CANCER

FIELD OF THE INVENTION:

The invention is directed to the treatment of prostate cancer by means of antibodies. Above all, the invention relates to the administration of an anti-alpha-v integrin (receptor) antibody to patients suffering from prostate cancer, especially castration- resistant prostate cancer (CRPC) without or after chemotherapy, optionally

accompanied by lymph node and bone tissue metastases (mCRPC). In particular, the invention relates to the therapy of said patients by means of the anti-angiogenic antibody DI17E6 and structural mutants thereof.

BACKGROUND OF THE INVENTION

Prostate cancer is the most commonly occurring cancer aside skin cancer in the US, and is the second most common cause of male cancer deaths.

Prostate cancer is classified in four stages: Stage I prostate cancer is found in the prostate only and cannot be felt during a digital rectal exam nor is it visible by imaging. In stage II prostate cancer, the tumor has grown inside the prostate but has not extended beyond it, whereas in stage III, the cancer has spread outside the prostate, but to a minimal extent only. Often, prostate cancer in stage III will have spread only to nearby tissues, such as the seminal vesicles. Finally, in stage IV, the cancer has spread outside the prostate to other tissues, such as the lymph nodes, bones, liver, and/or lungs or brain.

The spectrum of prostate cancers that are progressing despite castrate levels of testosterone includes tumors that have shown varying degrees and durations of response to primary hormone treatment, and clinical manifestations that range from a rising prostate-specific antigen (PSA) alone, a rising PSA with osseous and/or soft- tissue spread, or a predominantly visceral disease pattern.

Currently approved treatment of prostrate cancer includes surgical castration, chemical castration, or a combination of surgical and chemical castration. Removal of the testes, the primary testosterone producing organ, reduces the levels of circulating androgens, to less than 5% of normal levels. This reduction in androgen levels inhibits - - prostate tumor growth. Although the anti-tumor effects of surgical castration are direct, the anti-tumor effects can be temporary. Surgical castration often leads to clonal selection of androgen-independent prostate tumor cells. This results in re- growth of the prostate tumor in a form that proliferates without testosterone or DHT Stimulation. Chemical castration (also called medical castration) is often substituted for surgical castration, as an initial treatment. Despite its high prevalence, treatment options for men having prostate cancer remain relatively limited and typically depend on the stage of the cancer.

Treatment options include surgical treatments such as radical prostatectomy, in which the prostate is completely removed and radiation, applied through an external beam that directs the dose to the prostate from outside the body or via low-dose radioactive seeds that are implanted within the prostate to kill cancer cells locally. Anti-androgen hormone therapy also is used in the treatment of prostate cancer, either alone or in conjunction with surgery or radiation. Hormone therapy typically aims at blocking the pituitary from producing hormones that stimulate testosterone production by use of castration or administration of hormone analogs and requires that patients have injections of these hormone analogs for protracted periods. Finally, chemotherapeutic approaches have been used to treat advanced prostate cancer, usually as a last resort when other approaches have failed. Since a couple of years, the combination of docetaxel and prednisone was established as the new standard of care for patients who have progressed on androgen deprivation.

None of the treatments described above are curative and prostate cancer being androgen dependent at first, often will progress despite surgical and hormonal-based therapies, and become resistant over time, leading to a cancer type which is called "hormone refractory cancer" or "castration resistant cancer" (CRPC).

Clinical disease manifestations of CRPC are commonly related to bone metastases and may include pain, pathologic fractures, and spinal cord compression, with local recurrences that may be associated with pelvic discomfort, renal dysfunction due to ureteral compression, bladder outlet obstruction, and sexual dysfunction. Further, while bone cancer is the predominant result of CRPC, patients may develop soft- tissue metastases (lymph node(s)) and visceral metastasis in liver, lung, brain, and - - other organs. Patients with CRPC are minimally responsive to chemotherapy and the majority of patients die due to progressive prostate cancer within 20 months of initiating treatment. Bisphosphonates are commonly used in patients with castrate- resistant prostate cancer who have bone metastases. It has been shown that prostate tumors remain dormant and clinically undetectable until they begin to secrete angiogenic factors and down-regulate the expression of angiogenic inhibitors. In general, it can be stated that angiogenesis is critical to the genesis of prostate tumors. Therefore, it was not completely surprising that anti- angiogenic agents inhibit prostate cancer cell growth. In prostate cancer, tumor cells express an abnormal integrin repertoire and are surrounded by a markedly aberrant extracellular matrix (ECM). These changes have profound consequences, given the ability of each integrin to regulate specific cell functions. Expression of β3 and β1 subunits activates specific signaling pathways and support distinct cancer cell functions. β3 is uniquely required in cancer cells for increasing cdc2 levels as well as cdc2 kinase activity. These effects are specific for β3 and are not observed for β6. Up-regulation of β3 and β6 integrin variants has been described. Zheng et al. (Cancer Research 1999; 59, 1655-1664) used human prostate cancer cells isolated from sixteen surgical specimens, to show that these cells express ανβ3, whereas normal prostate epithelial cells do not. Similarly, ανβ6 was found to be expressed in adenocarcinoma (Li et al.; Molecular and Cellular Biology 2007; 27, 4444).

The use of integrin inhibitors is likely to affect both cancer cell survival and

angiogenesis since integrins are expressed by tumor cells as well as by endothelial cells. Although it is hard to discriminate between an effect on tumor growth and an effect on angiogenesis, a maximal response of these inhibitors can be predicted when the targeted integrin is expressed by both tumor and endothelial cells.

Bone is the most frequent metastatic site for prostate cancer. Bisanz et al. (Molecular Therapy 2005; 12, 634-643) illustrate a positive role for alpha-v integrins on prostate tumor survival in the bone. Analysis of human prostate cancer bone xenografts shows that intratumoral administration of liposome encapsulated human alpha-v siRNAs significantly inhibits the growth of PC3 tumors in bone and increases apoptosis of - - prostate tumor cells. Further studies (McCabe et al., Oncogene 2007; 26, 6238-6243) demonstrate that ανβ3 integrin activation on tumor cells is essential for the

recognition of key bone specific matrix proteins. These data suggest that the ανβ3 integrin modulates prostate cancer growth in distant metastasis. Since integrins mediate the interactions between tumor cells and bone

microenvironment and facilitate growth in bone, a potential application of the use of integrin inhibitors is to prevent prostate cancer bone lesions. These lesions are osteoblastic and/or osteolytic and are frequently detected in prostate cancer patients (over 80% of prostate cancer patients have established bone metastasis at autopsy). A recent study has shown that the ανβ3 integrin promotes bone gain mediated by prostate cancer cells that metastasize to the bone and point to ανβ3 as a potential therapeutic target to block prostate cancer osteoblastic lesions. Immunohistochemical analysis has demonstrated the presence of av integrin in a large proportion of human prostate cancer tissues samples. These and other results suggest that anti-integrin agents may have both direct and indirect antitumor activity. But there are only few clinical trials reporting that peptide or non-peptide integrin inhibitors are effective agents in prostate cancer therapy.

Therefore, there is a need to provide a potent anti-integrin agent for use in the therapy of prostate cancer, especially castration-resistant prostate cancer developing bone metastases.

SUMMARY OF THE INVENTION

It has been found by the inventors that the known monoclonal anti-alpha v antibody DI-17E6 (designated herein also as EMR62242 or EMD 525797) is highly effective in (i) a monotherapy based clinical setting and (ii) in a combinatorial clinical setting together with hormonal agents and / or chemotherapeutic agents and / or tyrosine kinase inhibitors or other angiogenesis inhibitors in the treatment of prostate cancer.

In a first aspect of the invention, it was shown in clinical trials that the known specifically engineered hybrid monoclonal anti-alpha-v antibody DI17E6 is well tolerated in prostate tumor patients without significant side effects at doses of at least - -

500mg mAb each two weeks administered by infusion during a treatment period of at least four months (Figure 3,Table 3).

In a second aspect of the invention it was shown, when administering DI17E6 with doses of at least 500mg each two weeks is effective in the treatment of prostate cancer, preferably castrate-resistant prostate cancer (CRPC). As a result, the number of prostate derived circulating tumor cells (CTC) in the blood of these patients can be significantly reduced within the treatment period, above all if this number was originally (at the beginning of the treatment) very high (Figure 1).

In a third aspect of the invention it was shown that the value of prostate specific antigen (PSA) can be significantly reduced in CRPC patients. The effect is dependent on the duration of the treatment and the disease status of the patient. In general after five or more months of treatment a PSA value can be obtained, which corresponds in less progressive cases to PSA values of healthy males, in other more severe cases, where the PSA value was dramatically high at the beginning of the antibody treatment, the PSA value after treatment can be reduced more than 10-fold (Figures 4, 5)

In a fourth aspect of the invention it could be demonstrated that by the administration of DI17E6 in a dose of at least 500mg each two weeks the metastatic affection of bone and lymph nodes can be significantly reduced. In a further aspect of the invention, it could be shown that DI17E6 reveals a dose- dependent response in prostate cancer patients, preferably castrate-resistant prostate cancer (CRPC) patients, with increasing efficacy at higher doses, wherein a dose of about 500mg and higher each two weeks is effective, whereas a dose of about 250mg DI17E6 or less is not effective in prostate cancer, preferably CRPC patients (Figure 3, 5).

It should be noted that DI17E6 is effective preferably in a monotherapy setting, wherein no further cytotoxic drug (such as cabazitaxel, docetaxel, doxorubicin, irinotecan etc.) was administered. This is the first time, where an engineered monoclonal antibody is effective in a tumor therapeutic approach without the necessity of the administration of a chemotherapeutic agent. - -

It should be further noted that according to the first results in said clinical trials DI17E6 seems to be the more effective the more progressive the disease is. Thus, DI17E6 elicits a stronger effect on PSA values, circulating tumor cells and metastases in patients after ineffective hormone- and / or chemotherapy and even after surgery impacts such as prostatectomy as compared to patients in a less progressive disease state without such surgery or even without pretreatment with chemotherapeutics and / or hormones. In summary, DI17E6 is very promising in the treatment of CRPC above all in patients with a progressive or end-stage disease status after chemotherapy treatment. A further result is that DI17E6 is able to induce tumor shrinkage and tumor lesion (Figure 6), especially in solid prostate tumors or tumor metastases deriving thereof, which are resistant to chemotherapy and / or radiotherapy.

Furthermore, there is evidence that treatment with DI17E6 (in monotherapy) reduces pain, which usually occurs in prostate cancer. Thus, the drug presents benefits in terms of pain and pain interference score (Figures 4B, 5B). It could be observed that the decrease of pain during treatment is correlated with the decreasing PSA level in said patients.

DI17E6 is well tolerated without premedication, and does not show clinically relevant does-related changes in assessed safety parameters, such as drug-related treatment emergent adverse events (TEAE) (Table 3).

In a further aspect of the invention, it could be shown that there is a strong correlation between bone metabolism biomarkers , including STC-1 , ADAMTS-1 , M30, M65, IL6 and IL8, and dose levels (Figure 7). At high doses > 1500mg a significant drop in osteopontin can be observed in the majority of CRCP patients. The pharmacokinetic profile of DI17E6 is dependent on dose after single and multiple doses with a half-life of approximately 250h at the 1500mg dose level.

The safety results of the phase 1 , open-label study show that repeated infusions of single-agent DI17E6 (EMD 525797) at each of four dose levels are generally well tolerated and appear to be safe in patients with mCRPC and progressive disease preferably following prior chemotherapy. There are no dose-limiting toxicities (DLT) - - and no infusion reactions. With regard to dose, no trends in the distribution of TEAEs, NCI-CTCAE (version 3.0) grade or drug relationship are observed. In addition, there is no evidence of accumulation of any specific event within individual cohorts. Eleven patients experienced TEAEs that are considered to be drug-related. In this regard, skin symptoms such as pruritus, erythema and rash, which are reported in a total of four patients, are predictable adverse events associated with DI17E6 (EMD 525797) given that integrins are responsible for the maintenance of the epithelial phenotype. Symptoms of mucosal inflammation and swollen tongue may also be characteristic of the mechanism of action of EMD 525797, but together with fatigue, might also be signs of the underlying disease. The hematologic and biochemic toxicity shifts observed in eight patients could also be explained by underlying disease, as well as concomitant medications.

PK assessment after single and multiple doses of study drug suggest that DI17E6 (EMD 525797) behaved in accordance with a receptor-mediated clearance model as described for other antibodies targeting membrane-associated receptors. Consistent with the findings of an earlier study in healthy volunteers, PKs of DI17E6 (EMD 525797) in mCRPC patients are dose-dependent with clearance determined predominantly by the availability of unbound receptors. At the doses used in the present study, it can be assumed that at doses of 1000mg or higher, almost all receptors are saturated and have a minor contribution to drug clearance.

Immunologically triggered antibodies directed against DI17E6 can be detected in some (16%) patients; however, no impact on PKs or safety could be found.

In castrate-resistant prostate cancer patients with bone metastases following prior chemotherapy, the median progression free survival is expected to be 8 to 10 weeks. Antitumor activity evaluations showed that DI17E6 (EMD 525797), as single-agent therapy, achieved an objective partial tumor response in a single patient in the 500mg cohort. In 9 of 18 patients (50%) receiving DI17E6 (EMD 525797) at a dose of 500mg or higher, no radiographic disease progression can be observed for 16 weeks or longer. In two patients, long-term treatment with DI17E6 (EMD 525797) at a dose of 500mg is associated with significant reductions in PSA levels and clinical benefit in terms of pain interference. At least one patient also showed primary tumor shrinkage - - and normalization of target lymph node size. Thus, DI17E6 (EMD 525797) appears to show single-agent activity in at least some patients with late-stage mCRPC.

In conclusion, single-agent EMD 525797 given as single and multiple doses is shown to be well tolerated in patients with mCRPC with bone metastases and progressive disease following prior chemotherapy with no spontaneous remissions. No safety concern can be identified and there is preliminary evidence of clinical benefit in numerous patients. Due to its target and safety profile, DI17E6 (EMD 525797) is a promising agent for combination therapy.

To sum up, the subject matter of this invention is directed to the following: · The use of anti-av integrin antibody DI17E6 or a biologically active variant, or modification thereof, for the treatment of patients, or for the manufacture of a medicament for the treatment of patients, said patients suffering from prostate cancer, preferably castrate-resistant prostate cancer (CRPC), preferably wherein said CRPC is accompanied by high levels of serum PSA in a range of 25 - 10.000ng/ml, more specifically in levels of more than 25ng/ml , or more than

50ng/ml, or more than 100ng/ml, or more than 250mg/ml, or more than

500ng/ml, or more than 1000ng/ml, or more than 2500ng/ml or more than 5000ng/ml, or more than 7500ng/ml.

• The respective use of DI17E6 antibody, wherein the cancer is metastasizing, preferably into bone and / or lymph node tissue.

• The respective use of DI17E6 antibody, wherein the prostate-specific antigen (PSA) value is declined more than 5-fold, 10-fold, 20-fold, preferably 10-fold during treatment compared to the value before starting antibody treatment.

• The respective use of DI17E6 antibody, wherein the reduction of the PSA value was achieved within 4 - 8 months of treatment, preferably after 4 months, more preferably after 6 months.

• The respective use of DI17E6 antibody, wherein the number of the circulating tumor cells (CTC) is declined during antibody treatment. - -

The respective use of DI17E6 antibody in a CRPC patient whose prostate has been removed, or alternatively, has been treated by radiation.

The respective use of DI17E6 antibody for reducing pain which occurs in prostate cancer, preferably in CRPC preferably accompanied by bone

metastasis.

The respective use of DI17E6 antibody, wherein the patient was pretreated with chemotherapeutics and / or hormonal agents, preferably when the cancer is progressive after said pretreatment with the chemotherapeutic and / or hormonal agent.

The respective use of DI17E6 antibody, wherein the effective dose of the antibody is 500mg - 1500mg per two weeks, preferably 500 - 1000mg per two weeks, or 1000 - 2000mg per month.

The respective use of DI17E6 antibody, wherein the effective dose of 500 - 1000mg is administered by a single infusion.

The respective use of DI17E6 antibody, wherein the antibody is administered in a monotherapy setting without additional chemotherapeutic agents.

The respective use of DI17E6 antibody, wherein the antibody is administered in a combinatory setting with a cytotoxic/cytostatic or an hormonal agent

sequentially or simultaneously.

The respective use of DI17E6 antibody, wherein in said combinatory setting the cytostatic or cxytotoxic agent is selected from the group consisting of: a chemotherapeutic agent, radiation, a tyrosine kinase inhibitor, and an

angiogenesis inhibitor; said tyrosine kinase inhibitor being an anti-ErbB antibody selected from the group consisting of an anti-EGFR antibody, an anti-Her2 antibody, and an anti-Her3 antibody, and said angiogenesis inhibitor being an alpha-v integrin inhibitor, preferably an RGD peptide, such as cilengitide.

The respective use of DI17E6 antibody, wherein the biologically active variant modification comprises the CDR regions and heavy and light chain variable - - regions, which are 80% - 95% identical in amino acid sequence compared to the variable regions of DI17E6.

The respective use of DI17E6 antibody, wherein the biological active variant or modification comprises a constant region, which is at least 80% - 98% identical with the amino acid sequence compared to the constant region of DI17E6.

The respective use of DI17E6 antibody, comprising one or more modifications within the heavy chain framework regions

FRl: QVQLQQSGAELAEPGASVKMSCKASGYTFS (SEQ ID No. 16)

FR2: WVKQRPGQGLEWIG (SEQ ID No. 17)

FR3 : KAT TADTSSSTAYMQLSGLTSEDSAVYYCAS (SEQ ID No. 18)

FR4 : WGQGTSVTVSS (SEQ ID No . 19),

wherein one or more of the bold and underlined positions are mutated and are different compared to the original respective sequence.

The respective use of a modified DI17E6 antibody comprising a human lgG1 constant region instead of human lgG2, or a human lgG2 hinge region instead of the human lgG1 hinge.

A method of treating castrate-resistant prostate cancer (CRPC) in a patient, preferably accompanied by bone metastases, comprising administering the anti- av integrin antibody DI17E6 or a biologically active variant, or modification thereof preferably in a dose of 500 - 1000mg each two weeks preferably for a period of at least three months.

A method of declining the pathologically increased PSA serum level of a patient suffering from prostate cancer, preferably castrate-resistant prostate cancer (CRPC) more than 5-fold, preferably more than 10-fold by administering to said patient the hybrid monoclonal antibody DI17 in an effective dose of at least 500mg each two weeks, or at least 1000mg per month, wherein the pathological increased PSA serum level before staring antibody treatment is at least 25ng/ml, preferably at least 50ng/ml.

A respective method, wherein the cancer is metastasizing in bone and / or lymph node tissue. - -

SHORT DESCRIPTION OF THE FIGURES:

Fig. 1 : shows a table summarizing the results of the measuring of circulating tumor cells (CTC) in six selected CRPC patients of dose level (1) 250mg, (2) 500mg, and (3) 1000mg. X-axis indicates days of treatment. Y-axis indicates circulating tumor cells (CTC).

Fig. 2: shows dose-depend pharmacokinetic profile of DI17E6 (EMD 525797) for 250mg, 500mg, 1000mg and 1500mg Ab administered per each 4 weeks. X-axis indicates time after first infusion (h); Y-axis indicates serum concentration (pg/ml).

Fig. 3: shows the treatment duration of a cohort of CRPC patients after the treatment with DI17E6.; X-axis: number of weeks treatment; Y-axis: Dose levels (dose level 1 : 250mg, dose level 2: 500mg, dose level 3: 1000mg, dose level 4: 1500mg)

Fig.4: (A) shows the change of the PSA level (ng/ml serum) in a specific CRPC patient (2001) without prostatectomy showing bone metastases during the treatment with DI17E6 (treatment start 24.08.2009). (A). X-axis indicates the duration of treatment (days); Y-axis indicates PSA level (ng/ml) (B) shows the average pain interference score as defined below of the same patient of (A). X-axis indicates the duration of treatment (days); Y-axis indicates average pain interference score.

Fig. 5: (A) shows the PSA course of a second CRPC patient with progressive disease after chemotherapy showing bone metastases and prostatectomy during the treatment with DI17E6 (treatment start 01.09.2009). (A). X-axis indicates the duration of treatment (days); Y-axis indicates PSA level (ng/ml) (B) shows the average pain interference score as defined below of the same patient of (A). X-axis indicates the duration of treatment (days); Y-axis indicates average pain interference score.

Fig. 6: shows a CT scan of a patient from the 500mg cohort showing significant shrinkage of primary tumor lesions: (a) before treatment with DI-17E6, (b) after the 17 ^th treatment with DI-17E6 (after 4 months).

Fig. 7: shows the occurrence of bone markers /circulating bone markers after the treatment with DI17E6. 1 = dose level 1 ; 2 = dose level 2 - -

DETAILED DESCRIPTION OF THE INVENTION

DI17E6 is an engineered specifically tailored lgG2 hybrid monoclonal antibody directed to alpha-v integrin (receptor). Cancer therapy by means of this antibody reduces side effects associated with this type of therapy, above all immune reactions, thereby reducing immunogenicity. The antibody is described in detail in WO

2009/010290.

Its hypervariable regions (CDRs) derive from murine mAb 17E6 (EMD 73034). This parent mouse lgG1 antibody is described, for example by Mitjans et al. (1995; J. Cell Sci. 108, 2825) and patents US 5,985,278 and EP 719 859. Mouse mAb 17E6 is produced by hybridoma cell line 272-17E6 and deposited under accession number DSM ACC2160.

Its light chain domains derive from humanized monoclaonal anti-EGFR antibody 425 (matuzumab). This antibody is described in detail for example in EP 0 531 472B1 , and derives from its murine counterpart 425 ( mouse MAb 425, ATCC HB9629), The antibody was raised against the human A431 carcinoma cell line and found to bind to a polypeptide epitope on the external domain of the human epidermal growth factor receptor (EGFR). Matuzumab has shown in clinical trials high efficacy.

Generally DI17E6 as used according to the invention comprises:

(i) a CDR light and a heavy chain region deriving from mouse monoclonal

anti-av integrin antibody 17E6

(ii) a light chain framework region which is taken from humanized monoclonal anti-EGFR antibody 425,

(iii) a heavy chain framework region deriving from mouse monoclonal

anti-av integrin antibody 17E6, optionally comprising one or more mutations of amino acids at specific positions, and

(iv) a heavy chain constant region deriving from human lgG2 and a human

constant kappa light chain region, wherein in said lgG2 domain the

lgG2 hinge region was replaced by the human lgG1 hinge domain, and;

wherein optionally one or more mutations within the lgG2 has been carried out. - -

Specifically, DI17E6 (designated as "DI-17E6y2h(N297Q)" or "EMD 525797") as used for the treatment as claimed and in the clinical trials as described above and below, has the following amino acid sequence:

(i) variable and constant light chain sequences (SEQ ID No. 1 ):

PIQMTQSPSSLSASVGDRVTITCRASQDISNYIAWYQQKPGKAPKLLIYYTSKIHS

GVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGNTFPYTFGQGTKVEIKRTVAA PSVFIFPPSDEQLKSGTASWCLLNNFYPREA VQW VDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC and

(ii) variable and constant heavy chain sequences (SEQ ID No. 2):

QVQLQQSGGELAKPGASVKVSCKASGYTFSSFWMHWVRQAPGQGLEWIGYINP RSGYTEYNEIFRDKATMTTDTSTSTAYMELSSLRSEDTAVYYCASFLGRGAMDY WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW SG ALTSGVHTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTC VDHKPSNTKVDKTV EPKSSDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQAQSTFRWSVLTWHQDWL GKEYKCKVS

NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTK QVSLTCLVKGFYPSDIAVE WESNGQPEN YKTTPPMLDSDGSFFLYS LTVDKSRWQQG VFSCSVMHEALH NHYTQKSLSLSPGK, wherein the underlined sequences represent the variable regions with the CDRs (in bold, identical with the parent mouse antibody). The modified lgG1 hinge region is represented by EPKSSDKTHTCPPCP (SEQ ID No. 3), and AQ is a substitution within the lgG2 domain.

However, as it was shown in WO 2009/010290, also variants of DI17E6 can be used according to the teaching of this invention. Thus, DI17E6 variants comprising one or more modifications within the heavy chain framework regions

FRl: QVQLQQSGAELAEPGASVKMSCKASGYTFS (SEQ ID No. )

FR2: WVKQRPGQGLEWIG (SEQ ID No. 17)

FR3: KATMTADTSSSTAY QLSGLTSEDSAVYYCAS (SEQ ID No. )

FR4: WGQGTSVTVSS (SEQ ID No . 19),

wherein one or more of the bold and underlined positions are mutated, can be used in the treatment of prostate cancer patients as described. In more detail, the following position heavy chain framework region is mutated at one, more or all of the following - - positions can be mutated: A9, E13, M20, K38, R40, A72, S76, Q82, G85, T87, S91 and S113. These variants show the same or very similar biological activity and efficacy as compared to DI17E6 defined by its sequences above.

In general, the invention as described includes also modifications and variants of the DI17E6 antibody that are functionally and / or pharmaceutically identical or similar to unmodified DI17E6, and wherein the CDR regions and heavy and light chain variable regions are at least 80%, or at least 85%, or at least 90%, or at least 95% identical in their amino acid sequence compared to the respective variable regions of DI17E6. In addition, the invention also includes modifications and variants of the DI17E6 antibody that are functionally and / or pharmaceutically identical or similar to unmodified DI17E6, and wherein the constant regions are at least 80%, or at least 85%, or at least 90%, or at least 98% identical in their amino acid sequence compared to the respective constant regions of DI17E6. Changes is the constant regions of the IgG chains of the antibody may improve specific properties like immunogenicity, ADCC, and so on.

In a further aspect, the DI17E6 antibody as used according to the invention can be fused to a cytotoxic agent, preferably a cytokine, such as IL2, IL8, IFNb, IFNa, TNFa, GM-CSF, G-CSF, or as specified below. The cytokine can be fuses via its N-terminal to the C-terminal of the antibody heavy chains, or via its C-terminal to the N-terminal of the heavy and light chain of the antibody.

According to the invention, DI17E6 is highly effective in patients suffering from prostate cancer, above all in patients whose tumor is progressive after chemotherapy and / or hormonal therapy and / or prostate surgery such as prostatectomy.

In order to strengthen or prolong efficacy of DI17E6 or its modifications described a combination therapy together with cytotoxic or cytostatic agents is applicable. The cytostatic or cytotoxic agent can be administered in sequential steps with the antibody or simultaneously in doses that are quite common and convenient in the clinical treatment of cancer. Preferred cytotoxic/cytostatic agents according to the invention are anti-angiogenic agents as described below in more detail, anti-EGFR or -Her3 agents including respective antibodies, such as Herceptin® (trastuzumab), Erbitux® (cetuximab) or panitumumab. In a preferred embodiment the combination therapy of - -

DI17E6 together with cetuximab is effective in prostate cancer, especially in CRPC according to this invention.

For the same purpose DI17E6 can be administered together (sequentially or simultaneously) with hormonal agents according to known regimen. The term "castrate-resistant" or "castration-resistant" us used herein refers to a status in prostate cancer wherein the disease is progressing with serum testosterone controlled below a castrate level, which is usually < 50ng/100ml. The term is synonymously used with "hormone-resistant" or "hormone-refractory" or "androgen- independent". The term "pain interference (total) score" means a score for pain as a result of the disease occurring during administration of the effective drug. Pain interference is assessed using the Brief Pain Inventory (BPI). BPI is used to evaluate pain interference with the following: (a) general activity, (b) mood, (c) walking ability, (d) normal work, (e) relations with other people, (f) sleep, and (g) enjoyment of life. Total score for pain interference is calculated as: (Mean score of non-missing questions) x (7/number of non-missing questions). If four or more questions are missing, the pain interference total score is set to missing.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells by causing destruction of cells. The term is intended to include radioactive isotopes, chemotherapeutic agents, and toxins such as

enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof. The term may include also members of the cytokine family, preferably IFNy as well as anti-neoplastic agents having also cytotoxic activity.

The term "cytostatic agent" as used herein refers to a substance that inhibits or suppresses cellular growth and multiplication without destroying cells.

The term "cytokine" is a generic term for proteins released by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; - - thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor (VEGF); integrin; thrombopoietin (TPO); nerve growth factors such as NGFB; platelet-growth factor; transforming growth factors (TGFs) such as TGFa and TGFB; erythropoietin (EPO); interferons such as IFNa, IFNB, and IFNy; colony stimulating factors such as M-CSF, GM-CSF and G-CSF; interleukins such as IL-1 , IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12; and TNFa or TNFB. Preferred cytokines according to the invention are interferons and TNFa.

An "anti-angiogenic agent" refers to a natural or synthetic compound which blocks, or interferes with to some degree, the development of blood vessels. The anti- angiogenic molecule may, for instance, be a biological molecule that binds to and blocks an angiogenic growth factor or growth factor receptor. The preferred anti- angiogenic molecule herein binds to an receptor, preferably to an integrin receptor or to VEGF receptor. The term includes according to the invention also integrin

(receptor) inhibitors.

The term "integrin inhibitors" or "integrin receptor inhibitors" refers to a natural or synthetic molecule that blocks and inhibit an integrin receptor. In some cases, the term includes antagonists directed to the ligands of said integrin receptors (such as for α _ν β3: vitronectin, fibrin, fibrinogen, von Willebrand's factor, thrombospondin, laminin; for a _v Q>5. vitronectin; for α _ν βι: fibronectin and vitronectin; for α _ν β _θ : fibronectin). Antagonists directed to the integrin receptors are preferred according to the invention. Integrin (receptor) antagonists may be natural or synthetic peptides, non-peptides, peptidomimetica, immunoglobulins, such as antibodies or functional fragments thereof, or immunoconjugates (fusion proteins). Preferred integrin inhibitors of the invention are directed to receptor of cc _v integrins (e.g. α _ν β3, α _ν β ₅ , α _ν ββ and subclasses). Preferred integrin inhibitors are a _v antagonists, and in particular α _ν β3

antagonists. Preferred a _v antagonists according to the invention are RGD peptides, peptidomimetic (non-peptide) antagonists and anti-integrin receptor antibodies such as antibodies blocking a„ receptors. Exemplary, non-immunological α _ν β3 antagonists are described in the teachings of US 5,753,230 and US 5,766,591. Preferred antagonists are linear and cyclic RGD-containing peptides. Cyclic peptides are, as a rule, more stable and elicit an enhanced serum half-life. The most preferred integrin antagonist of the invention is, however, cyclo-(Arg-Gly-Asp-DPhe-NMeVal) (EMD 121974, Cilengitide ^® , Merck KgaA, Germany; EP 0770 622) which is efficacious in blocking the integrin receptors α _ν β3, α _ν βι, a _v &6, tv&&, aii _b &3- combination therapy of DI17E6 together with Cilengitide in prostate cancer and preferably CRPC patients is effective according to the invention. The term "chemotherapeutic agent" or "anti-neoplastic agent" is regarded according to the understanding of this invention as a member of the class of

"cytotoxic agents", as specified above, and includes chemical agents that exert antineoplastic effects, i.e., prevent the development, maturation, or spread of neoplastic cells, directly on the tumor cell, and not indirectly through mechanisms such as biological response modification. Suitable chemotherapeutic agents according to the invention are preferably natural or synthetic chemical compounds, but biological molecules, such as proteins, polypeptides etc. are not expressively excluded. There are large numbers of anti-neoplastic agents available in commercial use, in clinical evaluation and in pre-clinical development, which could be included in the present invention for treatment of tumors / neoplasia by combination therapy with TNFa and the anti-angiogenic agents as cited above. It should be pointed out that the

chemotherapeutic agents can be administered optionally together with above-said antibody drug. Examples of chemotherapeutic or agents include alkylating agents, for example, nitrogen mustards, ethyleneimine compounds, alkyl sulphonates and other compounds with an alkylating action such as nitrosoureas, cisplatin and dacarbazine; antimetabolites, for example, folic acid, purine or pyrimidine antagonists; mitotic inhibitors, for example, vinca alkaloids and derivatives of podophyllotoxin; cytotoxic antibiotics and camptothecin derivatives. Preferred chemotherapeutic agents or chemotherapy include amifostine (ethyol), cabazitaxel, cisplatin, dacarbazine (DTIC), dactinomycin, docetaxel, mechlorethamine, streptozocin, cyclophosphamide, carrnustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), doxorubicin lipo (doxil), gemcitabine (gemzar), daunorubicin, daunorubicin lipo (daunoxome), procarbazine, ketokonazole, mitomycin, cytarabine, etoposide, methotrexate, 5- fluorouracil (5-FU), vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, camptothecin, CPT-11 , 10-hydroxy-7-ethyl-camptothecin (SN38), dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon alpha, interferon beta, irinotecan, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa,. uracil mustard, vinorelbine, chlorambucil and combinations thereof. Most preferred chemotherapeutic agents according to the invention in combination with DI17E6 are cabazitaxel, cisplatin, docetaxel, gemcitabine, doxorubicin, paclitaxel (taxol), irinotecan and bleomycin.

DI17E6 is administered usually by intravenous injection, however other administration forms convenient in the art for antibody / protein drugs are applicable. All standard infusion solutions and formulation are applicable, such as described in

WO 2005/077414 or WO 2003/053465, including liposomal formulations. It is, in addition, favorable to provide human serum albumin nanoparticles loaded with

DI17E6 and optionally (to increase cytotoxicity) chemotherapeutic drugs (Biomaterials 2010, 8, 2388-98; Wagner et al.). The following examples describe the invention in more details but do not limit the invention and its scope as claimed.

Examples

Example 1 : Study Design

A Phase I trial was initiated to determine safety, pharmacokinetics, and antitumor activity of CRPC patients treated with DI17E6 including effects on prostate-specific antigen, circulating tumor cells (CTC), and soft tissue and bone metastases. All patients were suffering from a progressing disease after chemotherapy. Patients were treated with iv-infusions of 250, 500, 1000 or 1500mg DI17E6 given over 1 hour.

Eligible patients were aged 18 years or older and had histologically or cytologically proven prostate cancer with evidence of bone metastases after prior chemotherapy. - -

Patients had either undergone bilateral orchiectomy or were receiving continuous androgen deprivation therapy with a gonadotropin releasing hormone agonist or antagonist and had stopped anti-androgen therapy for at least 4 weeks prior to enrolment. Patients were required to either be on stable (i.e. at least 3 months) ongoing bisphosphonate therapy or without any bisphosphonate therapy, with a total serum testosterone level less than 50 ng/dL. All patients had evidence of progressive disease, defined as at least two prostate-specific antigen (PSA) values above the individual nadir level with a minimum increase of 10% each determined at least two weeks prior to screening; nodal or visceral progression was sufficient for inclusion independent of PSA. In addition, patients had to have an Eastern Cooperative

Oncology Group (ECOG) score of 0 to 2, a life expectancy of at least 3 months, and adequate hematologic, renal and hepatic function. An institutional review board at each study center approved the study protocol, and all patients provided written informed consent. In this phase 1 , multicenter, open-label, dose-escalation study, mCRPC patients were administered three intravenous infusions of EMD 525797 at doses of 250, 500, 1000, or 1500 mg given over one hour every two weeks prior to response assessment at the end of week 6. Patients without evidence of progressive disease were eligible to receive further fortnightly doses until disease progression or unacceptable toxicity. Dose-limiting toxicities (DLTs) were assessed during the first six weeks and patients were followed for safety until four weeks after the last administration of EMD 525797. Patients were recruited in four sequential dose cohorts; after the last of six patients within a dose cohort had reached the end of week 6, a Safety Monitoring Committee determined subsequent dose escalation. Twenty-six male patients aged between 43 and 80 years (median age, 66 years) were enrolled and received at least one intravenous infusion of EMD 525797, constituting the safety population. All patients were of Caucasian origin. In general, demographic characteristics were comparable across the four dose cohorts (Table 1a), with a median time since first diagnosis of 5.2 years (range, 2-18 years) and a median time from diagnosis to first metastatic disease of 0.1 years (range, 0-16 years). Two patients withdrew before the end of the DLT period and were subsequently replaced, with 24 patients receiving three doses of EMD 525797 through treatment Week 6. Table 1a. Patient baseline demographics (safety population).

BMI, body mass index; TNM, tumor, node, metastasis.

In summary (Table b): - -

The clinical results from this Phase I clinical trial is being conducted at 3 sites in Germany and at one site in Belgium and show single agent and biological activity in subjects with castration-resistant prostate cancer with bone metastases and progressive disease after chemotherapy.

Example 2: Treatment duration

24 patients (43-80 years) received 3 doses (weeks 1 , 3 and 5) prior to response assessment at the end of week 6. Patients without progressive disease could receive further doses every 2 weeks. Dose-limiting toxicities (DLTs) were assessed over the - - first 6 weeks and patients were followed for safety until 4 weeks after the last administration of DI17E6.

Table 2 summarizes drug exposure per patient in each cohort. Patients had a mean EMD 525797 exposure duration of 117.5 days (median, 74.5 days; range, 14-534 days). Thirteen of 24 patients had a longer exposure time than expected (>84 days), with two patients in the 500 mg cohort remaining on treatment for 297 and 534 days, and one patient in the1000 mg cohort receiving treatment for 310 days. No DLTs were reported within the DLT period of 6 weeks. All patients experienced at least one TEAE and no dose-dependent relationship in TEAEs was observed. Table 2a: DI17E6 exposure per patient in each of the dose cohorts

+ == ongoing treatment; ^* dropped out pts (1 and 2 infusions only)

The study protocol stated, that subjects will receive every other week at least 3 doses (250, 500, 1000 mg / each 2 weeks) of DI17E6 and that subjects without evidence of progressive disease will receive further treatment doses every other week. - -

Treatment duration in all 4 cohorts (state: Aug 2010). Cohort 2 with 500mg/each 2 weeks, two patients (2001 and 2002) in late stage (estimated mOS < 12 months) are more than 10 months with unexpected tumor response on treatment.

Table 2b: This table shows the values of the treatment duration (state: August 2010). Dose level 1 : 250mg DI17E6; dose level 2: 500mg DI17E6; dose level 3: 1000mg DI17E6, and dose level 4: 1500mg DI17E6.

Example 3: Safety /Side effects

Table 3 summarizes drug-related TEAEs. Eleven patients (42.3%) experienced drug- related TEAEs, which were most commonly reported in the system organ class "Skin and subcutaneous tissue disorders" (4 patients total; pruritus generalized, erythema, rash), "General disorders and administration site conditions" (3 patients total; fatigue, mucosal inflammation, edema peripheral), "Gastrointestinal disorders" (2 patients total; dry mouth, swollen tongue, upper gastrointestinal hemorrhage), and "Infections and infestations" (2 patients total; rhinitis, septicemia). Only two patients (7.7%) had a drug-related grade 3 or 4 TEAE: one patient in the 500 mg cohort experienced a grade 3 increase of gamma-glutamyltransferase (GGT) and a single patient in the 1000 mg cohort experienced a grade 3 septicemia.

Two patients experienced serious TEAEs that were considered to be related to treatment. These were a grade 1 upper gastrointestinal hemorrhage in a patient in the 250 mg cohort and a grade 3 septicemia in one patient in the 1000 mg cohort. At screening, this latter patient had an ongoing diagnosis of urinary tract infection and recurrent events of septicemia of which the last event was considered related to DI17E6 (EMD 525797). Four patients died and investigators assessed all deaths as not reasonably related to EMD 525797. - -

Six patients (23.1%) permanently discontinued treatment due to TEAEs, including 4 patients in the 250mg cohort (upper gastrointestinal hemorrhage at day 12, muscular weakness at day 53, paraplegia at day 46, and ureteric obstruction at day 30), and 1 patient each in the 500mg (grade 3 GGT increase at day 534) and 1500mg

(metastases to central nervous system at day 85) cohorts. No administration site skin reactions were reported. Post-baseline hematology and biochemistry toxicity shifts to grade 3 or 4 occurred in 8 patients. However, there were no obvious trends in laboratory findings, vital signs or ECG recordings. Overall, 65.4% of patients had the same worst ECOG performance status on treatment compared with baseline. Table 3. Drug-related treatment emergent adverse events ^* (TEAEs).

^" MedD A Primary System Organ Class. MedDRA Preffered Term Version 13.0.

^† Grade 3 occurred after the first 6 weeks (dose limiting toxicity period). - -

Examples of observed side effect:

(i) A 62 year-old male experienced grade 1 upper gastrointestinal bleeding 13 days after the first and only infusion of DI17E6 (250 mg). The subject presented with non- serious hematemesis and was hospitalized. Gastroscopy showed a lesion in the distal esophagus. Active bleeding was excluded, the subject was treated with omeprazole, and the event resolved

(ii) A 79 year-old patient developed septicemia (grade 2) due to e. faecalis 1 days after the most recent and 9 weeks after the first infusion of DI17E6 (EMD 525797) (1000 mg). The patient was hospitalized and discharged with recovery. A month later, the patient developed a second septicemia episode (grade3) again due to e.

faecalis 4 days after the most recent and 2,5 months after the first infusion. The patient was hospitalized and discharged with recovery. Another month later, the patient developed a third septicemia episode (grade3) 4 days after the most recent and 3,5 months after the first infusion, which was attributed to EMD 525797. In summary:

^■ Accumulating safety data have been reviewed at all 4 SMCs

■ Overall no DLTs have been observed: DLT is defined as any grade 3 or 4

hematological or non-hematological toxicity occurring until end of week 6 suspected to be reasonably related to the investigational product by the

Investigator and/or Sponsor except for allergic/ hypersensitivity reactions and any out-of-range laboratory values without clinical correlate which are reversible within 7 days.

^■ No MTD reached until now

■ Overall only 2 SAEs have been observed as related to study medication

Example 4: Pharmacokinetics and pharmacodynamics

After single and multiple doses, EMD 525797 showed a dose-dependent, non-linear PK profile (Figure 2). After the first 1-hour intravenous infusion, C _max of DI17E6 (EMD 525797) was generally reached within 1-2 hours after the start of dosing. The elimination half-life increased with dose as a consequence of EMD 525797 clearance - - increasing with dose, whereas mean volume of distribution remained constant over the dose range (Table 4).

As in Figure 2 (table 4) depicted, administration of cohort 2 CRPC patients with 500 mg/ each 2 weeks reached serum levels with IC95, whereas patients from cohort 1 with 250 mg / each 2 weeks failed. The serum trough concentration of EMD 525797 in cohort 2, 500mg / each 2 weeks, is above the IC95 and reach the IC99 of the non-linear CL pathway (250mg / each 2 weeks failed).

Table 4:

AUCr, area under the concentration-time curve within one complete dosing interval; C _max - maximum serum concentration, C _mM through serum concentration; Ratio_AUC, relative area under the curve [AUC _T (Dose period3)/AUC _T (Dose period 1)]; Ratio_C _max - relative maxiumum serum concentration [Cmax(Dose period 3)/C _max (Dose period 1)]; t _max ' time to reach C _max <; V _5S' apparent volume of distribution at steady stage.

After multiple doses, DI17E6 (EMD 525797) maximal serum concentrations and exposure accumulated dose-dependently up to a maximum value (dosing period 3/dosing period 1) of 1.33 and 1.70, respectively, at 1500 mg.

In most patients, CTC concentrations remained stable around baseline values (data not shown). In two patients, considerable decreases in CTC concentrations were observed at around 14 and 42 days after the start of treatment, respectively.

Anti-EMD 525797 antibodies were detected in 4 of 25 (16.0%) evaluable patients. All four patients were in the 250 mg cohort; two patients reverted to seronegative status after two weeks, one patient had no follow-up, and one patient remained seropositive over the entire study period. - -

Example 5: Circulating tumor cells (CTC)

Assessment of circulating tumor cells (CTC) using CellSearch has been cleared by the Food and Drug Aministration as a prognostic factor for patients with metastatic prostate cancer. Several groups have shown that CTC are detected at high frequency in CRPC and are correlated with clinical outcomes (de Bono et al. 2008, Clin Cancer Res, Scher et al. The Lancet 2009).

In Figure 1 the effect of DI17E6 on circulating tumor cells (CTC) is depicted. Patient No. 2002 from cohort with a dose of 500 mg per each 2 weeks shows a decrease in CTC. Other patients had originally low level of CTC and therefore no significant change after 3 infusions at week 7. This effect was not observed in cohort 1 with 250mg per each two weeks, where a steep increase can be observed in 5 out of 7 patients at Week 7.

Example 6: Bone Markers

Bone resorption is responsible for the major morbitidy in Prostate cancer. Skeletal events are important in the progression of Prostate cancer. Markers to evaluate bone resorption and bone formation were assessed in the study. A pairwise correlation for all markers per cohort was performed and a potential dose effect for cohort II observed.

Bone markers are clustering together in cohort II and not in cohort I. The biological meaning is not completely, but this could indicate a dose effect in relation to the bone markers in the treatment of Prostate cancer with DI-17E6. It could be that DI-17E6 "normalizes" the bone activity so that the bone resorption markers and the bone formation markers are regulated in a more balanced way closer to a normal state than in the disease state. It has been shown that in mCRPC bone resorption markers are often overexpressed. Outstanding biomarkers which can be observed in context with the administration of DI17E6 are ADAMTS-1 , STC1 , IL6 and IL8.

As in Figure 7 depicted, two dose levels (250mg and 500mg) are compared. The 500mg dose shows three clusters (box right sight of dose level 2=500mg) of a pair- wise correlation. - -

Example 7: Change in PSA level and pain score bv treatment with DI17E6

Figures 4A and 5A show the change of serum PSA levels in two patients during treatment with 500mg / each 2 weeks DI17E6.

The level of PSA is related to tumour growth and has a positive correlation with tumor burden. A decrease of more than 50% in the PSA level as compared with ghe baseline has been proposed as a biological response criterion.

In clinical practice, PSA is measured accurately and easily. Most physicians base their decision about continuing treatment on a general impression of the patients well- being and on their PSA kinetics. Two patients (see charts from subjects 2001 and 2002) from the 500mg dose cohort show a more than 8fold decrease of the PSA values during treatment compared to baseline values (Fig. 4A, 5A).

Datient screening wldl w3dl5 w5d29 w7d43

1002 104,4 130,5 169,7 195,3 222

1004 5,7 5,9 11,3 8,7 6,6

1005 34,3 44,7 46,6 47 48,5

1006 1914 1622 1572 1531 1448

1001 60,1 68,1 88,9 123,9 167,2

1008 472 609,5 821,5

2001 77,5 83,3 81,5 129,6 111,3

2002 4774 6699 6500 5943 7878

2005 91,1 94,3 135,6 137,9 148,66

2007 28,9 25 33,3 40,4

2003 150,4 171,1 212,1 259,2

2009 1723 1788 2194 2694 3155

3003 83,00 84,40 102,60 108,50 109,50

3004 152,40 156,90 206,30 208,50 279,60

3001 354,80 450,50 539,60 528,00

3005 173,00 167,50 195,70 182,10 241,00

3006 2273,00 2337,00 2848,00 3430,00 3492,00

3002 49,90 59,70 84,10 100,00 63,80 -

4002 346,00 426,00 432,00 555,00 547,00

4003 2182,00 2495,00 2320,00 2731,00 2668,00

4004 238,00 290,00 294,00 236,00 319,00

4005 114,10 150,30 216,00 353,50 568,10

4009 1589,00 1458,00 1342,00 1167,00 1084,00

4011 481,20 534,70 690,40 627,40

Table 5 PSA values from all subjects from screening to week 7.

Pain interference was assessed using the Brief Pain Inventory (BPI). BPI was used to evaluate pain interference with the following: (a) general activity, (b) mood, (c) walking ability, (d) normal work, (e) relations with other people, (f) sleep, and (g) enjoyment of life. Total score for pain interference was calculated as: (Mean score of non-missing questions) x (7/number of non-missing questions). If four or more questions were missing, the pain interference total score was set to missing.

The pain score progression during administration is depicted in Figures 4B, 5B. It can be seen by comparing with the PSA progression (Fig. 4A, 5A) that during treatment with DI17E6 the improvement pain is correlated with the decrease of the PSA level. The increase of the pain score in Fig. 4B between days 276 and 296 was due to an arthralgia event independent on the drug effect.

Example 8: Decrease of primary tumor and soft tumor lesions (lymph nodes) Bone scans and computed tomography scans (CT) were performed to detect stable/response disease. Subjects with progression in bone or CT scan using PCWG2 and RECIST criteria stopped treatment.

Some patients showed a clinically significant decrease in the primary tumor (prostate) and target lesions (lymph nodes). The CT scan illustrating the shrinkage of both primary and target lesions is presented in Figure 6.

Table 6 : Patient 2001 , 500mg/q2w, partial remission in the primary tumor and in measurable lesions assessed in CT scans. - -

Response was assessed by CT/MRI scan and documented according to Response Evaluation Criteria in Solid Tumors (RECIST) criteria. At baseline, tumor lesions were categorized as measurable or non-measurable based on the ability to measure the lesion accurately in at least one dimension. Disease progression was defined as at least a 20% increase in the sum of the longest diameter of measurable lesions compared to nadir or the appearance of new lesions.

Secondary efficacy measures were assessed at screening and throughout the study (PSA and pain interference total score) or at week 6 (response assessment), and at 4- or 12-week intervals, respectively, thereafter for patients continuing treatment beyond week 6.