2, 40 mg/m2, 80 mg/m2, 120 mg/mz. 160 mg/mz. 200 mg/m2 were performed every 21 day, meaning on day 1, day 22, day 43. day 64. day 85, day 106, and so forth. The disease has been and will be monitored by physician's assessment of hematology, clinical symptoms and al chemistry as well as by measuring M-Protein leveis in the serum and urine of patients (in gidL) and free light-chain (FLC) levels in the serum of patients over time (results not shown). lmmunogiobulin Assessment The amount of lg antibodies including the determination of lgG ups was ed at screening.

M-protein Quantification and Serum Free Light Chain Assay Initially, the response to treatment was evaluated at day 1 of ent cycles 1-3 by M- protein quantification using immunoeiectrophoresis (iEP) and immunofixation electrophoresis (lFE) from serum and 24—hour urine coiiection. For treatment cycles 3 and beyond, ein quantification was performed at the Day 15 visit in order for the results to be available to assess response prior to initiating the next treatment cycle. A general tative immunoglobuiin assessment was done together with M—protein quantification.

Serum samples were used to perform FLC assays to examine multiple myeloma subjects with no detectabie M—protein (nonsecretory/oligoseoretory myeloma) and to allow for detection of early response to treatment. Therefore serum FLC assays were med on day 1, 2, 3, and 8 of treatment cycle 1, on day 2, 3. 8 and 15 of cycle 4, —133- 2012/074867 as well as on day 1. 8 and 15 of all other treatment cycles. M-protein and FLC were be analyzed at the screening and at the close-out visit. Evaluations at day i of cycie 1 served as baseline values.

Dose UrineISerum till-protein measurements and FLC measurements 2O > During first seven cycles disease stabilization based on clinical symptoms and serum FLC, > Urine M—Protein decreased after 8"1 treatment > ein criteria for Minor Response reached after 8th treatment > Decrease in Urine M~Protein level from baseline by more than 50% > es progression after Cycle 10 > Serum M-Protein between 0,06 and 0,1 g/dL (defined as not measurable 40 > Stable disease for 14 weeks > Serum M—Prctein decreased after 1Si treatment and stabilized for 14 weeks ‘> Diseases progression ed after treatment was held at the start of cycle 6 (day 105) > Urine M-Protein increased from 0 at screening to a maximum of about 16 mo/24h defined as not measurable 160 Serum FLC level increased during the screening period ng -21 days before day 1 of the treatment > Serum FLC level decreased very soon after 1St treatment and was already close to 25% decrease at day 8 > in comparison to baseline, FLC levels are reduced by about 40% during 15‘ cycle and by more than 50% after 2"", 3rd and 4th treatment VVV FLC criteria for Partial Response were reached very early Disease progression after the end of the 4‘" treatment cycle Serum M-F’rotein not measurable = 0; Urine M-Protein decreased from 140 mg/24h at baseline to 120 mg/24h before 2"d treatment d as not measurable) => non— secrete M eloma Table 17 provides observations made regarding Serum M-protein and serum FLC measurements in selected patients in a repeated single dose regime. in a repeated single dose regimen of BT062, DLTs were observed in the mucosa of patients treated in the 200 mg/m2 dose group. The target for BT062 (CD138) is expressed in the mucosa and toxicities in these tissues and organs con be considered as target related. s e events, not qualifying for DLT, were observed in eye patients. However, the eye toxicity is suggested to be rather related to the effector compound since this is a typical toxicity also found with other DM4 ates such as SAR3419, or lMGN388 that do not target CD138. This eye ty occurred in one —134- patient in the repeated single dose study 3 days after the 3rd cycle and in the other patient 4 days after the 4th cycle. in the maximal administered dose group (160 mg/kg) of the repeated single dose study, CD138 related toxicities occurred during the first days but also after repeated cycles, most of them were considered mild to moderate.

UrineISerum hit-protein ements and FLC measurements 3 x 50 mgImZ 20 Serum tilt-Protein decrease for 6 cycles: At least stable disease could be achieved over 6 cycles. with a decrease of serum M n by nearly 25% during/after 3rd and 5th treatment cycle 3 x 65 mgImZ 21 Free lambda-kappa light chain A strong decrease of the serum FLC ievei could be observed 3 x 120 mgim2 24 I afterjust a single treatment cycleUrine hit-Protein decrease A decease of urine M protein after first and repeated , with a reduction of more than 50% ed after 3rd, 7th and 10th cycle Tabie 18 provides observations made regarding Urine/Serum M-protein and serum FLC measurements in selected patients in a repeated muitipie dose regimen.

Determination of BTOGZ and DM4 from plasma To assess single dose PK properties of BT062, after N administration of BT062, extensive plasma sampling was med during the first treatment cycle. The same evaluation was performed during ent cycle 4. To a iesser extent plasma samples were also obtained at day 1 and 8 of ail other treatment cycles, as well as on close-out and follow-up visit. The amount of BT062 in the plasma is determined via a PK ELISA method described as s: Experimental description: The wells of a microtiter plate are first coated with anti-maytansinoid (anti-DM4) antibody overnight at 2-8 "‘0 and after blocking with assay buffer (0.5% S) incubated on the next day with plasma samples. These are diluted beforehand at least 1:100 in assay buffer. BTOBZ antibodies contained in the samples are bound by the -135— anti-0M4 antibody immobilized in the plates. After tion. unbound material is removed by washing. Then, a HRP-coniugated secondary antibody is added, which binds to the BTOBZ antibodies. Unbound secondary antibody is removed by r washing step. After this. TMB substrate solution is pipetted into all the wells.

A color reaction develops tional to the amount of BT062 bound during incubation of the . The color reaction is ended using a stop solution, which causes the color to change from blue to . The final measurement is carried out with a photometer at a wavelength of 450 nm.

The relationship between concentration and optical density is evaluated using an V6.6 software. if samples from clinical trials are measured (plasma samples from multiple a patients), for each patient an individual standard curve should be prepared in 1:100 diluted "predose" plasma (plasma before treatment with BT062). if in addition to the obligatory 1:100 dilution in assay buffer, a clinical test sample has to be diluted further (due to a high BT062 tration), this dilution should be prepared in 1:100 diluted predose plasma (of the patient ned). For stability tests (e.g. freezing/thawing stability, e stability), the BTOBZ standard and also corresponding samples or in—process controls are prepared in 1:100 diluted heparin plasma pool.

Determination of Shed CD138 and HAPA All pre-dose plasma samples were evaluated for levels of shed/soluble CD138 ($00138) to igate a potential correlation between levels of sCD‘l38 and antitumor activity. These measurements also allowed to determine that the lower than expected Cmax values are not dependent on the amount ofsCD138 present prior to administration of BT062 (see Figure 17). Predose plasma samples from day 1 of each treatment cycle and from close out and follow-up visit were evaluated for the presence of humoral responses against BT062 (drug product) by assessment of human antiproduct antibodies (HAPA).

Shed CD138 ements observed In Myeloma patients high levels of sCD138 can be observed and might be an indicator of prognosis of myeloma patients (Maisnar et al., 2005). -136— Patients with MGUS and MM might display high levels of soluble CD138 concomitant with higher levels of BZ-microglobulin and elevated plasma cell content in the bone marrow (Aref et at, 2003).

A kit was used for determining soluble CD138. Surprising, it was found that one patient (identified as 003-003) treated at 20 mg/m2 of BT062 displayed a minor response with regard to urine M-protein levels, although this t displayed high levels of sCD138 before treatment.

Soluble (s) CD138 values were ined in different subjects. sCD138 (ng/ml) 002-003 001-002 002-004 003-003 Table 19: Patient 003-003 (repeated single dose 20 mg/m )displayed very high values of sCD138. Nonetheless, this patient achieved a minor response in M-Protein level.

COMBINATION STUDIES in a Phase lllla multi-dose escalation study, BT062 was combined with Lenalidomide and Dexamethasone in ts with relapsed or relapsed/refractory le Myeloma.

One treatment cycle ted of 28 days, or in other words, 21 days of active treatment followed by 7 days without ent (resting period). BT062 was administered on days 1, 8 and 15 at a concentration of 80 mg/m2, lenalidomide (Len) (25 mg) was administered once daily on days 1 — 21 and dexamethasone (Dex) (40 mg) was administered on days 1, 8, 15 and 22. Day 1 treatment of BT062 at all cycles should concurred with day 1 of Len and dexamethasone. As can be seen from Fig. 34, a minor response was observed after the first treatment cycle and was ined at the start of the 4‘" cycle (day 99), even though start of cycle 2 and 3 was d by one week and treatment with BT062 was skipped on day 85 and treatment with Len was skipped on days 85 to 91 and the Dex dose was reduced to 20 mg/m2 during cycle WO 83817 3. As is clear to the person d in the art. either Lenalidomide, dexamethasone or BT062 concentrations may be d depending on toxicities and efficacy. Efficacy is assessed body fluids, preferably via efficacy blood parameters such as M-Protein or FLC ding on the MM disease type), or other markers from body fluids or bone marrow reflecting disease status.

With this treatment regime disclosed here, combination with Len/Dex is possible with lower toxicities, or in combination with this immunoconjugate, administration of the combination partners can be ed eg. lowered to minimize the toxicities associated with their administration. Since this regimen provides better tolerability it is applicable for combination with other drugs, having lower or at least not higher numbers of toxicities but the same or even better efficacy.

Possible anti-myeioma drug ates have been evaluated as combination partners for BT062 in cell lines: Cell Line Studies Combination studies in xenograph mouse models were preceded by studies in cell lines. The synergy determination in different cell lines was performed according to Chou and Tallay (1984), using the median effect analysis. Here. [050 values for the cytotoxic s for each drug and each cell line are calculated, and then lC50 ratios for each drug pair. The cells were then d to on series of either these drug mixtures, or the drugs alone. Experimental data were analyzed using the yn software (ComboSyn, lnc., Paramus, NJ). Combination indexes (Cl) for each independent experiment were calculated and reported separately. in the analysis, Cl less than 1, equal to 1 and more than 1 indicates synergy, additivity and antagonism, respectively. According to the classification of TC. Chou (CompuSyn, User’s guide, 2004), the author of the method, the scale of synergism and antagonism is as follows: Combination index Description < 0.1 Very strong synergism 0.1-0.3 Strong synergism 0.3-0.7 Synergism 0.7-0.85 Moderate synergism 9 Slight synergism ~138- 0.9-1.1 Nearly additive 1.1-12 Slight antagonism 1.2-1.45 Moderate antagonism 1.45—3.3 Antagonism 33-10 Strong antagonism > 10 Very strong antagonism RPMI 8226 Bortezomib Addifiv’e Slightly antagonistic Antagonistic s Thalidomide Additive to Additive to slightly nistic synergistic antagonistic_ _ Lenalidomide Slightly to Additive to Synergistic moderately synergistic antagonistic lan Slightly to Additive to Additive to slightly moderately synergistic synergistic antagonistic Dexamethasone Not determined Table 20: Estimates of synergistic results obtained in ceil lines according to the method of Chou and Talalay (1984).

In this example MOLP 8 cell lines were used for combination of BT062 with bortezomib, thalidomide. domide, melphalan and dexamethasone.

Combination with thalidomide or bortezomib, did neither result in a synergistic nor an additive effect, but rather an antagonistic effect. in contrast to these cell culture studies ation with omib was synergistic in the xenograft model described below.

Possible anti-myeloma drug candidates have been ted as combination partners for BT062 in aft studies using MOLPS human multiple myeloma cells. ~139— Example 1 Anti-Myeloma effect of combination y with BT062 and Lenalidomide Female SClD mice were subcutaneously inoculated with MOLP 8 human myeloma cells. Treatment with BT062 alone or in combination with Lenalidomide was initiated day 11 post tumor inoculation. BT062 was used in concentrations of 100 pg, 200 [.19 and 400 pg alone and in combination with Lenalidomide which was dosed intraperitoneally at 100 mg/kg on days 1 to 5 and days 8 to 12. A control group of animals ed phosphate ed saline (PBS) using the same schedule and route of administration. Tumor growth was monitored by measuring tumor size and calculated with the formula length x width x height x 1/2, ined on days 10, 14, 18 and 21.

Synergism was calculated as follows (Yu et al., 2001; Gunaratnam et al., 2009): RATlO (r) = expected FTV (combination)/observed FTV (combination) FTV: Fractional tumor volume = mean tumor volume (test)/mean tumor volume (control) A ratio > 1 is regarded as synergistic, whereas r < 1 is less than additive.

The ratio (r) is, when above 1, ed to herein as "SYNERGY RATIO." As can be seen from Table 21 synergism was observed after 28 days in concentrations of BT062 of 2001.19 and 4001.19: 440— BT062 100 + Len BT062 100 + ratio BTOGZ 100 Lenalidomide (observed Len expected exp/obs —_—_._,—m _fl_082 -n—I-'_Z_I—’ m 0,33 0,46 0,17 0,90 37062 200 Lenalidomide (observed) e-xpectedLen (expiobs) m-—021 1.02 0,73 17 0.13 0.45_-im 041 21 0,42 007— 0,45 24 0,11 0,55 0,06"3 1,08 28—--£E 1.86 -_—_BTGGZ400+Len —ratioBTOSZ400 ‘ Lenalidomide (observed) Len expected (explains , , 0,95 1,04 , 14 0,36 1.49 17 .112! o04 0,63 0,44 24__E3_ 0.02 0.80 28 1.43 Tahie 21: Fractional tumor volume in MOLP 8 xenografts.

Different concentrations of BT062 either alone or in combination with Lenalidomide have been administered into tumor bearing xenograft. FTV represents the relative tumor volume.

Synergistic effects are determined using Ratio values expected FTV versus observed FTV. A ratio >1 indicates synergy.

Dosage per TIC (%) Tumor tree Agent injection Total dose (DAY 17) Complete ors day 77 h(o2 m‘) ----—- BT062 100 ug/kg 100 ug/kg-—-—- From 200 ug/kg 200 --—- mom 400 ug/kg 400 ug/kg---— highly awe om'de 100 mg/kg 19mg ---"- _—-————— ------— ___III--12 2/6 0/6 0/6 Active __I..Il--5/6 4/6 0/6 highly active Table 22: domide BT062 combination: s at different dosages.

Figures 30 and 31 show the effect of the combination therapy on median tumor volume (TV) in a xenograft mouse model. The result in Figure 30 show additive effects of the combination. Notably the combination ed in a dose of 100 pg/kg of the immunoconjugate, when combined with a dose of 100 mg/kg lenalidomide. For synergy ratios, please refer to the table above.

Example 2 Anti-Myeloma effect of combination therapy with BT062 and VELCADE VELCADE has been evaluated as potentiai multiple myeloma drug combination r for BT062 in Xenograft studies using MOLPB multiple myeloma cells (IMGN lnc.).

Treatment with BTOGZ alone or in combination with VELCADE was initiated 11 days past tumor implantation. BT062 was used in trations of 100 pg, 200 pig and 400 ug alone and in combination with VELCADE which was dosed at 100 mg/kg on days 1. 4, 8 and 11. A control group of animals ed phosphate buffered saline (PBS) using the same schedule and route of administration. Tumor growth was monitored by measuring tumor size and calculated with the formula length x width x height x 1/2, determined on days 10, ‘14, 17, 21, 24 and 28, respectively.

Synergism was calculated as in Example 1 of the combination studies.

As can be seen from Table 23, synergy is observed in the combination BT062 with VELCADE at day 25 in all BT062 dose regimens. R values reported in the literature are even higher (Yu et al., 2001).

BT062 100 + BT062 100 Velcade Velcade (observed) expected ratio(()exp/obs 442- 0,48 0.95 0,26 0,46 1,75 BT062 200 + Vel (observed) BT062 400 + Vel synergy ratio Days BTOGZ 400 Velcade (observed) expected (exp/obs) 21 0.05 0.80 0.04 Table 23: Combination treatment with VELCADE.

Fractional tumor volume (FTV) ents the mean tumor volume (test) I mean relative tumour volume (control). Ratio of expected FTV nation) vs. observed FTV (observed). Ratio value > 1 indicate synergy, values less than 1 indicate an additive effect. -‘l43- WO 83817 2012/074867 Tumor Dosage Treatment days per (TX start date = (T—C) in log cell survivors injection day 10 post inoc.) T/C(%) days kill day 67 ---ml—- BT062 400 ug/kg Day1 7 31.5 2.8 4/6 2/6 0/6 highly' active Velcade days 1,4, 8. 11 --m 0/6 0/6 UN‘< ..\ Velcade 100m lk da 51.4.8‘11 - BT062 200 ug/kg Day 1 7 23 1/ Veicade 100 mglkg days 1, 4, 8. 11 active BTDGZ 400 ug/kg Day1 6 .5 Table 24: VELCADE BT062 combination: effects at different dosages.

Figure 31 shows the effect of the combination therapy on median tumor volume (TV) in a xenograft mouse model. The result show that in the model used, VELCADE treatment alone had no effect on the tumor volume. The combination with BT062 provided synergistic effects. y the synergism resulted in a dose of 100 ug/kg of the immunoconjugate, when combined with a dose of 100 mg/kg VELCADE. For synergy ratios, please refer to the table above.

Example 3: BT062/Meiphalan RPM! cells have been implanted subcutaneously into nude mice. Mice were randomized when tumor reached a total volume of approx 100 mms. BTOGZ was injected intravenously at 2 different concentrations: 400 ug/kg and 100 ug/kg; each based on the molecular weight of the linked DM4. PBS served as ve control. Per group, 8 mice with one tumor each (unilateral implantation) were used. BTOBZ was closed weekly followed by melphalan once weekly (3mg/kg) one day after BT062 injection intraperitonealiy (results not shown).

Example 4: In vivo drug combination studies BT862/lenaiidomideldexamethasone While in vitro different cell lines showed a concentration dependent CD138 decrease after 24h lenalidomide incubation (Figures 32(A) to (D)), in vivo drug combination studies showed that a combination of 4 mgi‘kg, 20mg/kg lenalidomide and 1.25 mg/kg dexamethasone was highly ive in a L363 MM xenografi model. -i44- In this model, a highly aggressive CD1 38 expressing plasma cell myeloma cell line L363 was subcutaneously implanted into NOD/SCID mice. Treatment started when tumors d a size of approx. 100 mm3. BTOGZ was injected intravenously once weekly on days 1, 8, 15, 22, 29 at trations of 2 mg/kg or 4 mg/kg either alone or in combination with lenalidomide, which was given orally on days 0—4, 7-11, 14-18, 21 - and 28—32 and Dexamethasone, which was given intraperitoneally on days 0, 7, 14, 21 and 28. Tumor size was measured once weekly. 4 mg/kg BT062 alone was active in reducing the tumor growth. Combination of 4 mg/kg BT062 with Len/Dex showed higher activity with regard to tumor growth inhibition leading than the single agents (Len/Dex alone; BT062 alone) (Figure 33).

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Claims (40)

WHAT IS CLAIMED IS

1. Use of an immunoconjugate in the manufacture of a medicament for treating a disease associated with target cells expressing CD138 in a human subject, the immunoconjugate comprising at least one engineered targeting antibody ing CD138 expressing cells, and at least one effector molecule, wherein said engineered targeting antibody is onally attached to said effector molecule to form said immunoconjugate, wherein at least a part of the engineered targeting antibody confers lgG4 isotype properties, n the disease associated with target cells expressing CD138 is a plasmaproliferative disorder or a solid tumor, wherein said treating comprises administering the immunoconjugate in a multiple dose regimen comprising at least two doses, wherein the aggregate dose to be administered within an active ent cycle is an aggregate maximum tolerable dose (AMTD) or a fraction of the AMTD and wherein said AMTD or said fraction exceeds the dose resulting in dose ng toxicity (DLT) when the immunoconjugate is administered as a single dose, including as part of a multiple single dose regimen or exceeds the maximum tolerable dose (MTD) when the immunoconjugate is stered as a single dose, including as part of a multiple single dose regimen within said active treatment cycle.

2. Use according to claim 1, wherein the immunoconjugate is to be administered in an active treatment cycle of 21 days.

3. Use according to claim 2, wherein the immunoconjugate is to be administered at least three times within said 21 days.

4. Use according to any one of the preceding , wherein the immunoconjugate is to be administered in equal doses.

5. Use according to any one of the preceding claims, wherein said multiple dose regimen is to last 3 weeks and is to be followed by a resting period.

6. Use ing to claim 5, wherein a level of said conjugate in a body fluid of said subject, during said resting period is to be at least 0.5 ug/ml, at least 1ug/ml, at least , at least 3 ug/ml, 4 ug/ml, 5 “9/ ml or 6 ug/ml, or wherein more than 80%, more than 90%, more than 95% of the CD138 of isolated target cells are to be occupied by said immunoconjugate within four to twenty—four hours after completion of administration of the immunoconjugate.

7. Use according to any one of the ing claims, wherein said or is a maytansinoid.

8. Use according to claim 7, wherein said maytansinoid is DM4.

9. Use according to any one of the preceding claims wherein the AMTD exceeds the dose of said DLT by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90%, at least 100% or at least 120% and said MTD by at least 30%, at least 40%, at least 50%, at least 60%, at least 80%, at least 90%, at least 100%, at least 120% or at least 140%.

10. Use according to any one of the preceding , wherein the AMTD is at least 240 mg/m2 and the dose resulting in said DLT is 200 mg/mz.

11. Use according to any one of the preceding claims, wherein the AMTD is at least 240 mg/m2 and said MTD is at least 160 mg/m2 or at least 180 mg/mz.

12. Use according to claim 10 or 11, wherein the AMTD is at least 300 mg/mz.

13. Use according to claim 10 or 11, wherein the AMTD is at least 360 mg/mz.

14. Use according to claim 10 or 11, wherein the AMTD is at least 420 mg/mz.

15. Use according to any one of the preceding claims, wherein said active ent cycle is to include said administering being med at least once a week.

16. Use according to claim 15, wherein said active treatment cycle is to include said administering being performed at equal doses for at least three weeks.

17. Use according to claim 16, wherein said active treatment cycle is to be followed by a resting period of at least one week, which together define a treatment cycle of at least 28 days.

18. Use according to claim 1 wherein the immunoconjugate is comprised in a pharmaceutical composition, wherein the active treatment cycle is to be followed by a resting , and wherein the dose of the immunoconjugate to be administered at least once a week is about 20 mg/m2, about 30 mg/m2, about 40 mg/m2, about 50 mg/m2, about 60 mg/m2, 70 mg/m2, about 80 mg/m2, about 90 mg/m2, about 100 mg/mz, about 110 mg/mz, about 120 mg/mz, about 130 mg/mz, about 140 mg/mz, about 150 mg/m2 or about 160 mg/mz, about 170 mg/mz, about 180 mg/mz, about 190 mg/mz, about 200 mg/mz, about 210 mg/mz, about 220 mg/mz, about 230 mg/mz, about 240 mg/mz, about 250 mglmz, about 260 mg/mz, about 270 mg/m2 or about 280 mg/m2 and the pharmaceutical composition is to be administered for at least three weeks alone or in combination with a cytotoxic agent.

19. Use ing to claim 18, wherein the active treatment cycle is to last at least 21 day and the conjugate is to be administered once a week at a dose from about 40 mg/m2 to about 140 mg/mz.

20. Use of an immunoconjugate in the manufacture of a ment for treating a disease associated with target cells expressing CD138 in a human subject, wherein the immunoconjugate comprises at least one engineered targeting antibody ing CD138 expressing cells, and at least one effector molecule, wherein said engineered targeting antibody is functionally attached to said effector molecule to form said immunoconjugate, wherein at least a part of the ered targeting antibody confers lgG4 isotype properties, wherein the engineered ing antibody comprises: (i) a heavy chain having at least 85% sequence identity with SEQ ID NO: 1, wherein the heavy chain comprises a variable region comprising amino acid residues 31 to 35 (CDR1), 51 to 68 (CDR2) and 99 to 111 (CDR3) of SEQ ID NO: 1, and (ii) a light chain having at least 85% sequence identity with SEQ ID NO: 2, wherein the light chain comprises a variable region comprising amino acid residues 24 to 34 (CDR1), 50 to 56 (CDR2) and 89 to 97 (CDR3) of SEQ lD NO: 2; n the effector le is at least one maytansinoid, wherein the disease associated with target cells expressing CD138 is a plasmaproliferative disorder or a solid tumor, wherein said treating comprises administering the medicament in an active treatment cycle of 21 days, at least three times within said 21 days, wherein said active treatment cycle comprises administering at least once a week, n the dose of the immunoconjugate to be administered at least once a week is between 80 mg/m2 and 120 mg/mz.

21. Use according to claim 20, wherein the immunoconjugate is to be administered at equal doses for at least three weeks and the active treatment cycle is to be ed by a resting period of at least one week, which together define a treatment cycle of at least 28 days.

22. Use according to any one of the of the preceding claims, wherein said administration is to be followed, after at least two 21 day treatment cycles, each optionally followed by a g period, by a further administration of said the immunoconjugate or pharmaceutical ition as a maintenance therapy.

23. Use according to claim 22, wherein the maintenance therapy is to comprise administering the immunoconjugate or a pharmaceutical composition comprising the same (i) once every three to six weeks or (ii) at repeated multiple doses, wherein each individual dose of immunoconjugate is to be about 10 mg/m2, about 20 mg/m2, about 30 mg/m2, about 40 mg/m2, about 50 mg/m2, about 60 mg/m2, 70 mg/m2, about 80 mg/m2, about 90 mg/m2 or about 100 mgIm2 lower than the individual dose of a primary therapy and/or wherein individual doses are to be administered in intervals exceeding the interval of the individual doses, e.g., by 1, 2, 3, 4, 5, 6, 7 days.

24. Use according to any one of the preceding claims wherein at least one cytotoxic agent is to be administered, optionally two or three, at least once a week or once in a treatment cycle.

25. Use according to claim 24, wherein said cytotoxic agent is lenalidomide, pomalidomide and/or dexamethasone.

26. Use according to claim 24 or claim 25, wherein said subject has not previously been exposed to an immunoconjugate comprising an antibody targeting (30138 sing cells, to lenalidomide and/or to dexamethasone.

27. Use ing to claim 24 or claim 25, wherein said t has usly been exposed to an immunoconjugate comprising an antibody targeting CD138 expressing cells, lenalidomide and/or dexamethasone. -‘163—

28. Use according to claim 27, wherein said subject relapsed after said administration.

29. Use according to any one of claims 24to 28, wherein lenalidomide is to be administered at a dose of 5 to 35 mg, or at a dose of less than 25, 20, 15 or 10 mg and/or wherein dexamethasone is to be administered at a dose of 20 to 50 mg, preferably at about 40 mg, or at a dose of less than 40 or 30 mg.

30. Use according to claim 29, wherein the lenalidomide dose is about 25 mg.

31. Use ing to claim 29 or 30, wherein the dexamethasone dose is about 40 mg.

32. Use according to any one of claims 29 to 31, wherein the lenalidomide or the dexamethasone are to be administered orally once a day for 21 days, or once a week.

33. Use according to any one of the preceding claims, wherein the plasmaproliferative disorder is multiple myeloma.

34. Use according to claim 33, wherein the multiple myeloma is relapsed or refractory multiple myeloma.

35. Use according to any one of claims 1 to 32, n said subject suffers from a solid tumor comprising target cells which express CD138 and wherein said solid tumor is refractory to cancer hormone therapy or herapy or the subject has relapsed after hormone therapy or chemotherapy, wherein said stration results in at least tumor growth delay or tumor .

36. Use according to any one of claims 1 to 32 or claim 35, wherein said solid tumor is estrogen receptor negative and/or progesterone receptor negative and/or Her2/neu negative.

37. Use according to claim 36, wherein a xic agent is to be administered, wherein the cytotoxic agent is a taxane.

38. Use according to any one of the preceding , wherein the ered targeting antibody comprises a light chain comprising SEQ ID N022 and a heavy chain comprising SEQ ID NO: 1. -164~

39. Use of an immunoconjugate in the manufacture of a medicament for treating a disease associated with target cells expressing CD138 in a human subject, wherein the immunoconjugate comprises at least one engineered targeting antibody ing CD138 expressing cells, and at least one effector molecule, wherein said engineered targeting antibody is functionally attached to said or molecule to form said immunoconjugate, wherein the engineered targeting antibody comprises a heavy chain having SEQ ID NO: 1 and a light chain having SEQ ID NO: 2, n the effector molecule is DM4, wherein the disease associated with target cells expressing CD138 is multiple a, wherein said treating comprises stering the medicament in an active treatment cycle of 21 days, at least three times within said 21 days, wherein said active treatment cycle comprises administering at least once a week, wherein the dose of the immunoconjugate to be administered at least once a week is between 80 mg/m2 and 120 mg/mz.

40. Use according to any one of claims 1 to 39, ntially as herein described with reference to any one of the Examples and/or