METHODS FOR INCREASING EFFICACY OF CD56-BASED THERAPY

FIELD OF THE I NVE NTION

[0001] The field of invention generally relates to increasing the efficacy of the treatment of cancers characterized by the overexpression of human CD56. More specifically, the invention concerns more effective treatment of patients susceptible to or diagnosed with cancers, for example tumors of neuroendocrine origin, small cell lung carcinoma (SCLC), ovarian cancers, and multiple myeloma, in which the cells overexpress CD56 as determined by a CD56 expression assay, e.g., an automated assay.

BACKGROUND OF THE INVENTION

[0002] Cancer is one of the leading causes of death in the developed world, with over one million people diagnosed with cancer and 500,000 deaths per year in the United States alone. Overall it is estimated that more than 1 in 3 people will develop some form of cancer during their lifetime.

[0003] Current treatments for cancers include surgery, radiation therapy, chemotherapy, and combinations thereof. However, current treatments are associated with considerable toxicity risks. In addition, many therapies fail because tumors are resistant to the available treatments.

[0004] Tumor cells of neuroendocrine origin express a protein called CD56 (also known as neural cell adhesion molecule, or NCAM). CD56 is a transmembrane protein of the immunoglobulin superfamily. It belongs to a family of membrane-bound glycoproteins that are involved in calcium-independent cell matrix and homophilic or heterophilic cell- cell interactions.

[0005] The CD56 antigen is expressed on the surface of small cell lung carcinomas

(SCLC), carcinoid tumors and Merkel cell carcinomas (MCC). CD56 is expressed on approximately 56% of ovarian tumors and is also expressed on approximately 70% of multiple myelomas. This expression profile suggests that CD56 represents a promising therapeutic target for a number of cancers, and currently, there is cleary an unmet medical need for improved cancer therapeutics. 6

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SUMMARY OF THE INVENTION

[0006] The present invention is based on the discovery of a dynamic range of expression of CD56 in cancer and the discovery that cancers with increased levels of CD56 expression are more responsive to treatment with therapeutic anti-CD56 antibodies or therapeutic anti-CD56 immunoconjugates. The present invention advantageously permits treatment of patients who have a greater likelihood of responding to treatment by administering therapeutic agents, i.e., anti-CD56 antibodies or anti-CD56 immunoconjugates, to patients who are found to have an increased expression level of CD56. Assays for CD56-expression that are accurate over a dynamic range of CD56 expression levels were necessary in order to accomplish this. Thus, assays using the conditions described herein were developed. The conditions of these assays allow for improved sensitivity and detection of a dynamic range of CD56 expression. The ability to detect the nuances in the expression of CD56 (i.e., a more dynamic range) allows the use of these assay conditions to identify patients and cancers with specific ranges of CD56 expression. These assays can be used for patient stratification, to monitor or determine therapeutic efficacy, or to evaluate the the likelihood of response to the treatment of cancers characterized by the overexpression of CD56.

[0007] In one embodiment, the invention provides methods for increasing the efficacy of cancer therapy with an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate), wherein the method comprises administering an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., an anti-CD56 immunoconjugate) to a subject having cancer, wherein the cancer overexpresses CD56. In some embodiments, overexpression of CD56 has been detected in a cancerous sample from the subject (using e.g., immunohistochemistry (IHC)).

[0008] In one embodiment, the invention provides methods for increasing the efficacy of cancer therapy with an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., an anti-CD56 immunoconjugate), wherein the method comprises administering active agents comprising anti-CD56 antibodies or antigen-binding fragments thereof (e.g., anti-CD56 immunoconjugates) to a population of cancer patients, wherein the patients have cancers that overexpress CD56. The population can comprise, for example, at least two or at least ten patients. In some embodiments, overexpression of CD56 has been detected (e.g., using IHC) in cancerous samples from the patients in the population.

[0009] In one embodiment, the invention provides methods for treating a patient having cancer, wherein the method comprises (a) detecting CD56 expression in a cancerous sample obtained from the subject (e.g., using IHC), (b) detennining a CD56 expression score for the cancerous sample; and (c) administering an active agent comprising an anti- CD56 antibody or antigen-binding fragment thereof (e.g., an anti~CD56 immunoconjugate) to the patient if the score indicates the patient will benefit from administration of the active agent comprising the anti-CD56 antibody or antigen-binding fragment thereof.

[0010] In one embodiment, the invention provides methods for treating a patient having cancer, wherein the method comprises (a) determining a CD56 expression score from a detection of CD56 expression obtained from a cancerous sample obtained from the patient (e.g., using IHC); and (b) administering an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., an anti-CD56 immunoconjugate) to the patient if the score indicates the patient will benefit from administration of the active agent comprising the anti-CD56 antibody or antigen-binding fragment thereof (e.g.,anti- CD56 immunoconjugate).

[0011] In one embodiment, the invention provides methods for treating a patient having cancer, wherein the method comprises (a) determining a CD56 expression score from a detection of CD56 expression obtained from a cancerous sample obtained from the patient (e.g., using IHC); and (b) instructing a healthcare provider to administer an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti- CD56 immunoconjugate) to the patient if the score indicates the patient will benefit from administration of the active agent comprising the anti-CD56 antibody or antigen-binding fragment thereof.

[0012J In one embodiment, the invention provides methods for treating a patient having cancer, wherein the method comprises (a) submitting a cancerours sample taken from a patient having cancer for detennining a CD56 expression score from a detection of CD56 expression (e.g., using IHC); and (b) administering an active agent comprising an anti- CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate) to the patient if the score indicates the patient will benefit from administration of the active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g.. anti-CD56 immunoconjugate).

In one embodiment, the invention provides methods for optimizing a therapeutic regimen with an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g.. anti-CD56 immunoconjugate) for a subject having cancer, wherein the method comprises administering an increased dose of an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate) to a subject having cancer wherein an increased expression of CD56 in a cancerouis sample from the subject has been detected (e.g., using IHC); and administering a decreased dose of an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate) to a subject having cancer wherein a decreased expression of CD56 in a cancerous sample from the subject has been detected (e.g., using IHC).

In one embodiment, the invention provides methods for optimizing a therapeutic regimen with an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate) for a subject having cancer, wherein the method comprises (a) detecting the level of CD56 expression in a cancerous sample obtained from the subject (e.g., using IHC); (b) determining a CD56 expression score for the cancerous sample; and (c) administering an increased dose of an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate) to the subject if the score is weak/low or administering a decreased dose of an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate) to the subject if the score is strong/high.

In one embodiment, the invention provides methods for identifying a cancer as sensitive to treatment with an active agent comprising an anti-CD56 antibody or antigen- binding fragment thereof (e.g., anti-CD56 immunoconjugate), wherein the method comprises (a) detecting the level of CD56 expression in a cancerous sample obtained from the cancer, wherein the detecting comprises the use of a method that distinguishes between staining intensity or staining uniformity in a CD56 expressing cancerous sample as compared to staining intensity or staining uniformity in one or more reference samples (e.g., IHC); (b) determining a CD56 staining intensity or staining uniformity score for the cancerous sample; and (c) comparing the CD56 staining intensity or staining uniformity score detemiined in step (b) to a relative value determined by measuring CD56 protein expression in at least one reference sample, wherein the at least one reference sample is a tissue, cell, or cell pellet sample which is not sensitive to treatment with an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate) and wherein a CD56 staining intensity score for the cancerous sample detennined in step (b) that is higher than the relative value identifies the cancer as being sensitive to treatment with an active agent comprising an anti-CD56 antibody or antigen- binding fragment thereof (e.g., anti-CD56 immunoconjugate). The methods can further comprise administering an active agent comprising an anti-CD56 antibody or antigen- binding fragment thereof (e.g., anti-CD56 immunoconjugate) to the subject from whom the cancerous sample was obtained.

[0016] In one embodiment, the invention provides methods for identifying a cancer likely to respond to an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate), wherein the method comprises (a) contacting a biological sample comprising cells from the cancer with an agent that binds CD56 protein on the cell surface; (b) detecting binding of the agent that binds CD56 protein on the cell surface of the biological sample of (a) (e.g., using IHC); (c) assigning a score to the binding of step (b), wherein the score is assigned based on comparison to one or more reference samples; and (d) comparing the score in step (c) to the score of a reference tissue or cell, wherein a score for the cancer CD56 level that is greater than the score for a normal or weak/low CD56 expressing reference sample or a score for the cancer CD56 level that is equal to or greater than the score for a strong/high CD56 expressing reference sample identifies the cancer as likely to respond to an active agent comprising an ai ti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate). The methods can further comprise administering an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate) to the subject from whom the biological sample was obtained.

[0017] In one embodiment, the invention provides methods for identifying a a subject having a cancer as likely to respond to a low dose treatment regimen using an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate), wherein the method comprises (a) contacting a biological sample comprising cells from the cancer with an agent that binds cell surface CD56 protein; (b) T U 2013/062376

- 6 - detecting binding of the agent to the biological sample of (a) (e.g., using IHC); (c) assigning a score to the binding of step (b). wherein the score is assigned based on comparison to one or more reference samples; and (d) comparing the score in step (c) to the score of a reference tissue or cell, wherein a score for the cancer CD56 level that is greater than the score for a normal or weak/low CD56 expressing reference sample or a score for the cancer CD56 level that is equal to or greater than the score for a strong/high CD56 expressing reference sample identifies the cancer as likely to respond to a low dose of an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g, anti-CD56 immunoconjugate). The methods can further comprise administering an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate) to the subject from whom the biological sample was obtained.

[0018] In one embodiment, the invention provides methods for detecting the expression of cell surface CD56 on cancer cells in a cancerous sample from a subject, wherein the method comprises (a) obtaining a cancerous sample, wherein the sample is formalin-fixed paraffin embedded; (b) contacting the sample with an antibody or antigen-binding fragment thereof that specifically binds cell surface CD56; (c) detecting the binding of the antibody or antigen-binding fragment thereof in (b) to the cell surface CD56 in the cancerous sample using a detection method (e.g., IHC) that can distinguish between staining intensity or staining uniformity in a CD56 expressing sample as compared to staining intensity or staining uniformity in one or more reference samples; and (d) assigning a CD56 expression score to the CD56 after comparing the level of cell surface CD56 staining intensity or staining uniformity in the tumor cancerous sample to one or more reference samples. The methods can further comprise administering an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate) to the subject from whom the cancerous sample was obtained.

[0019] In some embodiments, the CD56 detected is membrane bound cell surface CD56.

[0020] In some embodiments, the detecting is by immunohistochemistry (IHC). The IHC can be calibrated IHC that can distinguish different levels of CD56 expression.

[0021] In some embodiments, the detecting (e.g., IHC) produces a range of staining intensity for samples having weak/low cell surface CD56 expression. moderate/medium/intermediate CD56 cell surface expression, or strong/high CD56 cell surface expression.

[0022] In some embodiments, the detecting (e.g., IHC) distinguishes between staining intensity and staining uniformity in a CD56 expressing cancerous sample or biological sample as compared to a reference sample.

[0023] The cancerous sample or biological sample can have a staining intensity score of at least 2 or 3 for CD56 expression by immunohistochemistry. In some embodiments, the cancerous sample or biological sample has a staining intensity score of 2 or 3 for CD56 expression by immunohistochemistry on a formalin fixed paraffin embedded sample.

[0024] In some embodiments, the cancerous sample or biological sample has a staining uniformity for CD56 expression that is homogenous.

[0025] In some embodiments, the cancerous sample or biological sample has a staining intensity score of 2 or 3 for CD56 and a staining uniformity that is heterogeneous or homogenous.

[0026] In some embodiments, the cancerous or biological sample has a staining intensity of at least 2 hetero for CD56 expression by IHC. In some embodiments, the cancerous or biological sample has a staining intensity of at least 2 homo for CD56 expression by IHC. In some embodiments, the cancerous or biological sample has a staining intensity of at least 3 hetero for CD56 expression by IHC. In some embodiments, the cancerous or biological sample has a staining intensity of at least 3 homo for CD56 expression by IHC.

[0027] In some embodiments, the immunohistochemistry is performed manually. In some embodiments, the immunohistochemistry is performed using an automated system

[0028] In some embodimnets, the CD56 detection (e.g., using IHC) is determined relative to the CD56 level in one or more reference samples. In some embodiments, the reference sample is a positive reference sample or a negative reference sample. In some embodiments, the reference sample comprises cells, cell pellets, or tissue. In some embodiments, the refrence sample is a weak/low CD56-expressing sample, such as a sample comprising Namalwa cells. In some embodiments, the referece sample is a moderate CD56-expressing sample, such as a sample comprising H929 cells or OPM2 cells. In some embodiments, the reference sample is a strong/high CD56-expressing sample, such as a sample comprising H526 cells. [0029] Accordingly, a biological or cancerous sample can be said to overexpress CD56, have strong or high CD56, or elevated CD56 if the CD56 level in the biological or cancerous sample is greater than the CD56 level in a weak or low CD56-expresing reference sample. In addition, a biological or cancerous sample can be said to overexpress CD56, have strong or high CD56, or elevated CD56 if the CD56 level in the biological or cancerous sample is similar to or greater than the CD56 level in a moderate CD-56 expressing reference sample. In addition, a biological or cancerous sample can be said to overexpress CD56, have strong or high CD56, or elevated CD56 if the CD56 level in the biological or cancerous sample is similar to or greater than the CD56 level in a strong or high CD-56 expressing reference sample.

[0030] In some embodiments, the detection (e.g., using IHC) comprises detecting CD56 expression with a detecting antibody or antigen-binding fragment thereof that specifically binds cell surface CD56.

[0031 J In some embodiments, the detecting antibody is 1 B6. In some embodiments, the detecting antibody is an anti-CD56 antibody that is not 1 B6. In some embodiments, the detecting antibody is N901. In some embodiments, the detecting antibody is MRQ-42. In some embodiments, the detecting antibody is 1 B6, N901 , mAb735, BWSCLC1 , BWSCLC2, 123C3. or MRQ-42.

[0032] In some embodiments, the detecting antibody or antigen-binding fragment thereof further comprises a detection reagent selected from the group consisting of: an enzyme, a fluorophore. a radioactive label, and a luminophore.

1003 1 In some embodiments, the detection reagent is selected from the group consisting of: biotin, digoxigenin, fluorescein, tritium, and rhodamine.

[0034] In some embodiments, the cancer overexpresses CD56. In some embodiments, the cancer is selected from the group consisting of small cell lung carcinoma (SCLC), Merkel cell cancer, ovarian cancer, cervical cancer, breast cancer, and multiple myeloma. In some embodiments, the cancer is SCLC. In some embodiments, the cancer is a cancer that is not SCLC. In some embodiments, the SCLC is chemotherapy-nai ^' ve.

(0035] In one embodiment, the invention provides articles of manufacture comprising an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate). a container, and a package insert or label indicating that the active agent comprising the antibody or antigen-binding fragment thereof can be used 2376

- 9 . to treat a cancer characterized by the expression of CD56 at a level of 2 or 3 measured by IHC.

[0036] In one embodiment, the invention provides a combination diagnostic and pharmaceutical kit comprising an anti-CD56 detection antibody or antigen-binding fragment thereof for use in diagnosis and an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof (e.g., anti-CD56 immunoconjugate) for use in therapy. In some embodiments, the detection antibody or antigen-binding fragment is able to detect CD56 expression by IHC. In some embodiments, the kit further comprises a reference sample.

[0 37| In one embodiment, the invention provides a diagnostic kit comprising a detection antibody or antigen-binding fragment thereof that specifically binds to cell surface CD56, a reagent for immunohistochemistry (IHC), and one or more standardized reference samples, wherein the standardized reference samples comprise cells, cell pellets, or formalin fixed paraffin embedded tissue samples, and wherein the one or more standardized referenced samples are from non-CD56 expressing, weak/low-CD56 expressing, moderate CD56-expressing, or strong/high CD56 expressing cells, cell pellets, or tissues.

[0038] In some embodiments, the IHC is calibrated IHC that can distinguish different levels of CD56 expression. In some embodiments, the calibrated IHC produces a range of staining intensity for samples having weak/low cell surface CD56 expression, moderate/intermediate cell surface CD56 expression, or strong/high cell surface CD56 expression.

[0039] In some embodiments, the IHC distinguishes between staining intensity and staining uniformity in a CD56 expressing sample as compared to a reference sample.

[0040] In some embodiments, the IHC is performed on a formalin fixed paraffin embedded sample.

[0041] In some embodiments, the IHC is performed manually. In some embodiments, the IHC is performed using an automated system.

[0042] In some embodiments, the detection antibody is 1B6. In some embodiments, the detection antibody is an anti-CD56 antibody that is not 1B6. In some embodiments, the detecting antibody is N90L In some embodiments, the detecting antibody is N901. In some embodiments, the detecting antibody is MRQ-42. In some embodiments, the detecting antibody is 1B6, N901 , mAb735, BWSCLC 1 , BWSCLC2, 123C3, or MRQ-42.

[0043] In some embodiments, the therapeutic active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof comprises an anti-CD56 antibody or antigen-binding fragment thereof, a linker, and a cytotoxin (i.e., is an imm noconjugate).

[0044] In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, and a dicarboxylic acid based linker. In some embodiments, the linker is selected from the group consisting: N- succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) or N-succinimidyl 4-(2-pyridyldithio)- 2-sulfopentanoate (sulfo-SPP); N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) or N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB); N-succinimidyl 4- (maleimidoniethyl) cyclohexanecaiboxylate (SMCC); N-sulfosuccinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC); N-succinimidyl-4- (iodoacetyl)-aminobenzoate (SIAB); and N-succinimidyl-[(N-maleimidopropionamido)- tetraethyleneglycol] ester (NHS-PEG4-maleimide). In some embodiments, the linker is N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP).

[0045] In some embodiments, the cytotoxin is selected from the group consisting of a maytansinoid, maytansinoid analog, benzodiazepine, taxoid, CC-1065, CC-1065 analog, duocamiycin, duocamiycin analog, calicheamicin, dolastatin, dolastatin analog, auristatin, tomaymycin derivative, leptomycin derivative, cisplatin, and etoposide or a prodrug of the cytotoxin. In some embodiments, the cytotoxin is a maytansinoid. In some embodiments, the maytansinoid is is N(2')-deacetyl-N(2')-(3-niercapto-l -oxopropyl)- maytansine or N(2')-deacetyl-N2-(4-mercapto-4-niethyl-l-oxopeiityl)-maytan sine. In some embodiments, the maytansinoid is is N(2')-deacetyl-N(2')-(3-mercapto-l - oxopropyl)-maytansine (DM1).

[0046] In some embodiments, the active agent comprising the anti-CD56 antibody comprises the antibody huN901 , the linker SPP, and the maytansinoid DM1.

[00471 In some embodiments, the reference sample is a positive reference sample or a negative reference sample or a sample that is known to have strong/high, moderate/medium, or weak/low levels of CD56 expression. In some embodiments, the reference sample comprises cells, cell pellets, or tissue. [0048] In some embodiments, the detection antibody further comprises a detection reagent selected from the group consisting of: an enzyme, a fluorophore, a radioactive label, and a luminophore. In some embodiments, the detection reagent is selected from the group consisting of: biotin, digoxigenin, fluorescein, tritium, and rhodamine.

[0049] In some embodiments, the weak/low CD56-expressing control is Namalwa cells or cell pellets. In some embodiments, the moderate CD56-expressing control is H929 or OPM2 cells or cell pellets. In some embodiments, the strong/high CD56-expressing control is H526 cells or cell pellets.

[0050] In some embodiments, the overexpression of CD56 in the cancer is sufficient to produce an antibodies bound per cell (ABC) value of at least about 50,000. In some embodiments, the overexpression of CD56 in the cancer is sufficient to produce an ABC value of at least about 70,000. In some embodiments, the overexpression of CD56 in the cancer is sufficient to produce an ABC value of at least about 160,000. In some embodiments, the overexpression of CD56 in the cancer is sufficient to produce an ABC value of at least about 270,000.

[0051] Provided herein are also methods of treating patients with SCLC. In some embodiments, the method comprises administering a therapeutically effective amount of an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof to a subject having SCLC, wherein overexpression of CD56 has been detected in the SCLC using IHC. In some embodiments, the SCLC has a staining intensity of at least 2 hetero for CD56 expression. In some embodiments, the SCLC has a staining intensity of at least 2 homo for CD56 expression. In some embodiments, the SCLC has a staining intensity of at least 3 hetero for CD56 expression. In some embodiments, the SCLC has a staining intensity of at least 3 homo for CD56 expression.

[0052] In some embodiments, the SCLC CD56 staining intensity is determined relative to the CD56 staining intensity of a reference sample. In some embodiments, the reference sample comprises tissue, control cells, or control cell pellets. In some embodiments, the reference sample has weak/low CD56-expression (e.g., a reference sample comprising Namalwa cells). In some embodiments, the refrence sample has moderate/medium CD56-expression (e.g., a reference sample comprising OPM2 or H929 cells). In some embodiments, the reference sample has strong/high CD56-expression (e.g.. a reference sample comprising H526 cells). [0053] In some embodiments, the overexpression of CD56 in the SCLC is sufficient to produce an ABC value of at least about 50,000. In some embodiments, the overexpression of CD56 in the SCLC is sufficient to produce an ABC value of at least about 70,000. In some embodiments, the overexpression of CD56 in the SCLC is sufficient to produce an ABC value of at least about 160,000. In some embodiments, the overexpression of CD56 in the SCLC is sufficient to produce an ABC value of at least about 270,000.

[CW54] In some embodiments, the SLCL is chemotherapy-naive SCLC. In some embodiments, the active agent comprises the antibody huN901 , the linker SPP, and the maytansinoid DM 1.

[0055] Also provided herein are active agents for use in methods of increasing the efficacy of cancer therapy. In some embodiments, the active agent comprising an anti- CD56 antibody or antigen-binding fragment thereof, and the active agent is administered to a subject having cancer, wherein the cancer overexpresses CD56. In some embodiments, overexpression of CD56 has been detected in a cancerous sample from the subject.

[0056] In some embodiments, an active agent comprises an anti-CD56 antibody or antigen-binding fragment thereof and is for use in a method of increasing the efficacy of cancer therapy, wherein the active agent is administered to a population of patients having cancers that overexpress CD56. In some embodiments, the population comprises at least 2 patients. In some embodiments, the population comprises at least 10 patients. In some embodiments, overexpression of CD56 has been detected in cancerous samples from the patients in the population.

[0057] In some embodiments, an active agent comprises an anti-CD56 antibody or antigen-binding fragment thereof and is for use in a method of treating cancer in a patient, comprising: (a) detecting CD56 expression in a cancerous sample obtained from the patient; (b) determining a CD56 expression score for the cancerous sample; and (c) administering the active agent to the patient if the score indicates the patient will benefit from administration of the active agent.

[0058] In some embodiments, an active agent comprises an anti-CD56 antibody or antigen-binding fragment thereof and is for use in a method of treating cancer in a patient, comprising: (a) determining a CD56 expression score from a detection of CD56 expression obtained from a cancerous sample obtained from the patient; and (b) administering the active agent to the patient if the score indicates the patient will benefit from administration of the active agent.

[0059] In some embodiments, an active agent comprises an anti-CD56 antibody or antigen-binding fragment thereof and is for use in a method of treating cancer in a patient, comprising: (a) determining a CD56 expression score from a detection of CD56 expression obtained from a cancerous sample obtained from the patient; and (b) instructing a healthcare provider to administer the active agent to the patient if the score indicates the patient will benefit from administration of the active agent.

[0060] In some embodiments, an active agent comprises an anti-CD56 antibody or antigen-binding fragment thereof and is for use in a method of treating cancer in a patient, comprising: (a) submitting a cancerours sample taken from a patient having cancer for determining a CD56 expression score from a detection of CD56 expression; and (b) administering the active agent to the patient if the score indicates the patient will benefit from administration of the active agent.

[0061 j In some embodiments, an active agent comprises an anti-CD56 antibody or antigen-binding fragment thereof and is for use in a method of treating cancer in a patient, comprising the step of optimizing the therapeutic regimen of the active agent comprising: (a) administering an increased dose of the active agent to a subject having cancer wherein an increased expression of CD56 in a cancerours sample from the subject has been detected; and (b) administering a decreased dose of the active agent to a subject having cancer wherein a decreased expression of CD56 in a cancerous sample from the subject has been detected.

[0062] In some embodiments, an active agent comprises an anti-CD56 antibody or antigen-binding fragment thereof and is for use in a method of treating cancer in a patient, comprising the step of optimizing the therapeutic regimen of the active agent comprising: (a) detecting the level of CD56 expression in a cancerous sample obtained from the subject; (b) determining a CD56 expression score for the cancerous sample; and (c) administering an increased dose of the active agent to the subject if the score is weak/low or administering a decreased dose of the active agent to the subject if the score is strong/high. [0063] Also provided herein is an anti-CD56 antibody or antigen-binding fragment thereof for use in a method for diagnosing whether a cancer is sensitive to treatment with an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof, the method comprising: (a) detecting the level of CD56 expression in a cancerous sample obtained from the cancer, wherein the detecting comprises the use of a method that distinguishes between staining intensity or staining uniformity in a CD56-expressing cancerous sample as compared to staining intensity or staining uniformity in one or more reference samples; (b) determining a CD56 staining intensity or staining uniformity score for the cancerous sample; and (c) comparing the CD56 staining intensity or staining uniformity score determined in step (b) to a relative value determined by measuring CD56 protein expression in at least one reference sample, wherein the at least one reference sample is a tissue, cell, or cell pellet sample which is not sensitive to treatment with the active agent and wherein a CD56 staining intensity score for the cancerous sample determined in step (b) that is higher than the relative value identifies the cancer as being sensitive to treatment with the active agent.

[0064] Also provided herein, is an anti-CD56 antibody or antigen-binding fragment thereof for use in a method for identifying a cancer likely to respond to an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof, the method comprising: (a) contacting a biological sample comprising cells from the cancer with an agent that binds CD56 protein on the cell surface; (b) detecting binding of the agent that binds CD56 protein on the cell surface of the biological sample of (a); (c) assigning a score to the binding of step (b), wherein the score is assigned based on comparison to one or more reference samples; and (d) comparing the score in step (c) to the score of a reference tissue or cell, wherein a score for the cancer CD56 level that is greater than the score for a normal or weak/low CD56-expressing reference sample or a score for the cancer CD56 level that is equal to or greater than the score for a strong/high CD56- expressing reference sample identifies the cancer as likely to respond to the active agent.

[0065] Also provided herein, is an anti-CD56 antibody or antigen-binding fragment thereof for use in a method for diagnosing whether a subject having a cancer is likely to respond to a low dose treatment regimen using an active agent comprising an anti-CD56 antibody or antigen-binding fragment thereof, the method comprising: (a) contacting a biological sample comprising cells from the cancer with an agent that binds cell surface P T/US2013/062376

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CD56 protein; (b) detecting binding of the agent to the biological sample of (a); (c) assigning a score to the binding of step (b), wherein the score is assigned based on comparison to one or more reference samples; and (d) comparing the score in step (c) to the score of a reference tissue or cell, wherein a score for the cancer CD56 level that is greater than the score for a normal or weak/low CD56-expressing reference sample or a score for the cancer CD56 level that is equal to or greater than the score for a strong/high CD56-expressing reference sample identifies the cancer as likely to respond to a low dose of the active agent.

[0066] Also provided herein is an anti-CD56 antibody or antigen-binding fragment thereof for use in a method for detecting the expression of cell surface CD56 on cancer cells in a cancerous sample from a subject, the method comprising: (a) obtaining a cancerous sample, wherein the sample is formalin-fixed paraffin embedded; (b) contacting the sample with an antibody or antigen-binding fragment thereof that specifically binds cell surface CD56; (c) detecting the binding of the antibody or antigen- binding fragment thereof in (b) to the cell surface CD56 in the cancerous sample using a detection method that can distinguish between staining intensity or staining uniformity in a CD56-expressing sample as compared to staining intensity or staining uniformity in one or more reference samples; and (d) assigning a CD56 expression score to the CD56 after comparing the level of cell surface CD56 staining intensity or staining uniformity in the tumor cancerous sample to one or more reference samples.

[0067] In some embodiments, the anti-CD56 antibody or antigen-binding fragment thereof is for use according to the methods provided herein, wherein the method further comprises administering an active agent comprising an anti-CD56 antibody or antigen- binding fragment thereof to the subject from whom the cancerous sample or biological sample was obtained.

[0068] In some embodiments, the active agent for use as provided herein comprises an anti-CD56 antibody or antigen-binding fragment thereof conjugated to a cytotoxin.

[0069] In some embodiments, the active agent or anti-CD56 antibody or antigen-binding fragment thereof is for uses as provided herein, wherein the detecting is by immunohistochemistry (IHC). In some embodiments, the IHC is calibrated IHC that can distinguish different levels of CD56 expression. 2013/062376

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[0070] In some embodiments, the active agent or anti-CD56 antibody or antigen-binding fragment thereof is for uses as provided herein, wherein the detecting produces a range of staining intensity for samples having weak/low cell surface CD56 expression, moderate CD56 cell surface expression, or strong/high CD56 cell surface expression.

[0071 J In some embodiments, the active agent or anti-CD56 antibody or antigen-binding fragment thereof is for uses as provided herein, wherein the detecting distinguishes between staining intensity and staining uniformity in a CD56-expressing cancerous sample or biological sample.

[0972] In some embodiments, the active agent or anti-CD56 antibody or antigen-binding fragment thereof is for uses as provided herein, wherein the cancerous sample or biological sample has a staining intensity score of at least 2 or 3 for CD56 expression by immunohistochemistry. In some embodiments, the cancerous sample or biological sample has a staining intensity score of at least 2 or 3 for CD56 expression by immunohistochemistry on a formalin fixed paraffin embedded sample. In some embodiments, the cancerous sample or biological sample has a staining uniformity for CD56 expression that is homogenous. In some embodiments, the cancerous sample or biological sample has a staining intensity score of at least 2 or 3 for CD56 and a staining uniformity that is heterogeneous or homogenous.

[0073| In some embodiments, the active agent or anti-CD56 antibody or antigen-binding fragment thereof is for uses as provided herein, wherein the cancerous sample or biological sample has a staining intensity of at least 2 hetero for CD56 expression by immunohistochemistiy. In some embodiments, the cancerous sample or biological sample has a staining intensity of at least 2 homo for CD56 expresion by immunohistochemistry. In some embodiments, the cancerous sample or biological sample has a staining intensity of at least 3 hetero for CD56 expression by immunohistochemistry. In some embodiments, the cancerous sample or biological sample has a staining intensity of at least 3 homo for CD56 expresion by immunohistochemistry.

[0074] In some embodiments, the active agent or anti-CD56 antibody or antigen-binding fragment thereof is for uses as provided herein, wherein the immunohistochemistiy is performed manually. In some embodiments, the immunohistochemistry is performed using an automated system. [0075] In some embodiments, the active agent or anti-CD56 antibody or antigen-binding fragment thereof is for uses as provided herein, wherein the CD56 detection is determined relative to the CD56 level in one or more reference samples. In some embodiments, the reference sample comprises cells, cell pellets, or tissue. In some embodiments, the reference sample is a weak/low CD56-expressing sample (e.g., a sample comprising Namalwa cells). In some embodiments, the the reference sample is a moderate CD56- expressing sample (e.g., a sample comprising H929 cells or OPM2 cells). In some embodiments, the reference sample is a strong/high CD56-expressing sample (e.g., a sample comprising H526 cellsV

[0076] In some embodiments, the active agent or anti-CD56 antibody or antigen-binding fragment thereof is for uses as provided herein, wherein the detection comprises detecting CD56 expression with a detecting antibody or antigen-binding fragment thereof that specifically binds cell surface CD56. In some embodiments, the detecting antibody is 1 B6. In some embodiments, the detecting antibody is N901. In some embodiments, the detecting antibody is N901. In some embodiments, the detecting antibody is MRQ-42. In some embodiments, the detecting antibody is N901 , mAb735, BWSCLC 1 , BWSCLC2, 123C3, or MRQ-42. In some embodiments, the detecting antibody or antigen-binding fragment thereof further comprises a detection reagent selected from the group consisting of: an enzyme, a fluorophore, a radioactive label, and a luminophore. In some embodiments, the detection reagent is selected from the group consisting of: biotin, digoxigenin, fluorescein, tritium, and rhodamine.

[0077] In some embodiments, the active agent or anti-CD56 antibody or antigen-binding fragment thereof is for uses as provided herein, wherein the cancer is a CD56-positive cancer. In some embodiments, the cancer is selected from the group consisting of small cell lung carcinoma (SCLC), Merkel cell cancer, ovarian cancer, cervical cancer, breast cancer, and multiple myeloma. In some embodiments, the cancer is SCLC. In some embodiments, the SCLC is chemotherapy-na ^' ive.

[0078] In some embodiments, the active agent or anti-CD56 antibody or antigen-binding fragment thereof is for uses as provided herein, wherein the anti-CD56 antibody or the antigen-binding fragment thereof of the active agent is conjugated to the cytotoxin via a linker. In some embodiments, the linker is selected from the group consisting of a cleavable linker, a non-cleavable linker, a hydrophilic linker, and a dicarboxylic acid T U 2013/062376

- 1 8 - based linker. In some embodiments, the linker is selected from the group consisting: N- succinimidyl 4-(2-pyridyldithio)pentanoate (SPP) or N-succinimidyl 4-(2-pyridyldithio)- 2-sulfopentanoate (sulfo-SPP); N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) or N-succinimidyl 4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB); N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC); N-sulfosuccinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (sulfoSMCC); N-succinimidyl-4- (iodoacetyl)-aminobenzoate (SIAB); and N-succinimidyl-[(N-maleimidopropionamido)- tetraethyleneglycol] ester (NHS-PEG4-maleimide). In some embodiments, the linker is N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP).

007 | In some embodiments, the active agent or anti-CD56 antibody or antigen-binding fragment thereof is for uses as provided herein, wherein the cytotoxin is selected from the group consisting of a maytansinoid, maytansinoid analog, benzodiazepine, taxoid, CC- 1065, CC-1065 analog, duocarmycin, duocarmycin analog, calicheamicin, dolastatin, dolastatin analog, auristatin, tomaymycin derivative, leptomycin derivative, cisplatin, and etoposide or a prodrug of the cytotoxin. in some embodiments, the cytotoxin is a maytansinoid. In some embodiments, the maytansinoid is N(2')-deacetyl-N(2')-(3- mercapto-1 -oxopropyl)-maytansine or N(2')-deacetyl-N2-(4-mercapto-4-methyl-l - oxopentyl)-maytansine. In some embodiments, the maytansinoid is N(2')-deacetyl-N(2')- (3-mercapto-l-oxopropyl)-maytansine (DM1 ).

[0080] In some embodiments, the active agent or anti-CD56 antibody or antigen-binding fragment thereof is for uses as provided herein, wherein the active agent comprises the antibody huN901 , the linker SPP, and the maytansinoid DM1.

[0081] Also provided here an active agents comprising an anti-CD56 antibody or antigen- binding fragment thereof for use in methods of treating a patient with SCLC, the methos comprising administering a therapeutically effective amount of the active agent to a subject having SCLC, wherein overexpression of CD56 has been detected in the SCLC using IHC. In some embodiments, the SCLC has a staining intensity of at least 2 hetero for CD56 expression. In some embodiments, the SCLC has a staining intensity of at least 2 homo for CD56 expression. In some embodiments, the SCLC has a staining intensity of at least 3 hetero for CD56 expression. In some embodiments, the SCLC has a staining intensity of at least 3 homo for CD56 expression. 2013/062376

- 19 -

[0082] In. some embodiments, the SCLC CDS 6 staining intensity is determined relative to the CD56 staining intensity of a reference sample, m some embodiments, the reference sample comprises tissue, control cells, or control ceil pellets. In some embodiments, the reference sample has weak/low CD56-expression (e.g., a reference sample comprising Namaiwa ceils), in. some embodiments, the refrence sample has moderate CD56- exprcssion (e.g., a reference sample comprising OPM 2 or H929 ceils). In some embodiments, the reference sample has strong/high CD56-expression (e.g. a reference sample comprising 1:1526 cells), in some embodiments, the the SLCL is chemotherap - naive SCLC.

[0083] In some embodiments, the overexpression of CD56 in the SCLC is sufficient to produce an ABC vah.se of at least about. 50,000. In some embodiments, the overexpression of€D56 in the SCLC is sufficient to produce an ABC value of al least, about 70.000. In some embodiments, the overexpression of CDS 6 in the SCLC is sufficient, io produce an. ABC value of at least about 160.000. In some embodiments, the overexpression of CDS in the SCLC" is sufficient to produce an ABC value of at least about 270.000.

[0084] In some embodiments, the active agent comprises the antibody huN90 L the linker

SPP, and the maytansmoid DM1 .

BRIEF DESCRIPTIONS OF THE. DRAWINGS

[0085] Figure 1. SCLC core scores for Biomax TMA LC I 0010 at three concentrations.

[0086| Figure 2. Distribution of staining scores from the manual method versus the current automated staining method for CD56.

[0087] Figure 3. SCLC in vivo studies: IMGN901 in combination with cisplatin plus etoposide.

[0088] Figure 4. Maximum percent change in sum of target lections in evaluable patients

(N=30) at Phase 1.

[0089] Figures 5A-D. Iimnunoreactivity of anti-CD56 clone 1B6 with FFPE H526 cells

(A), OPM2 cells (B), Namaiwa cells (C), and H929 cells (D). DETAILED DESCRIPTION OF THE INVENTION

[0090] The present invention provides methods for treating cancers and for increasing the efficacy of or the likelihood of response to the treatment for diseases characterized by the overexpression of CD56. The present invention is based on the discovery of methods of detecting a dynamic range of expression of CD56 in cancer as compared to normal tissue and the discovery that cells with increased levels of CD56 expression are more responsive to treatment with anti-CD56 antibodies or anti-CD56 immunoconjugates. Kits comprising one or more reagents useful for practicing the methods of the invention are further provided.

I. Definitions

[0091] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0092] As used interchangeably herein. "CD56" or "NCAM-1 " refers to neural cell adhesion molecule 1 , which is a multifunctional member of the Ig superfamily. It belongs to a family of membrane-bound glycoproteins that are involved in calcium-independent cell matrix and homophilic or heterophilic cell-cell interactions.

[0093] The terms "elevated expression," "increased expression" or "overexpression' of

CD56 refer to a sample which contains strong/high levels of CD56 expression as compared to a reference sample or control. The CD56 can be elevated, increased, or overexpressed as compared to a control value (e.g., expression level in a biological sample, tissue, or cell from a subject without cancer, a sample or cancer known to express no or weak/low CD56, a normal sample, or a cancer that does not have elevated CD56 values). For example, a sample with increased expression can contain an increase of at least 2-fold, at least 3-fold, or at least 5-fold relative to a control values.

[0094] In one example, the CD56 expression is measured by IHC and given a staining intensity score or a staining uniformity score by comparison to calibrated controls exhibiting defined scores (e.g., an intensity score of 3 is given to the test sample if the intensity is comparable to the level 3 calibrated control or an intensity of 2 is given to the test sample if the intensity is comparable to the level 2 calibrated control). For example, a score of 2 or 3 (3+), by immunohistochemistry indicates an increased expression of CD56. In one example, the CD56 expression is measured by IHC and compared to a reference sample with a known CD56 IHC intensity score of 1 , 2, or 3. If the CD56 IHC intentsity score for the test sample is similar to the CD56 IHC intensity score of a reference sample that has a score of 2 or 3, then the test sample is considered to have an elevated level of CD56. Similarly, if the CD56 IHC intensity score for the test sample is greater than the CD56 IHC intensity score of a reference sample that has a score of 1 , then the test sample is considered to have an elevated level of CD56.

[0095] A staining uniformity that is heterogeneous or homogeneous is also indicative of increased CD56 expression. The staining intensity and staining uniformity scores can be used alone or in combination (e.g., 2 homo, 2 hetero, 3 homo, 3 hetero, etc.). In one example, the CD56 intensity and uniformity is measured by iHC and compared to a reference sample with a known CD56 IHC intensiiy and uniformity score of greater than 2 homogeneous, greater than 2 heterogeneous, greater than 3 homogeneous or greater than 3 homogeneous. If the CD56 IHC intensity and uniformity score for the test sample is similar to the CD56 IHC intensity and uniformity score of a reference sample that has a score of greater than 2 homogeneous, greater than 2 heterogeneous, greater than 3 homogeneous or greater than 3 homogeneous, then the test sample is considered to have an elevated level of CD56. In another example, an increase in CD56 expression can be determined by detection of an increase of at least 2-fold, at least 3-fold, or at least 5-fold relative to control values (e.g., expression level in a biological sample, tissue, or cell from a subject without cancer or with a cancer that does not have elevated CD56 values).

[0096] A "reference sample" can be used to correlate and compare the results obtained in the methods of the invention from a test sample. Reference samples can be cells (e.g., cell lines, cell pellets) or tissue. The CD56 levels in the "reference sample" can be an absolute or relative amount, a range of amount, a minimum and/or maximum amount, a mean amount, and/or a median amount of CD56. The "reference sample" can include a prior sample or baseline sample from the same patient, a normal reference with a known level of CD56 expression, or a reference from a relevant patient population with a known level of CD56 expression. The methods of the invention involve a comparison between expression levels of CD56 in a test sample, e.g., a cancerous sample or a biological sample, and a "reference value." In some embodiments, the reference value is the expression level of the CD56 in a reference sample. A reference value can be a predetermined value and can also be determined from reference samples (e.g., control biological samples) tested in parallel with the test samples. A reference value can be a single cut-off value, such as a median or mean or a range of values, such as a confidence interval. Reference values can be established for various subgroups of individuals, such as individuals predisposed to cancer, individuals having early or late stage cancer, male and/or female individuals, or individuals undergoing cancer therapy. Examples of normal reference samples or values and positive reference samples or values are described herein.

[0097] In some embodiments, the reference sample is a sample from a healthy tissue, in particular a corresponding tissue which is not affected by cancer, or a tissue (normal or tumor) with a known level of CD56 expression. These types of reference samples are referred to as negative control samples or "normal" reference samples. In other embodiments, the reference sample is a sample from a tumor or healthy tissue that expresses detectable CD56. These types of reference samples are referred to as positive control or positive reference samples. Both normal and positive control or reference samples can also be used as a comparative indicator for the type (hetero versus homo) and/or degree (0, 1 , 2, 3) of staining intensity, which correlates with the level of CD56 expression. Positive control comparative samples are also referred to as calibrated reference samples. Positive controls include, for example, H526 cells, H929 cells, nerves in human normal esophagus, muscle in human normal esophagus, nerves in human normal pancreas, and/or islets in human normal pancreas. As shown in the Examples, weak/low or non-CD56 references include, for example, Namalwa (Burkitt's lymphoma) cell pellets, and epithelial cells in human normal esophagus and acinar cells in human normal pancreas. Appropriate positive and negative reference levels of CD56 for a particular cancer can be determined by measuring levels of CD56 in one or more appropriate subjects, and such reference levels may be tailored to specific populations of subjects (e.g., a reference level may be age-matched so that comparisons may be made between CD56 levels in samples from subjects of a certain age and reference levels for a particular disease state, phenotype, or lack thereof in a certain age group). Such reference levels may also be tailored to specific techniques that are used to measure levels of CD56 in biological samples where the levels of CD56 may differ based on the specific technique that is used.

[0098] As used herein, "immunohistochemistry" refers to histochemical and immunologic methods used to analyze, for example, cells or tissues. Thus, the terms P T/US2013/062376

"iinniunohistochemistry," "immunocytochemistry," and "immunocheniistry" are used interchangeably.

[0099] The term "primary antibody" herein refers to an antibody that binds specifically to the target protein antigen in a sample. A primary antibody is generally the first antibody used in an immunohistochemical (IHC) procedure. In one embodiment, the primary antibody is the only antibody used in an IHC procedure. The term "secondary antibody" herein refers to an antibody that binds specifically to a primary antibody, thereby forming a bridge or link between the primary antibody and a subsequent reagent, if any. The secondary antibody is generally the second antibody used in an immunohistochemical procedure.

[00100] A "sample" of the present invention is of biological origin, in specific embodiments, such as from eukaryotic organisms. In preferred embodiments, the sample is a human sample, but animal samples may also be used in the practice of the invention. Non-limiting sources of a sample for use in the present invention include solid tissue, biopsy aspirates, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tumors, organs, cell cultures and/or cell culture constituents, for example. A "cancerous sample" is a sample that contains a cancerous cell. The present invention is useful for solid tissue samples where the amount of available material is small. The method can be used to examine an aspect of expression of CD56 or a state of a sample, including, but not limited to, comparing different types of cells or tissues, comparing different developmental stages, and detecting or determining the presence and/or type of disease or abnormality.

[00101] For the puiposes herein, a "section" of a tissue sample refers to a single part or piece of a tissue sample, e.g., a thin slice of tissue or cells cut from a tissue sample. It is understood that multiple sections of tissue samples may be taken and subjected to analysis according to the present invention. In some cases, the selected portion or section of tissue comprises a homogeneous population of cells. In other cases, the selected portion comprises a region of tissue, e.g., the lumen as a non-limiting example. The selected portion can be as small as one cell or two cells, or could represent many thousands of cells, for example.

[00102] By "correlate" or "correlating" is meant comparing, in any way, the performance and/or results of a first analysis with the performance and/or results of a second analysis. For example, one may use the results of a first analysis in carrying out the second analysis and/or one may use the results of a first analysis to determine whether a second analysis should be performed and/or one may compare the results of a first analysis with the results of a second analysis. In one embodiment, increased expression of CD56 correlates with increased likelihood of effectiveness of a CD56-targeting anti-cancer therapy.

[00103] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab'. F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl , IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[00104] A "blocking" antibody or an "antagonist" antibody is one which inhibits or reduces biological activity of the antigen it binds, such as CD56. In a certain embodiment blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. Desirably, the biological activity is reduced by 10%, 20%. 30%, 50%. 70%, 80%. 90%, 95%, or even 100%.

[00105] The term "anti-CD56 antibody" or "an antibody that binds to CD56" refers to an antibody that is capable of binding CD56 with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting CD56.

[00106] The extent of binding of an anti-CD56 antibody to an unrelated, non-CD56 protein can be less than about 10%) of the binding of the antibody to CD56 as measured. 13 062376

- 25 - e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD56 has a dissociation constant (Kd) of <1 μΜ, <100 nM, <10 nM, <1 nM, or <0.1 nM.

[00107] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.

[00108] A "monoclonal antibody" refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[00109] The term "humanized antibody" refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986. Nature. 321 :522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239: 1534-1536). In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas ail or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc). typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5.225,539.

[001 lO ^' j Humanizaiion, resurfacing or engineering of antibodies of the present invention can be performed using any known method, such as but not limited to those described in. Winter (Jones ei ah, Nature 321 :522 ( 1986); Riechrnann et aL Nature 332:323 (1988 ); Verhoeyen et ah. Science 239: 1 534 ( 1988)), Sims et ah, J. Immunol . 151 : 2296 ( 1993): Chothia and Lesk, .1. Mol. Biol 196:901 (1987), Carter et ah, Proc. Nail. Acad. Sci. U.S.A. 89:4285 { 1992}; Presta et ah, .1. Immunol. 1 51 :2623 (1993). U.S. Pat. Nos. 5,639,641 , 5,723323; 5,976,862; 5,824,514; 5,817.483: 5,814,476; 5,763,192; 5,723323: 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5.530, 101 : 5,585.089: 5,225.539: 4,816,567; PC17: US98/1 6280; US96/18978; US91/09630; US91/05939; US94/01234; GB89/01334; GB91 /01 1 34; GB92/01 755; WO90/.14443; WO90/M424; WO90/14430; EP 229246; 7,557, 189: 7.538, 195; and 7,342, 1 10, each of which is entirely incorporated herein by reference, including the references cited therein. In addition, resurfacing methods have been described, for example, in Roguska et ah, Proc. Natl. Acad. Sci,, USA, 91(3):969-973 (1994 ? and Roguska et ah, Protein Eng. 9(10):895-904 (1996), which are incorporated in their entirety herein by reference.

[00111] A "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the F s and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs; (1) an approach based on cross-species sequence variability (i.e., abat et al. Sequences of Proteins of Immunological Interest, (5th ed,, 1 91. National Institutes of Health, Bethesda Md.)); and (2) an approach based on crysta!lographic studies of antigen-antibody complexes (Al-lazikani et al ( 1 97) J. Moke, Biol. 273:927- T U 2013/062376

- 27 -

948)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[00112] The abat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-1 13 of the heavy chain) (e.g. Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[00113] The amino acid position numbering as in Kabat, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g.. residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901 -917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. Loop Kabat AbM ChoLhia

LI L24-L34 L24-L34 L24-L34

L2 L50-L 6 L50-L56 L50-L56

L3 L89-L97 L89-L97 L89-L97

H I H31 -H35B H26-H35B H26-H32..34

(Kabat Numbering)

H I H31-H35 H26-H35 II26-II32

(C othia Numbering)

112 H50-H65 H50-H58 II52-H56

H3 H95-H102 H95-H 102 H95-H 102

[00114] The term "human antibody" means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the an. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.

[00115] The term "chimeric antibodies" refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.

[00116] The term "epitope" or "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation.

[00117] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" 2013/062376

- 29 - refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high- affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.

[00118] "Or better" when used herein to refer to binding affinity refers to a stronger binding between a molecule and its binding partner. "Or better" when used herein refers to a stronger binding, represented by a smaller numerical Kd value. For example, an antibody which has an affinity for an antigen of "0.6 i M or better", the antibody's affinity for the antigen is <0.6 nM, i.e., 0.59 nM. 0.58 nM, 0.57 nM etc. or any value less than 0.6 nM.

[00119] By "specifically binds," it is generally meant that an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody "A" may be deemed to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind to epitope "C" with a higher specificity than it has for related epitope "D."

[00120] The phrase "substantially similar," or "substantially the same", as used herein, denotes a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody of the invention and the other associated with a reference/comparator antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristics measured by said values (e.g., Kd values). The difference between said two values is less than about 50%, less 6

- 30 - than about 40%, less than about 30%. less than about 20%, or less than about 10% as a function of the value for the reference/comparator antibody.

[00121] A polypeptide, antibody, polynucleotide, vector, cell, or composition which is "isolated" is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cell or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

[00122] As used herein, "substantially pure" refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[00123] The term "immunoconjugate" or "conjugate" as used herein refers to a compound or a derivative thereof that is linked to a cell binding agent (i.e., an anti-CD56 antibody or fragment thereof) and is defined by a generic formula: C-L-A. wherein C = cytotoxin, L = linker, and A = ceil binding agent or anti-CD56 antibody or antibody fragment. Immunoconjugates can also be defined by the generic formula in reverse order: A-L-C.

[00124] A "linker" is any chemical moiety that is capable of linking a compound, usually a drug, such as a maytansinoid, to a cell-binding agent such as an anti CD56 antibody or a fragment thereof in a stable, covalent manner. Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase- induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Linkers also include charged linkers, and hydrophilic forms thereof as described herein and know in the art.

[00125] The terms "cancer," "cancerous," "malignant," and "malignancies" refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of "cancer," "malignant" or "tumorigenic" diseases include, for example, CD56 positive cancers or cancers of unknown primary (CUP). Non-limiting examples include neuroendocrine tumors, small cell lung carcinoma (SCLC), Merkel cell cancer, ovarian cancers, cervical cancer, breast cancer, and multiple myeloma. CD56 positive cancers include cancers that have been identified in the literature and/or are known in the art to express CD56 on the cell surface as well as those cancers that have been shown to express CD56, for example, using IHC in the present application.

[00126] CD56 positive cancers also include cancers that have been previously treated with anti-cancer agents such as chemotherapeutic agents, as well as cancers that have not been previously treated with anti-cancer agents. "Chemotherapy-naive" refers to patients, cancers, cancer cells, or turmors that have not previously been treated with chemotherapy.

[00127] "Tumor" and "neoplasm" refer to any mass that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including precancerous lesions.

[00128] The terms "cancer cell," "tumor cell," and grammatical equivalents refer to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term "tumor cell" will be modified by the term "non-tumorigenic" when referring solely to those tumor cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.

[00129] The term "subject" refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[00130] The term "population" refers to a group of at least two animals (e.g., mammals).

A population can, for example, comprise at least 2, at least 3, at least 5, at least 10, at least 15, at least 25, at least 50, at least 100, or at least 250 subjects or patients.

[00131] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[00132] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulation can be sterile. [00133] An "effective amount" of an antibody as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose.

[00134] The term "therapeutically effective amount" refers to an amount of an antibody or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent or stop) cancer cell infiltration into peripheral organs; inliibit (i.e., slow to some extent or stop) tumor metastasis; inliibit, 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 "treating." To the extent the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic.

[00135] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[00136] The term "respond favorably" generally refers to causing a beneficial state in a subject. With respect to cancer treatment, the term refers to providing a therapeutic effect on the subject. Positive therapeutic effects in cancer can be measured in a number of ways (See, W.A. Weber, J. Nucl. Med. 50T S-10S (2009)). For example, tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging techniques can all be used to assess therapeutic efficacy of an anti-cancer therapeutic. With respect to tumor growth inhibition, according to NCI standards, a T/C < 42% is the minimum level of anti-tumor activity. A T/C <10% is considered a high anti-tumor activity level, with T/C (%) = Median tumor volume of the treated / Median tumor volume of the control x 100. A favorable response can be assessed, for example, by increased progression free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), or, in some cases, stable disease (SD), or a decrease in progressive disease (PD), or any combination thereof. These can be assessed using, for example, the Response Evaluation Criteria for Solid Tumors (RECIST) 1.1 guidelines (see e.g. , Eisenhauer et al., European Journal of Cancer 45:228- 6

- 33 -

247 (2009)); see also ECIST Compared available at http://www.recist.com/recist- comparative/index.html (visited September 24. 2012)).

[00137] PFS, DFS, and OS can be measured by standards set by the National Cancel- Institute and the U.S. Food and Drug Administration for the approval, of new drags. See

Johnson et ah (2003) J. C!in. Oncol. 21(7): 1404-141 1. PFS.. DFS, and OS can also be assessed using RECIST 1.1 standards.

(00138] "Progression free survival" (PFS) refers to the time from enrollment to disease progression or death. PFS is generally measured using the Kaplan-Meier method, and

RECIST 1 .1 standards. Generally, progression free survival refers to the situation wherein a patient remains alive, without the cancer getting worse.

100139] "Time to Tumor Progression" (TTP) is defined as the time from enrollment to disease progression. TTP is generally measured using the RECIST ^' 1 .1 criteria.

[00140] A "complete response" or "complete remission" or "CR" indicates the disappearance of all signs of tumor or cancer in response to treatment. This does not always mean the cancer has been cured.

(001. 1 J A "partial response" or "PR" refers to a decrease in the size or volume of one or more tumors or iesions, or in the extent of cancer in the body, in response to treatment.

[00142] "Stable disease" refers to disease without progression or relapse. In stable disease there is neither sufficient tumor shrinkage to quality for partial response nor sufficient tumor increase to qualify as progressive disease.

[00143] "Progressive disease" refers to the appearance of one more new lesions or tumors and/or the unequivocal progression of existing non-target, lesions. Progressive disease can also rever to a tumor growth of more than 20 percent since treatment began, either due to an increases in mass or in spread of the tumor.

[00144] "Disease free survival" (DFS) refers to the length of time dining and after treatment that, the patient remains free of disease.

[001451 "Overall Survival" (OS) refers to the time from patient enrollment to death or censored at the date last known alive. OS includes a prolongation in life expectancy as compared to naive or untreated individuals or patients. Overall survival refers to the situation wherein a. patient remains alive for a defined period of time, such as one year. five years, etc., e.g., from the time of diagnosis or treatment, [00146] The word "label" when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. The label can be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can catalyze chemical alteration of a substrate compound or composition which is detectable.

[00147] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkyating agents, antimetabolites, spindle poison plant alkaloids, cytoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in "targeted therapy" and conventional chemotherapy.

[00148] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already diagnosed with or suspected of having the disorder. In certain embodiments, a subject is successfully "treated" for cancer according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity, tumori genie frequency, or tumorigenic capacity, of a tumor; reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non-tumorigenic state; or some combination of effects.

[00149] Prophylactic or preventative measures refer to therapeutic measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of prophylactic or preventative measures include those prone to have the disorder and those in whom the disorder is to be prevented.

[00150] As used herein, the term "healthcare provider" refers to individuals or institutions which directly interact and administer to living subjects, e.g., human patients. Non- limiting examples of healthcare providers include doctors, nurses, technicians, therapist, pharmacists, counselors, alternative medicine practitioners, medical facilities, doctor's offices, hospitals, emergency rooms, clinics, urgent care centers, alternative medicine clinics/facilities, and any other entity providing general and/or specialized treatment, assessment, maintenance, therapy, medication, and/or advice relating to all, or any portion of, a patient's state of health, including but not limited to general medical, specialized medical, surgical, and/or any other type of treatment, assessment, maintenance, therapy, medication and/or advice.

I J In some aspects, a healthcare provider can administer or instruct another healthcare provider to administer a therapy to treat a cancer. "Administration" of a therapy, as used herein, includes prescribing a therapy to a subject as well as delivering, applying, or giving the therapy to a subject. A healthcare provider can implement or instinct another healthcare provider or patient to perform the following actions: obtain a sample, process a sample, submit a sample, receive a sample, transfer a sample, analyze or measure a sample, quantify a sample, provide the results obtained after analyzing/measuring/quantifying a sample, receive the results obtained after analyzing/measuring/quantifying a sample, compare/score the results obtained after analyzing/measuring/quantifying one or more samples, provide the comparison/score from one or more samples, obtain the comparison/score from one or more samples, administer a therapy or therapeutic agent (e.g. , a CD56 binding agent), commence the administration of a therapy, cease the administration of a therapy, continue the administration of a therapy, temporarily interrupt the administration of a therapy, increase the amount of an administered therapeutic agent, decrease the amount of an administered therapeutic agent, continue the administration of an amount of a therapeutic agent, increase the frequency of administration of a therapeutic agent, decrease the frequency of administration of a therapeutic agent, maintain the same dosing frequency on a therapeutic agent, replace a therapy or therapeutic agent by at least another therapy or therapeutic agent, combine a therapy or therapeutic agent with at least another therapy or additional therapeutic agent. These actions can be performed by a healthcare provider automatically using a computer-implemented method (e.g. , via a web service or standalone computer system). [00152] As used in the present disclosure and claims, the singular forms "a." "an," and "the" include plural forms unless the context clearly dictates otherwise.

[00153] It is understood that wherever embodiments are described herein with the language "comprising," otherwise analogous embodiments described in terms of "consisting of and/or "consisting essentially of are also provided.

[00154] The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both "A and B," "A or B," "A," and "B." Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

I f . Biological samples

[00155] Biological samples are often fixed with a fixative. Aldehyde fixatives such as formalin (fonnaldehvde) and glutaraldehyde are typically used. Samples fixed using other fixation techniques such as alcohol immersion (Battifora and Kopinski, J. Histochem. Cytochem. (1986) 34: 1095) are also suitable. The samples used may also be embedded in paraffin. In one embodiment, the samples are both formalin-fixed and paraffin-embedded (FFPE). In another embodiment, the FFPE block is hematoxylin and eosin stained prior to selecting one or more portions for analysis in order to select specific area(s) for the FFPE core sample. Alternatively, biological samples are flash frozen (performed by immersing the sample in isopentane/dry ice or isopentane/liquid nitrogen) and embedded in optimal cutting temperature (OCT) compound at subzero temperature. Flash frozen samples require no fixation prior to embedding in OCT compound. Methods of preparing tissue blocks from these particulate specimens have been used in previous IHC studies of various prognostic factors, and/or is well known to those of skill in the art (see, for example, Abbondanzo et al., Am J Clin Pathol. 1990 May;93(5):698-702; Allred et al., Arch Surg. 1990 Jan;125(l): 107-13).

[00156] Briefly, any intact organ or tissue may be cut into fairly small pieces and either incubated in various fixatives (e.g., formalin, alcohol, etc.) for varying periods of time until the tissue is "fixed" or flash frozen and embedded in OCT compound. The samples may be virtually any intact tissue surgically removed from the body. The samples may be cut into reasonably small piece(s) that, fit on the equipment routinely used in 2013/062376

- 37 - histopathology laboratories. The size of the cut pieces typically ranges from a few millimeters to a few centimeters.

Ι Π . Detection Antibody Conjugates

[00157] The present invention further provides antibodies that bind to CD56, generally of the monoclonal type, that are linked to at least one agent to form a detection antibody conjugate. In order to increase the efficacy of antibody molecules as diagnostics, it is conventional to link or covalently bind or complex at least one desired molecule or moiety. Such a molecule or moiety may be, but is not limited to, at least one reporter molecule. A reporter molecule is defined as any moiety that may be detected using an assay. Non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or ligands, such as biotin.

[00158] Any cell binding agent (e.g., an antibody or polypeptide) of sufficient selectivity, specificity or affinity may be employed as the basis for detection of the CD56 polypeptide. Such properties may be evaluated using conventional immunological screening methodology known to those of skill in the art. Sites for binding to biological active molecules in the antibody molecule, in addition to the canonical antigen binding sites, include sites that reside in the variable domain that can bind the antigen. In addition, the variable domain is involved in antibody self-binding (Kang et al., 1988) and contains epitopes (idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

[00159] Certain examples of protein binding (e.g., antibody) conjugates are those conjugates in which the protein binding agent (e.g., antibody) is linked to a detectable label. "Detectable labels" are compounds and/or elements that can be detected due to their specific functional properties, and/or chemical characteristics, the use of which allows the protein binding agent (e.g., antibody) to which they are attached to be detected, and/or further quantified if desired.

[00160] Many appropriate imaging agents are known in the art, as are methods for their attachment to antibodies (see, e.g., U.S. Pat. Nos. 5,021 ,236; 4,938,948; and 4,472,509, each incorporated herein by reference). The imaging moieties used can be paramagnetic 13 062376

- 38 - ions; radioactive isotopes; fluorochromes; NMR-detectable substances; and/or X-ray imaging, for example.

[00161] Exemplary fluorescent labels contemplated for use as as protein binding (e.g., antibody) conjugates include Alexa 350, Alexa 430, Alexa 488, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY- TRX, Cascade Blue, Cy3, Cy5,6-FAM, Dylight 488, Fluorescein Isothiocyanate (FITC), Green fluorescent protein (GFP), HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phycoerytlirin, REG, Rhodamine Green, Rhodamine Red, tetramethyl rhodamine (TMR), Renographin, OX, TAMRA, TET, Tetramethylrhodaniine, Texas Red, and derivatives of these labels (i.e., halogenated analogues, modified with isothiocynate or other linker for conjugating, etc.), for example. An exemplary radiolabel is tritium.

£00162] Protein binding (e.g., antibody) detection conjugates contemplated in the present invention include those for use in vitro, where the antibody is linked to a secondary binding ligand and/or to an enzyme (an enzyme tag) that will generate a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase and/or glucose oxidase. In some embodiments, secondary binding ligands are biotin and/or avidin and streptavidin compounds. The use of such labels is well known to those of skill in the art and are described, for example, in U.S. Pat. Nos. 3,817,837; 3,850.752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241 ; each incorporated herein by reference.

[00163] Molecules containing azide groups may also be used to form covalent bonds to proteins through reactive nitrene intermediates that are generated by low intensity ultraviolet light (Potter & Haley, 1983). In particular, 2- and 8-azido analogues of purine nucleotides have been used as site-directed photoprobes to identify nucleotide binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). The 2- and 8-azido nucleotides have also been used to map nucleotide binding domains of purified proteins ( hatoon et al., 1989; King et al„ 1989; and Dholakia et al., 1989) and can be used as antibody binding agents.

[00164] Several methods are known in the art for the attachment or conjugation of an antibody to its conjugate moiety. Some attachment methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a 13 062376

- 39 - diethylenetriaminepentaacetic acid anhydride (DTP A); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/or tetrachloro-3a-6a-diphenylglycouril-3 attached to the binding agent (e.g., antibody) (U.S. Pat. Nos. 4,472,509 and 4,938,948, each incoiporated herein by reference). Monoclonal antibodies may also be reacted with an enzyme in the presence of a coupling agent such as glutaraldehyde or periodate. Protein binding (e.g., antibody) conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with an isothiocyanate. In U.S. Pat. No. 4,938,948, imaging of breast tumors, for example, is achieved using monoclonal antibodies, and the detectable imaging moieties are bound to the antibody using linkers such as methyl-p- hydroxybenzimidate or N-succinimidyi-3-(4-hydroxyphenyl)propionate.

[00165] In other embodiments, derivatization of immunoglobulins by selectively introducing sulfhydryl groups in the Fc region of an immunoglobulin using reaction conditions that do not alter the antibody combining site are contemplated. Antibody conjugates produced according to this methodology are disclosed to exhibit improved longevity, specificity and sensitivity (U.S. Pat. No. 5,196,066, incoiporated herein by reference). Site-specific attachment of effector or reporter molecules, wherein the reporter or effector molecule is conjugated to a carbohydrate residue in the Fc region, have also been disclosed in the literature (O'Shannessy et al., 1987).

[00166] In other embodiments of the invention, immunoglobulins are radiolabeled with nuclides such as tritium. In additional embodiments, nanogold particles (such as sizes from about 0.5 nm-40 nm) and/or Quantum Dots (Hayward, Calif.) are employed.

IV. Enzymes and Substrates (Chromagens)

[00167] The use of substrates and indicators is contemplated for detection of CD56, such as the exemplary embodiments provided below, for example.

[00168] Horseradish peroxidase (HRP) is an enzyme that first forms a complex with hydrogen peroxide and then causes it to decompose, resulting in water and atomic oxygen. Like many other enzymes, HRP and some HRP-like activities can be inhibited by excess substrate. The complex formed between HRP and excess hydrogen peroxide is catalytically inactive and in the absence of an electron donor (e.g., chromogenic substance) is reversibly inhibited. It is the excess hydrogen peroxide and the absence of an electron donor that brings about quenching of endogenous HRP activities. 2013/062376

- 40 -

[00169] When used in assays systems, HRP can also be used to convert a defined substrate into its activated chromagen, thus causing a color change. The HRP enzyme may be conjugated to an antibody, protein, peptide, polymer, or other molecule by a number of methods. Such methods are known in the art. Adding glutaraldehyde to a solution containing an admixture of HRP and antibody will result in more antibody molecules being conjugated to each other than to the enzyme. In the two-step procedure, HRP reacts with the bifunctional reagents first. In the second stage, only activated HRP is admixed with the antibody, resulting in much more efficient labelling and no polymerization. HRP is also conjugated to (strept)avidin using the two-step glutaraldehyde procedure. This form is used in procedures where LAB and LSAB are substrate, for example. Conjugation with biotin also involves two steps, as biotin must first be derivatized to the biotinyl-N- hydroxysuccinimide ester or to biotin hydrazide before it can be reacted with the epsilonamino groups of the HRP enzyme.

[00170] 3,3'-diaminobenzidine (DAB) is a substrate for enzymes such as HRP that produces a brown end product that is highly insoluble in alcohol and other organic solvents. Oxidation of DAB also causes polymerization, resulting in the ability to react with osmium tetroxide, and thus increasing its staining intensity and electron density. Of the several metals and methods used to intensify the optical density of polymerized DAB, gold chloride in combination with silver sulfide appears to be the most successful.

[00171] 3-Amino-9-ethylcarbazole (AEC), is a substrate for enzymes such as HRP, and upon oxidation, forms a rose-red end product that is alcohol soluble. Therefore, specimens processed with AEC must not be immersed in alcohol or alcoholic solutions (e.g.. Harris' hematoxylin). Instead, an aqueous counterstain and mounting medium should be used. 4-Chloro-l-naphthol (CN) is a substrate for enzymes such as HRP that precipitates as a blue end product. Because CN is soluble in alcohol and other organic solvents, the specimen must not be dehydrated, exposed to alcoholic counterstains, or coverslipped with mounting media containing organic solvents. Unlike DAB, CN tends to diffuse from the site of precipitation.

[00172] p-Phenylenediamine dihydrochloride/pyrocatechol (Hanker- Yates reagent) is a substrate for enzymes such as HRP that gives a blue-black reaction product that is insoluble in alcohol and other organic solvents. Like polymerized DAB, this reaction U 2013/062376

- 41 . product can be osmicated. Varying results have been achieved with Hanker- Yates reagent in imniunoperoxidase techniques.

[00173] Calf intestine alkaline phosphatase (AP) (molecular weight 100 kD) removes (by hydrolysis) and transfers phosphate groups from organic esters by breaking the P-0 bond; an intermediate enzyme-substrate bond is briefly formed. The chief metal activators for AP are Mg++, Mn++ and Ca++.

[00174] AP had not been used extensively in immunohistochemistry until publication of the unlabeled alkaline phosphataseantialkaline phosphatase (APAAP) procedure. The soluble immune complexes utilized in this procedure have molecular weights of approximately 560 kD. The major advantage of the APAAP procedure compared to the peroxidase anti-peroxidase (PAP) technique is the lack of interference posed by endogenous peroxidase activity. Endogenous peroxidase can be blocked using a dilute solution of hydrogen peroxide. Because of the potential distraction of endogenous peroxidase activity on PAP staining, the APAAP teclinique is recommended for use on blood and bone marrow smears. Endogenous alkaline phosphatase activity from bone, kidney, liver and some white cells can be inhibited by the addition of 1 mM levamisole to the substrate solution, although 5 mM has been found to be more effective. Intestinal alkaline phosphatases are not adequately inhibited by levamisole.

[00175] In the immunoalkaline phosphatase staining method, the enzyme hydrolyzes naphthol phosphate esters (substrate) to phenolic compounds and phosphates. The phenols couple to colorless diazonium salts (chromogen) to produce insoluble, colored azo dyes. Several different combinations of substrates and chromogens have been used successfully.

[00176) Naphthol AS-MX phosphate AP substrate can be used in its acid form or as the sodium salt. The chromogen substrate Fast Red TR and Fast Blue BB produce a bright red or blue end product, respectively. Both are soluble in alcoholic and other organic solvents, so aqueous mounting media must be used. Fast Red TR is preferred when staining cell smears.

[00177] Additional exemplary substrates include naphthol AS-BI phosphate, naphthol AS- TR phosphate and 5-bromo-4-chloro-3-indoxyl phosphate (BCIP). Other possible chromogens include Fast Red LB, Fast Garnet GBC, Nitro Blue Tetrazolium (NBT) and iodonitrotetrazolium Violet (INT), for example. V, Immunodetection Methods

[00178] In still further embodiments, the present invention concerns immunodetection methods for binding, purifying, removing, quantifying and/or otherwise generally detecting biological components such as CD56 as contemplated by the present invention. The antibodies prepared in accordance with the present invention may be employed to detect CD56. Some immunodetection methods include immunohistochemistry, flow cytometry, radioimmunoassay (RIA), immunoradiometric assay, fluoroimniunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few. The steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g., Doolittle M H and Ben-Zeev O, Methods Mol Bioi. 1999; 109:215-37; Gulbis B and Galand P, Hum Pathol. 1993 Dec;24(12): 1271 -85; and De Jager R et al.. Semin Nucl Med. 1993 Apr;23(2): 165-79, each incorporated herein by reference.

[00179] In general, the immunobinding methods include obtaining a sample suspected of comprising ligand protein, polypeptide and/or peptide, and contacting the sample with a first ligand binding peptide (e.g., an anti-ligand antibody) in accordance with the present invention, as the case may be, under conditions effective to allow the formation of immunocomplexes.

[00180] Contacting the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of imjnune complexes (primary immune complexes) is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any ligand protein antigens present. After this time, the sample-antibody composition, such as a tissue section, dot blot or western blot, will generally be washed to remove any non- specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.

[00181] In general, the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags. U.S. Patents concerning the use of such labels 6

- 43 - include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated herein by reference. Of course, one may find additional advantages through the use of a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art.

100182] The anti-ligand antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined. Alternatively, the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding agent that has binding affinity for the antibody. In these cases, the second binding agent may be linked to a detectable label. The second binding agent is itself often an antibody, which may thus be termed a "secondary" antibody. The primary immune complexes are contacted with the labeled, secondary binding agent, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes. The secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.

[00183] Further methods include the detection of primary immune complexes by a two- step approach. A second binding agent, such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above. After washing, the secondary immune complexes are contacted with a third binding agent or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.

[00184] In another embodiment, a biotinylated monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complexed biotin. In that method the sample to be tested is first incubated in a solution comprising the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex. The antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex. The amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution comprising the second step antibody against biotin. This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate. With suitable amplification, a conjugate can be produced that is macroscopically visible.

[00185] In the clinical diagnosis and/or monitoring of patients with various forms of disease, the detection of CD56 and/or an alteration in the levels of CD56, in comparison to the levels in a corresponding biological sample from a normal subject or a biological sample from a non-diseased tissue is indicative of a patient with the disease. The detection of CD56 at a level comparable to the levels in a corresponding biological sample from a subject known to have a CD56 overexpressing cancer is also indicative of disease. However, as is known to those of skill in the art, such a clinical diagnosis would not necessarily be made on the basis of this method in isolation. Those of skill in the art are very familiar with differentiating between significant differences in types and/or amounts of biomarkers, which represent a positive identification, and/or low level and/or background changes of biomarkers. Indeed, background expression levels are often used to form a "cut-off" above which increased detection will be scored as significant and/or positive.

[00186] In one embodiment, immunohistochemistry (IHC) is used for immunological detection. Using IHC, detection of CD56 in a sample can be achieved by targeting a sample with a probe, e.g., an anti-CD56 antibody. The probe can be linked, either directly or indirectly to a detetable lable or can be detected by another probe that is linked, either directly or indirectly to a detectable lable.

[00187] In some embodiments, the IHC can distinguish between different levels of protein expression, e.g., calibrated IHC. In some embodiments, the IHC can distinguish staining intensity for samples having weak/low ceH surface CD56 expression, moderate/ intermediate/medium CD56 cell surface expression, or strong/high CD56 cell surface expression. [00188] In some embodiments, the IHC can distinguish between staining intensity and staining uniformity. In one embodiment, immunological detection (by immunohistochemistry) of CD56 is scored for both intensity and unifonnity (percent of stained cells - membrane only). Comparative scales for CD56 expression for intensity correlate as 0 - Negative, 0-1 - Very Weak, 1 - Weak, 1 -2 - Weak to Moderate, 2 - Moderate, 2-3 - Moderate to Strong, 3 - Strong, 3+ - Very Strong. Quantitatively, Score 0 represents that no staining is observed. Score 1 represents that a faint/barely perceptible membrane staining is detected, and the cells are only stained in part of their membrane. For Score 2, a weak to moderate complete membrane staining is observed. Lastly, Score 3 (or 3+) represents that a moderate to strong complete membrane staining is observed. Those samples with 0 or 1 score for CD56 expression can be characterized as not overexpressing CD56, whereas those samples with 2 or 3 scores can be characterized as overexpressing CD56. Comparative scales for CD56 percent cell membrane staining unifonnity con-elate as follows: 0 - Negative, Focal - <25%, heterogeneous (hetero) - 25- 75%, and homogeneous (homo) - >75%. As described in the Examples below, 2 homo or 2 hetero scores were associated with stable disease after treatment with the anti-CD56 immunoconjugate, and 3 homo or 3 hetero scores were associated with partial responses (as well as some stable disease or progressive disease) after treatment with the anti-CD56 immunoconj ugate.

[00189] Samples overexpressing CD56 can also be rated by immunohistochemical scores corresponding to the number of copies of CD56 molecules expressed per cell, or antibodies bound per cell (ABC), and have been determined biochemically. In some embodiments, the detection method is sufficiently sensitive to distinguish between cells with ABC values of about 70.000, about 160,000, and about 270,000. Thus, in some embodiments, the detection method results in a greater staining intensity and unifonnity in cells with ABC values of about 270,000 than in cells with ABC values of about 160,000. In some embodiments, the detection methods results in a greater staining intensity and unifonnity in cells with ABC values of about 270,000 than in cells with ABC values of 70,000. In some embodiments, the detection methods results in a greater staining intensity and uniformity in cells with ABC values of about 160,000 than in cells with ABC values of 70,000. [00190] IHC can be performed manually or using an automated system (e.g., using an automated stainer). IHC can be performed on cells, cell pellets, tissues etc., to detect membrane bound CD56. In some embodiments, the samples are fixed samples. In some embodiments, the samples are paraffin embedded samples. In some embodiments, the samples are formalin fixed and paraffin embedded samples. In some embodiments, the samples are fresh frozen samples.

[00191] In one embodiment, flow cytometry is used for immunological detection. Thus, for example, the number of antibodies bound per cell (ABC) can be assessed using flow cytometry. A high number of anti-CD56 antibodies bound per cell can indicate strong/high CD56 expression levels and a high likelihood to be susceptible to treatment with an anti-CD56 antibody or immunoconjugate thereof. In some embodiments, an ABC value of at least about 10,000, 25,000, 50,000, 60,000, 70,000. 80,0000, 90,000. 100.000, 1 10,000, 120,000, 125,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 225,000, 250.000, or 270,000 indicates a high likelihood to be susceptible to treatment with an anti-CD56 antibody or immunoconjugate thereof. In one embodiment, the N901-PE antibody is used for ABC measurements.

VI. Detection Kits and Compositions

[00192] Also provided by the invention are kits for use in the practice of the present invention as disclosed herein. Such kits may comprise containers, each with one or more of the various reagents (typically in concentrated form) utilized in the methods, including, for example, one or more binding agents (antibodies), already attached to a marker or optionally with reagents for coupling a binding agent to an antibody or nucleic acid molecule (as well as the marker itself); buffers, the appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, dUTP, ATP, CTP, GTP and UTP), reverse transcriptase, DNA polymerase, RNA polymerase, and one or more sequence-specific or degenerate primers for use in detection of nucleic acid molecules by amplification; and/or reagents and instrumentation for the isolation (optionally by microdissection) to support the practice of the invention. A label or indicator describing, or a set of instructions for use of, kit components in a ligand detection method of the present invention, will also be typically included, where the instructions may be associated with a package insert and/or the packaging of the kit or the components thereof. [00193] In still further embodiments, the present invention concerns immunodetection kits for use with the immunodetection methods described above. As the antibodies are generally used to detect CD56, the antibodies will preferably be included in the kit. The immunodetection kits will thus comprise, in suitable container means, a first antibody that binds to CD56, and/or optionally, an immunodetection reagent and/or further optionally, a CD56 protein or cell or sample containing a CD56 protein.

[00194] The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with and/or linked to the given antibody. Detectable labels that are associated with and/or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.

[00195] Further suitable immunodetection reagents for use in the present kits include the two-component reagent that comprises a secondary antibody that has binding affinity for the first antibody, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label. As noted above, a number of exemplary labels are known in the art and/or all such labels may be suitably employed in connection with the present invention.

[00196] The kits may further comprise one or more therapeutic agents for the treatment of cancer, such as an anti-CD56 immunoconjugate and/or a chemotherapeutic agent.

The kit may further comprise an a CD56 detection reagent used to measure CD56 expression in a subject comprising a CD56 detection reagent, and instructions for use. In one embodiment, the CD56 detection reagent comprises a CD56 binding peptide, protein or a molecular probe. In another embodiment, the CD56 detection reagent is an anti- CD56 antibody. In one embodiment, the CD56 detection reagent is the anti-CD56 antibody 1 B6 (Novocastra/Leica). In another embodiment, the CD56 detection reagent is an anti-CD56 antibody that is not 1 B6. In some embodiments, the detection reagent is 1B6, N901 , mAb735, BWSCLC 1 , BWSCLC2, 123C3. or MRQ-42. In another embodiment, the kit further comprises a secondary antibody which binds the anti-CD56 antibody. In one embodiment the CD56-specific antibody is included at a concentration of about 0.15 to about 0.50 μg/mL, about 0.15 to about 0.40 μg/mL, about 0.15 to about 0.30 ^tg/mL, about 0.15 to about 0.25 g/nlL, about 0.20 to about 0.25 μg/mL, or about 0.22 μg/nlL. In another embodiment, the antibody is included in concentrated solution with instructions for dilutions to achieve a final concentration of about 0.15 to about 0.50 μg/mL, about 0.15 to about 0.40 μg/mL, about 0.15 to about 0.30 μ ηύ,, about 0.15 to about 0.25 μg/mL, about 0.20 to about 0.25 μg/mL, or about 0.22 μg/mL. In another embodiment, the kit further comprises a detection reagent selected from the group consisting of: an enzyme, a fluorophore, a radioactive label, and a luminophore. In another embodiment, the detection reagent is selected from the group consisting of: biotin, digoxigenin, fluorescein, tritium, and rhodamine.

[00197] The kit can also include instructions for detection and scoring of CD56 expression. The kit can also include one or more control or reference samples or photographs of control or reference samples. Non-limiting examples of control or reference samples include cell pellets or tissue culture cell lines derived from normal (normal control) or tumor (positive control) samples. Exemplary positive control cell lines include H526 (SCLC), OPM2 (multiple myeloma), H929 (multiple myeloma) and cell lines stably or transiently transfected with an expression vector that expresses CD56. An exemplary negative control is Namalwa cells.

VII. CD56-binding agents

[00198] Any antibody that binds CD56 can be used in the detection methods of the present invention. For example, the anti-CD56 antibody can be mouse, chimierc, or humanized. The full-length amino acid sequence for CD56 is known in the art and also provided herein as represented by SEQ ID NO: 1. An exemplary useful antibody for detection of CD56 is the Novocastra/Leica antibody 1 B6 (Catalog # NCL-CD56-1B6 or NCL-L- CD56-1B6). In some embodiments, the detecting antibody is 1 B6, N901 , mAb735, BWSCLC1 , BWSCLC2, 123C3 (Dako), or MRQ-42 (ventana). See e.g., 5,593,898 and US RE37,596, both of which are herein incorporated by reference in their entirties. An example of a therapeutically effective anti-CD56 antibody is huN901-SPP-DMl (IMGN901 , lorvotu umab mertansine).

[00199] The antibody portion of IMGN901 was originally derived from N901. N901 is an IgGl murine monoclonal antibody (also called anti-N901) that is reactive with CD56. See e.g., Griffin el al, J. Immunol. 130:2947-2951 (1983) and U.S. Patent No. 5,639,641.

[00200] The preparation of different versions of humanized N901 , is described, for example, by Roguska et al Proc. Natl. Acad. Sci. USA, 91 :969-973 (1994), and Roguska et al, Protein Eng., 9:895:904 (1996), the disclosures of which are incorporated by reference herein in their entirety. To denote a humanized antibody, the letters "hu" or "h" appear before the name of the antibody. For example, humanized N901 may be referred to as huN901 or h ^' N901. The sequences for huN901 are provided as SEQ ID NOs: 2 and 3. The CDRl -3 sequences of the light chain of huN901 correspond to amino acids 24- 39, amino acids 55-61 , and amino acids 94-102 of SEQ ID NO:2, respectively. The CDRl-3 sequences of the heavy chain of huN901 correspond to amino acids 31-35, amino acids 50-59, and amino acids 99-107 of SEQ ID NO:3, respectively.

|002011 IMGN901 is an antibody-drug conjugate (ADC) comprised of the CD56-binding monoclonal antibody, huN901 , an SPP linker, and the maytansinoid cytotoxic agent, DM1. See, U.S. Patent No. 7,303,749, Example 1, for an exemplary description of huN901/DMl conjugation. The entirety of U.S. Patent No. 7,303.749 (Inventor: R.V.J. Chari; Issued Dec. 4, 2007) is incorporated by reference herein. Additional information regarding maytansinoid compounds is also discussed further herein.

[00202J IMGN9 l binds with high affinity to CD56 expressed on the surface of tumor cells. Once bound, the conjugate is internalized and the DM1 is released.

[00203] The SPP linker is N-succinimidyl 4-(2-pyridyldithio) pentanoate (SPP) (see, e.g., CAS Registry number 341498-08-6).

[00204] DM1 is an antimitotic agent that disrupts tubulin polymerization and microtubule assembly. See, Remillard S. et al, 1975, Science 189: 1002-1005). See also, U.S. Patent No. 7,303,749, Example 1, describing that "Ansamitocin P-3, provided by Takeda (Osaka, Japan) was converted to the disulfide-containing maytansinoid DM1 , as described herein and in U.S. Pat. No. 5,208,020." The entirety of U.S. Pat. No. 5,208,020 (Inventors: Chari et al ; Issued May 4, 1993) is incorporated by reference herein.

VIII. CD56 Immunoconjugates

[00205] The present invention also includes methods for increasing the efficacy of conjugates (also referred to herein as immunoconjugates), comprising the anti-CD56 antibodies, antibody fragments, functional equivalents, improved antibodies and their aspects as disclosed herein, linked or conjugated to a cytotoxin (drug) or prodrug. A particularly effective therapeutic immunoconjugate of the invention comprises the anti- CD56 antibody, huN901 , as described above. T/US2013/062376

- 50 -

[00206] Suitable drugs or prodrugs are known in the art. In certain embodiments, drugs or prodrugs are cytotoxic agents. The cytotoxic agent used in the cytotoxic conjugate of the present invention can be any compound that results in the death of a cell, or induces cell death, or in some manner decreases cell viability, and includes, for example, maytansinoids and maytansinoid analogs, benzodiazepines, taxoids, CC-1065 and CC- 1065 analogs, duocarmycins and duocarmycin analogs, enediynes, such as calicheamicins, dolastatin and dolastatin analogs including auristatins, tomaymycin derivaties, leptomycin derivaties, methotrexate, cisplatin, carboplatin, daunorubicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, morpholino doxorubicin, and etoposide. In certain embodiments, the cytotoxic agents are maytansinoids and maytansinoids analogs. In another embodiment, the cytoxic agent is etoposide. In another embodiment, the cytoxic agent is cisplatin. In another embodiment, the cytoxic agents are etoposide and cisplatin.

[00207] The drug or prodrug can, for example, be linked to the anti-CD56 antibody, such as huN901 , or fragments thereof through a disulfide bond. The linker molecule or crosslinking agent comprises a reactive chemical group that can react with the anti-CD56 antibody or fragment thereof. In certain embodiments, reactive chemical groups for reaction with the cell-binding agent are N-succinimidyl esters and N-suIfosuccinimidyl esters. Additionally the linker molecule comprises a reactive chemical group, in certain embodiments a dithiopyridyl group that can react with the drug to form a disulfide bond. In certain embodiments, linker molecules include, for example, vV-succinimidyl 3-(2- pyridyldithio) propionate (SPDP) (see, e.g.. Carlsson et al., Biochem, J , 173: 723-737 (1978)), iV-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB) (see, e.g., U.S. Patent No. 4,563,304), yV-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB) (see US Publication No. 20090274713), N-succinimidyl 4-(2-pyridyldithio) pentanoate (SPP) (see, e.g., CAS Registry number 341498-08-6), 2-iminothiolane, or acetylsuccinic anhydride.

[00208] Antibody-maytansinoid conjugates with non-cleavable links can also be prepared.

Such crosslinkers are described in the art (see ThermoScientific Pierce Crosslinking Technical Handbook and US Patent Application Publication No. 2005/0169933) and include but are not limited to, /V-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane- l-carboxy-(6-amidocaproate), which is a "long chain" analog of SMCC (LC-SMCC), κ- maleinudoundecanoic acid .V-suceimmidyl ester (KMUA), β-maleimidopropanoic acid A- succininiidyl ester iBMPS), γ-maleimidobutyric acid jY-succinimidyl ester (GMBS), ε- nmleirnidocaproic acid A ^r -hydroxysuccmimkie ester (EMCS), m-maleimidobenzoyi-A'- hydroxysuccirsimide ester (MBS), A-(a-maleimidoacetoxv)-su cininude ester (AMAS), succij\i.tnidyl"6~(f5-maieimidopropionamido)hexanoate (SMPH), A-suceinimidyi 4-(p- maleimidopheny ij-butyrate ( SMPB), and N-(p-ma!eimidophenyi)isocyanate (PM ΡΪ). A ^' - succinimidyl~4-(iodoacetyl)~aminobenzoate (SIAB). A ^r -succlnimidyl iodoacetate (Si A), A'-succinimidyl bromoaeetate (SBA), and A ^r -succirrin idyl 3-(brorooacetamido)propionate (SBAP). In certain embodiments, the antibody is modified with crosslinktng reagents such as suceinm idyi 4-( -maleimidornethyl)-cyclohexane-l-carboxylate (SMCC), s ifo- SMCC, maleirnidobeiizoyl-N-hydfoxysuccinimide ester (MBS), su!fo-MBS or succinimidy!-iodoacetate. as described in the literature, to introduce 1. -10 reactive groups (Yoshitake et. a!. Eur. J. Biochera., 101 :395-399 (1979); Hashida et ah J. Applied Biochem., 56-63 ( 1984): and Liu et ah Biochem., 18:690-697 ( 1979)).

] The present invention includes aspects wherein the average molar ratio of the cytotoxic agent (e.g. , maytansinoid) to the cell-binding agent in the cell-binding agent cytotoxic agent conjugate is about 1 to about 10. The terms "MAR," "Maytansinoid- Ab Ratio." "drug load." "DAR," and "Drug-Ab Ratio" can be used herein to characterize the ratio of cytotoxic agent to cell-binding agent in a conjugate comprising a maytansinoid compound as the cytotoxic agent and an antibody or fragment thereof as the cell binding agent. Thus, in some embodiments, the MAR is about 1 to about 10, about 2 to about 7. about 3 to about 5, about 2.5 to about 4.5 (e.g. , about 2.5, about 2.6. about 2.7, about 2.8. about 2.9, about 3.0, about 3. 1 , about 3.3. about 3.4, about. 3.5, about 3.6, about 3.7, about 3.8. about 3.9, about 4.0, about 4.1. about 4.2, about 4.3. about 4.4, about 4,5), about 3.0 to about 4.0, about 3.2 to about 4.2, about 4.5 to 5.5 (e.g. , about 4.5, about 4.6, about 4.7, about 4.8. about 4.9. about 5.0, about 5.1 , about 5.2, about 5.3, about 5.4, about 5.5). In one aspect, the number of drug molecules that can be attached to a cell binding agent can average from about 2 to about 8 (e.g. , 1 .9, 2.0, 2. L 2.2. 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0. 3.1, 3.2, 3.3, 3.4. 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1. 4.2, 4.3, 4.4. 4.5, 4,6, 4.7. 4.8, 4.9. 5.0, 5.1, 5.2, 5,3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9. 6.0, 6.1 , 6.2, 6.3, 6.4, 6,5, 6.6, 6.7, 6,8, 6.9, 7.0. 7.1 , 7,2, 7.3, 7.4, 7.5. 7.6, 7.7. 7,8, 7.9, 8.0, 8.1 ), in certain embodiments, the drug is A ² -deacetykV" ~{ 3-mereapto- 1 -oxopropyi)~maytansine (DM1 ) or A' ^"' -deacety !-N ^" -(4- mercapto-4-methyl-l-oxopentyl) maytansine (DM4). In another embodiment, the antibody huCD56-N901 is conjugated to DM1 or DM4.

IX. Correlation of CD56 expression and therapeutic efficacy

[00210] In certain embodiments, the invention provides a method for identifying subjects with an increased likelihood for responding to CD56-targeting anti-cancer therapies. The invention is based, in part, on the discovery that elevated CD56 expression levels correlates with efficacy of CD56-targeting anti-cancer therapeutics and the discovery of methods of detecting a dynamic range of CD56 expression in tissue samples.

[00211] Evaluation of patient samples and correlation to in vivo efficacy using xenograft models and results with human patients demonstrates the power of the expression analysis for selecting subjects more likely to respond to treatment. IHC provides a score for CD56 expression on tumor cells: 0 (no expression) to greater than or equal to 3 (very strong/high levels of expression). Samples scoring 2, or 3 for CD56 expression (or 2 or 3) have an increased likelihood to respond to CD56-targeted anti-cancer therapies at clinically-relevant doses of CD56 immunoconjugates (e.g., 0.1 to 10 or more mg/kg xenograft dose of a CD56 immunoconjugate can approximate a 3.0 to 400 mg/m in patients). In some embodiments a clinically relevant dose of a CD56 immunoconjugate (e.g., IMGN901 ) is 1 to 150 mg/ m ² , (e.g. , 30 mg/m ² , 36 mg/ m ² , 48 mg/m ² , 60 mg/m ² ,

7 7

75 mg/m", 90 mg/m ^" , or 1 12 mg/m"). In one embodiment, the dose of the CD56

7

immunoconjugate is 90 mg/m . In another embodiment, the dose of the CD56 immunoconjugate is 1 12 mg/m ² . Thus, identification of individuals having an elevated CD56 score would help identify those individuals who might respond to a clinically relevant dosage. As described in more detail below, sensitivity to CD56 therapeutics correlated with CD56 scoring of 2 or higher, and in one example, with a score of 3 or greater. In addition, , a homogenous staining uniformity or a combination of increased staining with heterogenous staining uniformity can indicate increased CD56 expression. For example, scores of greater than 2 hetero or greater than 2 homo can be used as a patient selection criterion for treatment with a CD56 therapeutic agent. In another example, scores of greater than 3 heter or greater than 3 homo can be used as a patient selection criterioun for treatment with a CD56 immunoconjugate. [00212] in some embodiments, samples that are obtained from a paitent and have an ABC value of at least about 10,000, 25,000, 50,000, 60,000, 70,000, 80,0000, 90,000, 100,000. 1 10,000, 120,000, 125,000, 150.000, 160,000, 170,000, 180,000, 190,000, 200.000, 225,000, 250,000, or 270,000 have an increased likelihood to respond to CD56-targeted anti-cancer therapies at clinically-relevant doses of CD56 immunoconjugates.

[00213] CD56 expression analysis also identifies patients in whom decreased levels of a CD56-targeting anti-cancer therapy ("low dose thereapy") can be effective to cause antitumor responses. As is appreciated in the art, compounds are generally administered at the smallest dosage that achieves the desired therapeutic response. This is specifically important for therapeutics that cause clinical, and often undesired, side effects. The ability to recognize those subjects with elevated CD56 expression levels allows for minimization of the dosage of the CD56-targeting therapeutic, thus decreasing possible side effects, while maintaining therapeutic efficacy.

X. Pharmaceutical compositions and therapeutic methods

[00214] CD56-binding agents (including antibodies, immunoconjugates, and polypeptides) are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as the treatment of cancer. In certain embodiments, the agents are useful for inhibiting tumor growth, inducing differentiation, reducing tumor volume, and/or reducing the tumorigenicity of a tumor. The methods of use may be in vitro, ex vivo, or in vivo methods. In certain embodiments, the CD56-binding agent (antibody or immunoconjugate, or polypeptide) is an antagonist of the human CD56 to which it binds.

[00215] In certain embodiments, the disease treated with the CD56-binding agent or antagonist (e.g., IMGN901 ) is a cancer. In certain embodiments, the cancer is characterized by tumors expressing CD56 to which the CD56-binding agent (e.g., antibody) binds.

[00216] The present invention provides for methods of treating cancer comprising administering a therapeutically effective amount of a CD56-binding agent to a subject (e.g., a subject in need of treatment). For example, the cancer can be a CD56 positive cancer or cancer of unknown primary (CUP). In certain embodiments, the cancer is selected from the group consisting of neuroendocrine tumors, small cell lung carcinoma (SCLC), Merkel cell cancer, ovarian cancers, cervical cancer, breast cancer, and multiple myeloma. In certain embodiments, the subject is a human.

[00217] The present invention further provides methods for inhibiting tumor growth using the antibodies, immunoconjugates, or other agents described herein. In certain embodiments, the method of inhibiting the tumor growth comprises contacting the cell with a CD56-binding agent (e.g., antibody) in vitro. For example, an immortalized cell line or a cancer cell line that expresses CD56 is cultured in medium to which is added the antibody or other agent to inhibit tumor growth. In some embodiments, tumor cells are isolated from a patient sample such as, for example, a tissue biopsy, and cultured in medium to which is added an CD56-binding agent to inhibit tumor growth.

[00218] In some embodiments, the method of inhibiting tumor growth comprises contacting the tumor or tumor cells with the CD56-binding agent (e.g., antibody) in vivo. In certain embodiments, contacting a tumor or tumor cell with a CD56-binding agent is undertaken in an animal model. For example. CD56-binding agents can be administered to xenografts expressing CD56 that have been grown in immunocompromised mice (e.g., NOD/SCID mice) to inhibit tumor growth. In some embodiments, the CD56-binding agent is administered at the same time or shortly after introduction of tumoi igenic cells into the animal to prevent tumor growth. In some embodiments, the CD56-binding agent is administered as a therapeutic after the tumorigenic cells have grown to a specified size.

[00219] In certain embodiments, the method of inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of a CD56-binding agent. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or has had a tumor removed.

[00220] Thus, in certain embodiments the invention provides methods of treating cancer using anti-CD56 antibodies (e.g., chimeric, humanized, or fully human) and immunoconjugates (anti-CD56 antibody- linker- cytotoxic agent), wherein the cancer is identified, using the methods described herein, as having increased or elevated CD56 expression (e.g., with an IHC score of greater than or equal to 2 hetero, greater than or equal to 2 homo, greater than or equal to 3 hetero, or greater than or equal to 3 homo.) In one particular embodiment, the cancer is identified as having a level of CD56 expression and distribution of greater than or equal to 3 hetero. In a certain embodiment, the CD56 immunconjuage is huN901-SPP-DMl . [00221] In certain embodiments, formulations are prepared for storage and use by combining a purified antibody or agent of the present invention with a phannaceutically acceptable vehicle (e.g., carrier, excipient) (Remington, The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000). Suitable phannaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and w-cresol); low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG).

[00222] The pharmaceutical compositions of the present invention can be administered in any number of ways for either local or systemic treatment. Administration can be topical (such as to mucous membranes including vaginal and rectal delivery) such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidennal and transdennal); oral; or parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration.

[00223] An antibody or immunoconjugate of the invention can be combined in a phamiaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having anti-cancer properties. The second compound of the phamiaceutical combination formulation or dosing regimen preferably has complementary activities to the immunoconjugate of the combination such that they do not adversely affect each other. [00224] In some embodiments, the therapeutic CD56-binding agent (e.g., antibody) is administered at a dose of at least 45 mg/m\ e.g., over 12 weeks. In some embodiments, the therapeutic CD56-binding agent (e.g., antibody or immunoconjugate) is administered in a dosing range of 60-1 12 mg/m ² . Thus in some embodiments, 60 mg/m ² of the CD56- binding agent (e.g., antibody or immunoconjugate) is administered. Thus in some embodiments, 70 mg/m ² of the CD56-binding agent (e.g., antibody or immunoconjugate) is administered. Thus in some embodiments, 75 mg/m' of the CD56-binding agent (e.g., antibody or immunoconjugate) is administered. Thus in some embodiments, 80 mg/m ² of the CD56-binding agent (e.g., antibody or immunoconjugate) is administered. Thus in some embodiments, 90 mg/m ² of the CD56-binding agent (e.g., antibody or immunoconjugate) is administered. Thus in some embodiments, 100 mg/m" of the CD56-binding agent (e.g., antibody or immunoconjugate) is administered. Thus in some embodiments, 1 10 mg/m ² of the CD56-binding agent (e.g., antibody or immunoconjugate) is administered. Thus in some embodiments, 1 12 mg/m ² of the CD56- binding agent (e.g., antibody or immunoconjugate) is administered. Thus in some embodiments, 120 mg/m ^" of the CD56-binding agent (e.g., antibody or immunoconjugate) is administered. For any of the above embodiments, one exemplary CD56 immunoconjugate includes the following: huN901 -SPP-DMl .

[00225] In some embodiments, the therapeutic CD56-binding agent is used in combination with carboplatin (e.g., at an AUC of about 4-7, 5-6, 5, or 6). In some embodiments, the therapeutic CD56-binding agent is used in combination with etoposide (e.g., about 50-150 mg/m ² , 75-125 mg/m ² , or 100 mg/m ² ). In some embodiments, the therapeutic CD56- binding agent is used in combination with carboplatin (e.g., at an AUC of 4-7, 5-6, 5, or 6) and etoposide (e.g., about 50-150 mg/m ² , 75-125 mg/m ² , or 100 mg/m ² ). In some embodiments, the therapeutic CD56-binding agent is administered at 90 mg/m" in combination with carboplatin at an AUC of 5 and etoposide at 100 mg. In some embodiments, the therapeutic CD56-binding agent is administered at 90 mg/m ^" on day 1 and day 8 of a three week cycle in combination with carboplatin at an AUC of 5 on day 1 every 21 days and etoposide at 100 mg on days 1 , 2, and 3 every 21 days.

[00226] In some embodiments, the therapeutic CD56-binding agent is used in combination with a chemotherapeutic agent selected from the group consisting of a gerncitabine compound such as GEMZAR® (e.g., 900 mg/rn ² , 800 mg/m ² , 750 mg/m ² , 700 mg/m ² , 600 mg/m ² , 500 mg/m ² , or 300 mg/m ² ), a topotecan compound such as HYCAMTIN® (e.g., 1 mg/m , 0.5 mg/m ^" , or 0.25 mg/m"), and a doxorubicin compound such as DOXIL®, ADRIAMYCIN®, RUBEX®, hydroxydaunorubicin, PEGylated liposomal doxorubicin, or anthracycline doxorubicin encapsulated in liposomes (e.g., 70 mg/m ² , 65 mg/m ² , 60 mg/m ² , 50 mg/m ² , 45 mg/m ² , 30 mg/m ² , or 25 mg/m ² ), as described, for example, in U.S. Application No. 13/434.419 (filed March 29, 2012), which is herein incorporated by reference in its entirety. In some embodiments, the CD56-binding agent is used in combination with a gemcitabine compound, a gemcitabine compound, and a doxorubicin compound.

[00227] In some embodiments, the CD56-binding agent is used in combination with at least two chemotherapeutic agents. For example, the CD56-binding agent can be used in combination with a taxane compound (such as paclitaxel or docetaxel) and a platinum compound (such as a carboplatin, a cisplatin, an oxaliplatin, an iproplatin, an ormaplatin, or a tetraplatin compound) as described, for example, in U.S. Published Application No. 201 1/0177064 (filed January 21 , 201 1), which is herein incorporated by reference in its entirety.

[00228] In some embodiments, the therapeutic CD56-binding agent is used in combination with lenalidomide (e.g., in an amount of about 25 mg daily on days 1 to 21 , every four weeks). In some embodiments, the therapeutic CD56-binding agent is used in combination with dexamethsone (e.g., in an amount of about 40 mg daily on day 1 , day 8, day 15 and day 22, every four weeks). In some embodiments, the therapeutic CD56- binding agent is used in combination with lenalidomide (e.g.. in an amount of about 25 mg daily on days 1 to 21 , every four weeks) and dexamethsone (e.g., in an amount of about 40 mg daily on day 1 , day 8, day 15 and day 22, every four weeks).

[00229] For the treatment of the disease, the appropriate dosage of an antibody or agent of the present invention depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the antibody or agent is administered for therapeutic or preventative purposes, previous therapy, patient's clinical history, and so on all at the discretion of the treating physician. The antibody or agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved (e.g., reduction in tumor size). Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual antibody or agent. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. In certain embodiments, dosage is from 0.01 g to 100 mg per kg of body weight, and can be given once or more daily, weekly, monthly or yearly. In certain embodiments, the antibody or other CD56-binding agent is given once every two weeks or once every three weeks. In certain embodiments, the dosage of the antibody or other CD56-binding agent is from about 0.1 mg to about 20 mg per kg of body weight. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.

[00230] The combination therapy can provide "synergy" and prove "synergistic", i.e., the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect can be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be attained when the compounds are administered or delivered sequentially, e.g., by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.

[00231] In some embodiments, CD56 levels can be measured before, during, and /or after treatment with a therapeutic CD56 binding molecule. The efficacy of treatment can be monitored on the basis of CD56 expression levels. For example, an anti-CD56 therapeutic can be administered to a subject until CD56 levels in samples from the subject are reduced or eliminated.

[00232] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure, 2376

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EXAMPLES

Example 1

An immunohistochemical (IHC) staining method in formalin fixed paraffin embedded (FFPE) samples for detecting varying expression levels of CD56

[00233] The IHC staining assay uses in vitro diagnostic (IVD) reagents including the Novocastra CD56 antibody (Novocastra/Leica, clone 1B6) as the test article and the Leica Bond RX automated stainer. Protocols for manual or automated IHC are described in detail below.

[00234] Bound test or control article were detected by incubation with the Leica Bond Refine detection system which includes a post primary reagent (rabbit anti-mouse IgG), followed by a polymer reagent (goat anti-rabbit polymer) and 3.3-Diaminobenzidine tetrahydrochloride (DAB) chromogen. FFPE samples were stained with the ready to use (RTU) CD56 or specified concentration(s) of primary antibody (prepared by diluting CD56 concentrate in Leica diluent) as outlined below. Test and control antibodies used in the assay are listed in Table 1.

Table 1 : IHC Antibodies

Leica RTU CD56 antibody (Cat. # PA0191) murine, clone CD564

Lyophilized Novocastra CD56 antibody (Cat. # NCL-CD56-

Test articles

1B6,) stock concentration 88μg/ml, murine, clone 1B6 Liquid Novocastra CD56 antibody (Cat. #NCL-L-CD56- 1B6,) stock concentration 44μg/mL, murine, clone 1B6

Beckman Coulter IgGl (Cat.# 6602872,) stock

concentration: ! mg/ml, murine, clone 2T8-2F5

Leica/Novocastra IgGl (Cat. #MOPC21Ab,) stock

Control articles

concentration: 50C^g/mL, murine,

Leica RTU Negative Control (Mouse) (Cat.# PA0996,) murine, clone MOPC-21

[00235] The Leica Bond RX automatically tracks and records the lot numbers for all reagents used in the staining process with the exception of bulk reagents. The bulk reagents used in the assays for the optimization of the clinical protocol are summarized in Table 2. Reagents used to fix, process, and embed samples are listed in Table 3. Test articles were prepared by diluting in Leica diluents. 62376

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Table 2: Bulk Reagents

Reagent Vendor

Bond Dewax Leica

Solution

Bond Epitope Leica

Retrieval Solution

1 (ER 1 )

I Bond Wash Leica

I Ethanol, 100% Pharmco-Aaper

Ethanol, 95% Pharmco-Aaper

Xvlenes Thermo/Richard

Allen

Cytoseal Thermo/Richard

Allen

Table 3 : Fixation. Processing, and Embedding Reagents

Assay conditions for manual and automated (Bond) IHC protocol may vary. For example, steps using the fully automated assay include the following automated steps: bake and dewax, a heat induced epitope retrieval step (HIER), and a staining step that includes application of blocking, primary antibody application, washes, polymer detection, and counterstaining. A similar sequence of steps is required for the manual method where each step must be performed manually. The manual method takes longer to perform and requires different assay conditions than the automated method such as incubation time, temperature, and buffers. The detection method used in the manual method was the Avidin-Biotin Complex detection method which is less sensitive than the polymer detection and requires additional blocking steps. [002371 Cell pellet samples shown to exhibit varying ranges of CD56 expression by flow cytometry (Table 4) and nonnal human tissues (nonnal esophagus, nonnal pancreas) and small cell lung carcinoma were used in this study to characterize positive and negative controls and for analysis of specificity. To prepare the cell pellets, cell lines supplied by American Tissue Culture Collection (ATCC), Dr. Thompshon (University of Texas Medical Branch), or the Gennan Collection of Microorganisms and Cell Cultures (DSMZ) were cultured using standard tissue culture methods at ImmunoGen. For each cell line, enough cells were harvested for both the preparation of an FFPE cell pellet and for quantification of CD56 expression. Antibodies bound per cell (ABC) values were detennined for CD56 for each of the positive control cell lines using flow cytometry methods and are listed in Table 4. The methods used to determine ABC are provided in Example 10. Staining conditions were optimized for CD56 so that H526, OPM2, and H929 positive control cells, which each express a unifonn level of CD56 and a different ABC value by flow cytometry, show differential staining scores.

Table 4: Cell Pellet Samples

cell lines using anti- CD56 phycoerythrin, BD Quantibrite™ beads and flow cytometry. ABC values differed from one experiment to the next and the value provided are examples from a single experiment.

[00238] Tumor tissue microarrays (TMAs) were used as test samples for either optimization or validation experiments. The TMAs included a Universal Control (Pantomics) and Small Cell Lung Carcinoma (SCLC) TMAs (Biomax and Cybrdi). Whole sections of SCLC were also used as test samples for optimization and validation experiments. [00239j Whole sections of 5 micron (μιη) thickness were prepared from the tissue and cell pellet blocks using a microtome and were mounted onto positively charged glass slides, air dried overnight and then stored in slide boxes until stained. Sections of TMAs (6) containing tumors were freshly cut by the respective vendor, at a thickness of 5 μηι, and sent to ImmunoGen. Sections prepared from each sample were stained using the staining process outlined above. Sections of either CD56-positive cell pellets that exhibit varying levels of membrane-associated CD56 expression (4) and/or human normal tissue samples of pancreas and esophagus (5) were included in addition to test samples. For each experiment, samples were stained with the specified concentration(s) of primary antibody (prepared by diluting in Leica diluent). When multiple concentrations of primary antibody were included in an experiment, serial dilutions were performed to obtain the final concentrations. H&E staining of all normal tissues, all whole tumor sections, and one TMA were performed according to standard procedures.

[00240] All samples were evaluated as follows unless otherwise noted: all tissue sections stained by H&E and/or IHC were evaluated by a board-certified pathologist to confirm the anatomic site, tumor type, and integrity of the tissue. Samples of poor quality (e.g., necrotic, crushed) and tumor samples that did not contain invasive tumor (not present, NP) were not evaluated further. The immunoreactivity of the test and control article within cells and tissues was determined by the consulting pathologist. Control samples were first evaluated followed by test samples (whole sections and individual cores from the TMAs). For each tumor tissue or cell pellet evaluated, a description of the staining intensity and respective proportion of tumor cells stained was reported as described in Table 5. Membrane associated staining was recorded for every sample. Final scores specify the extent of membrane staining only unless otherwise stated. In normal tissue, only the defined substructures were evaluated when calculating intensity and proportion.

Table 5: Intensity Score and Unifomiity Scales

Used to Assess the Immunoreactivity of CD56

Intensity (brightness of stain) Uniformity (percent of stained cells)

0 Negative 0 Negative

1 Weak Focal <25%

2 Heterogeneous

Moderate 25-75%

(hetero) Intensity (brightness of stain) Uniformity (percent of stained c<

3 Strong Homogeneous (homo) i >

[00241] Data analysis was performed on final scores returned from each core of all stained

TMAs at the specified test article concentration. When duplicate cores were evaluated from one patient, only the higher score was included in the analysis. Intensity values from each core were categorized as listed in Table 5.

[00242] Optimization of the automated assay was performed by first assessing the Leica ready to use CD56 antibody with the Leica recommended protocol on CD56 positive and negative cell pellets, normal tissues and SCLC samples (Step 1 ). Step 1 was performed to test the feasibility using the ready to use (RTU) CD56 antibody Leica recommended protocol. In Step 2, the Leica concentrated stock antibody (CD56) was tested over a range of CD56 dilutions to select conditions that exhibit an appropriate level of sensitivity and have the ability to distinguish varying levels of CD56 staining. In Step 2. a panel of tumor samples was stained, and a qualitative assessment of all staining results was first performed by comparing the relative staining intensities for each dilution. (See Example 2.) The criteria for identification of an optimal antibody dilution were set forth as follows: no background staining seen in samples stained with isotype control or in negative tissue substructure controls stained with test article, and a maximum dynamic range of staining observed among tumor test samples. These conditions ensure the specificity of staining for CD56 as well as the ability to distinguish between strong/high, moderate, and weak/low CD56 levels. Staining scores were then given to the samples stained with each test article concentration.

[00243] The results of the experiments demonstrated that the ready to use automated Leica assay, while specific, did not exhibit the sensitivity or dynamic range of expression levels required to distinguish ranges of CD56 expression. The CD56 RTU Leica recommended protocol showed targeted staining with high signal to noise in positive and negative test samples in which tissues substructures known to express CD56, including nerves and islets in pancreas, and muscle and nerves in esophagus, were positive. Positive cell pellet controls (confirmed by flow cytometry) also showed positive staining. Of the two antigen retrieval conditions, antigen retrieval with ER1 (Leica) for 10 minutes and antigen retrieval with ER1 for 20 minutes, both produced crisp membrane staining. ER1 for 20 minutes was selected as the antigen retrieval protocol to ensure complete antigen retrieval during the HIER step of this staining protocol. However, as noted above, the staining associated with the CD56 RTU antibody and ER l for 20 minutes did not exhibit an optimal dynamic range. The staining intensity trends observed from the CD56 positive cell pellets do not correspond to the reported Antibodies Bound per Cell (ABC) values determined by flow cytometry where a higher ABC value of 160,000 (OPM2 cells) resulted in a lighter staining intensity and uniformity score (2-3 hetero) than a lower ABC value of 70,000 (H929 cells: 3 homo), in sum, although the staining assay using the CD56 RTU test article was specific, the staining results did not exhibit the sensitivity or dynamic range in cell pellet controls known to express varying levels of CD56.

Example 2

Assay design for dynamic range staining of CD56 levels

[00244] After discovering that the CD56 ready to use (RTU) assay did not satisfy the required criteria that the staining reflect the dynamic range of antigen concentration present in the controls, various working concentrations of the CD56 stock antibody test article and the Coulter IgG l stock antibody control article were assayed to identify the optimal concentration exhibiting a dynamic range of staining in a small panel of CD56 positive cell pellets and small cell lung carcinoma samples and no staining in CD56 negative ceil pellets. The cell pellets and test samples included three CD56 positive cell pellets, one CD56 negative cell pellet, two whole sections of small cell lung carcinoma, and a TMA containing 80 SCLC cores from 40 patients. These samples were stained with three concentrations of test article prepared in Leica diluent [0.44 ig/m\ ( 1 :200 dilution), 0.22μ /ηι1 (1 :400 dilution), and 0.1 1 ng/ml (1 :800 dilution)].

[00245] All samples stained with control article exhibited no staining. A concentration of 0.1 1 μg/mL showed no level 3 staining intensity in either the calibrated cell pellet controls or in any SCLC cores evaluated and was therefore not selected as an optimal concentration. A greater differential in staining scores was observed for the calibrated cell pellets at a concentration of 0.22 μg/mL than at 0.44 μg/mL. Furthermore, a concentration of 0.22 μg/mL showed a greater distribution of staining scores among the panel of SCLC cores than at 0.44 μg mL as depicted in Figure 1 .

[00246] An automated IHC assay using the Leica Bond RX bake and dewax, antigen retrieval with ERl for 20 minutes, the "IHC Protocol F- With Extra Rinses" protocol and the Novocastra CD56 antibody at a concentration of 0.22 g/mL provided a dynamic range of staining in a small panel of CD56 positive and negative cell pellets and SCLC tumor samples. These conditions showed specificity and the appropriate sensitivity in SCLC tumor tissues. Staining was localized to tumor tissue, and normal components of the tumor sample such as stroma and blood vessels were negative. A dynamic range of intensity and uniformity scores were observed across a large panel of SCLC samples and show that this assay performs with increased sensitivity over the prior methods.

[00247] An IHC staining assay using the Leica Bond RX with bake and dewax, antigen retrieval with ER l for 20 minutes, and a test article concentration of 0.22 μg/mL was shown to exhibit comparable performance among different vendors and patient populations and also exhibits a broad distribution of scores within the SCLC samples.

Example 3

Determination of relative expression levels in positive control

cell pellets and tissue samples

[00248] IHC assays currently used in the field have not successfully demonstrated dynamic levels of CD56 expression in tissues. The assay, described in Example 2, was developed to allow for detection of varying levels of CD56. Cell pellets and tissues expressing varying levels of CD56 expression were therefore identified for use as controls in this assay. (Staining of cell pellets is shown in Figure 5.) CD56 expression variability from passage to passage was commonly observed in many cell lines. The identification of cell lines that showed relatively consistent expression from passage to passage included H526, OPM2, RH30, 300-19 cell transfected with CD56, and H929 cells. Among these cell lines, H526, OPM2, and H929 cells were selected as optimal controls since they grow uniformly and exhibited the appropriate range of CD56 expression levels, confirmed by results generated from the automated IHC assay described in Example 2 and data generated from a calibrated flow cytometry method (a method using Quantibrite beads™ as standards and a saturating concentration of PE-labeled anti-CD56 clone N901 ). Nerve is the predominant tissue that expresses strong/high levels of CD56 and is therefore used as a control in IHC staining assays. However, nerve tissue is not effective for a control when a dynamic range of staining is required. Therefore, normal tissue and structures within these normal tissues showing strong/high and weak/low staining were identified. These tissues and structures were used as calibrated controls to demonstrate that a dynamic range of staining was obtained when performing the assay. For example, in the normal human esophagus, the nerve structure was identified as a strong/high level control, muscle was identified as moderate level control, and epithelium was identified as a negative control structure within this tissue. The second example was human normal pancreas where again, the nerves were identified as a strong/high level control, islets were identified as the moderate control, and acinar cells were identified as the negative control. Human nonnal esophagus, human normal pancreas. H526 cells, OPM2 cells, H929 cells, and Namalwa cells and the respective staining acceptance criteria are listed in Table 6 for use in the assay described in Example 2.

Table 6: Control Samples and Respective Staining Acceptance Criteria

Normal Tissues (n=15) Acceptance Criteria (scores)

j Human Normal Pancreas Nerves: 3 homo

(P-N-PAN- 121610-1 ) Islets: 2 homo

Acinar Cells: negative

Human Normal Esophagus Nerves: 3 homo

(P-N-ESP-04281 1 -1 ) Muscle: 2 hetero

Epithelium: 0

Verification Controls (n=3) Acceptance Criteria (scores)

H526 cells (P-H526-031610-1 )

H526 staining intensity> OPM2 staining

OPM2 cells (P-OPM2-08101 1- 1 )

intensity > H929 staining intensity

r; H929 cells (P-H929-082510-1 )

Example 4

Manual methods for detection of CD56 ] Control samples and Merkel cell carcinomas (MCC) tissue samples received as FFPE blocks were cut into 5 μηι sections using a microtome. Two sections from each sample were collected onto pre-charged 'plus' microscope glass slides. Slides were air dried then baked for 30 min at 60° C in a drying oven. All slides were de-paraffinized and rehydrated by sequential immersions for 2 min each in the following solvents (number of immersions for each solvent in parentheses): xylene (4), absolute ETOH (2), 95% ETOH (2), followed by five 1 min immersions in distilled water. Antigen retrieval was performed for all tissues sections using a decloaker (Biocare medical, Cat# DC2002- CE). Briefly, slides immersed in Reveal buffer, pH 6, were kept at a pressure of 17 psi and a temperature of 120° C for 3 min. After the temperature cooled to 90 ^° C. slides were removed from the decloaker and immersed in 90° C water for 10 min. Slides were rinsed with PBS, and sections were blocked in PBS containing 2% normal mouse serum for 15 min. Slides were drained. The test article, anti-CD56 clone 1 B6, and the control article, mulgGl isotype, were diluted to 0.75 g/mL in PBS containing 2% mouse serum. One duplicate of each tissue section was incubated 60 min with either test or control reagent. Slides were washed. Sections or cores from each slide were incubated for 30 min with biotinylated horse anti-mouse secondary antibody (diluted to 10 μg/mL in PBS containing 2% mouse serum). Slides were washed in PBS and sections or cores from each slide were incubated for 40 min with ABC, prepared according to manufacturer's instructions. Slides were washed and sections or cores were developed with DAB according to manufacturer's instructions. Sections were incubated with DAB and hydrogen peroxide substrate for 5 min and rinsed with cold water. Slides containing developed tissue sections or cores were counterstained by immersion in a container filled with Hematoxylin for 2 min followed by a quick immersion in Scotts Buffer (0.135 M L12CO3 aqueous solution) which enhances the blue nuclear staining. Slides were cleared of excess stain and dehydrated by rinsing twice with 95% ETOH, twice with 100% ETOH, and four times with xylenes. Each rinse was 1 min. Coverslips were mounted onto the slides using Mounting medium. Scoring of samples was as described in Table 5, above.

Example 5

Automated staining assay shows a superior dynamic range of sensitivity

as compared to the manual method j Staining methods that include manual detection (as described above) of CD56 Merkel Cell Carcinoma samples have been previously described (AACR 2010 Abstract 5335, poster presented on Apr 21, 2010. 'The Antigen Target of Lorvotuzumab Mertansine (IMNG901)), CD56, is expressed at significant levels of Merkel Cell Carcinoma'). The previously described manual methods were compared to the automated staining method described herein using the SCLC TMAs described above. As shown in Figure 2, the automated methods described herein produced a surprisingly improved distribution of staining scores over the manual method.

Example 6

Use of the automated staining assay for additional disease indications f 0251| The automated staining methods discovered and described herein are used to tair! a panel of FFPE tissues including ovarian, merkel, neuroendocrine, and other CD56- positive tumors (at least 10 samples from each indication). The same cell pellet and tissue controls listed in Table 6 are included in these studies to ensure the dynamic range of staining is obtained.

Example 7

Correlation of CD56 staining levels with therapeutic efficacy

[00252] Experiments were conducted to evaluate huN901-SPP-DMl (IMGN901) as a novel single agent and in combination with standard first line of care for SCLC (cisplatin and etoposide). For each experiment, IHC was performed using the methods and scoring described above to evaluate the staining level for CD56. Two studies were performed: one using NCI-H526 (SCLC cell line, ABC value approximately 270,000) xenograft efficacy models in female nude mice and one using NCI-69 (SCLC line, ABC value undetermined) xenograft efficacy models in female nude mice. As shown in Table 7, the activity of IMGN901 (300 μg kg dosed on day 1 and day 8) in combination with cisplatin (5 mg/kg dosed on day 1 ) plus etoposide (8 mg/kg dosed on days 1-5) was highly active in both models. However, a greater number of partial regressions (PRs), complete regressions (CRs), and Tumor Free Survival (TFS) were seen for the NCI-H526 efficacy model, having the CD56 ABC value of -270,000, than for the NCI-H69 model. IHC studies on the NCI-H526 model also showed a higher staining score of 3 homogeneous than the NCI-H69 showing a score of 2-3 hetero-homo (Figure 3) using the automated method described above. These results suggest a correlation between CD56 levels, assayed using the IHC methods described herein, and efficacy of IMGN901 in combination with cisplatin and etoposide. Table 7

*Treatment-to-Control Ratio, determined according to NCI guidelines.

Example 8

Assessment of CD56 levels in a Phase 1 clinical trial

[00253] Lorvotuzumab mertanisine (LM or IMGN901 ) is a novel antibody-drug conjugate (ADC) composed of a CD56-targeting antibody attached via a disulfide linker to the maytansinoid, DM 1. During a Phase 1 study of IMGN901 , samples were assayed, initially by manual IHC staining methods as described above, to determine the level of CD56 expression in patient tumor samples and to determine the relationship between CD56 expression and response rates. Enrollment during Phase 1 was open to patients with advanced solid tumors for whom carboplatin/etoposide was a reasonable treatment option. The Phase 1 portion of the study was designed to identify the recommended phase 2 combination regimen when IMGN901 was provided IV on Days 1 and 8 with carboplatin IV on Day 1 and etoposide IV on Days 1 -3 of a 21 -day cycle. IMGN901 doses ranged from 60- 1 12mg/m ² with carboplatin dosed at an AUC of 5 or 6 and etoposide at 100mg/m ² . Combination therapy could continue for 4 cycles with up to 6 allowed. Patients with responding or stable disease (SD) could continue on single agent IMGN901 until progressive (PD) or intolerable toxicity.

[00254] Response assessments were to be performed every 6 weeks per the Response Evaluation Criteria for Solid Tumors (RECIST) 1.1 guidelines (see e.g. , Eisenhauer et al., European Journal of Cancer 45:228-247 (2009)); see also RECIST Compared available at http://www.recist.com/recist-comparative/index.html (visited September 24, 2012)). {00255] Manual IHC staining was performed as described above. Scoring was performed also as described above. Treatment response as measured by percent reduction in tumor from baseline is provided in Figure 4, Staining of all 30 patient, samples or of small cell lung cancer patient samples was scored and summarized in Tables 8 and 9 below.

Table 8 : IHC Staining of Archived Tumor Samples of All Patients in Figure 4

Table 9: IHC Staining of Archived Tumor Samples of SCLC Patients in Figure 4

[00256] There were no responders in patients with limited or no expression of CD56.

Patients from whom samples showed CD56 expression levels of 2 homo or 2 hetero showed stable disease, and patients from whom samples showed CD56 expression levels of 3 homo or 3 hetero showed a partial response (as well as some stable disease or progressive disease).

[00257] The data suggest a con-elation between elevated CD56 expression (e.g., 3 homo, 3 hetero, 2 homo, or 2 hetero) and treatment response rate. Additional automated studies are also performed on patient samples.

Example 9

Assessment of CO56 levels in a Phase 2 clinical trial

[00258] A Phase 2 study is underway to determine the efficacy of IMGN901 in combination with carboplatin and etoposide in patients with extensive disease SCLC and 6

- 71 - who have not received any prior systemic chemotherapy for the treatment of SCLC (i.e., were chemonaive). In the Phase 2 study, IMGN901 dosing was initiated at the recommended phase 2 dose (RP2D) of 1 12 mg/nr on day 1 and day 8 of a three week cycle and subsequently decreased to 90 mg/m ² on day 1 and day 8 of a thr ee week cycle. IMGN901 was given IV in combination with carboplatin at an AUC of 5 on day 1 every 21 days and etoposide at 100 mg IV on days 1 , 2, and 3 every 21 days. Combination therapy could continue for 4 cycles with up to 6 allowed. Patients with responding or stable disease (SD) could continue on single agent IMGN901 until progressive (PD) or intolerable toxicity.

[00259] Patient tissue samples are assayed by automated IHC staining methods, as described above, to determine the level of CD56 expression in patient samples. To date, 71 patient samples have been evaluated and the results are provided in Table 10 below.

Table 10: Patient IHC Staining Results

<2 hetero 1 3/71 4%

[00260] The relationship between CD56 expression and response rates are determined for the patients in the trial. Response assessments are performed every 6 weeks per the Response Evaluation Criteria for Solid Tumors (RECIST) 1.1 guidelines (see Eisenliauer et al., European Journal of Cancer 45:228-247 (2009); see also RECIST Compared available at http://www.recist.com/recist-comparative/index.html (visited September 24, 2012)). While the analysis of the Phase 2 sample data is not complete, results from the SCLC patients in the Phase 1 trial suggest a con-elation between elevated CD56 expression (e.g., 3 homo, 3 hetero, 2 homo, or 2 hetero) and treatment response rate (see Table 9). In addition, all three patients in the Phase 1 trial who were chemonaive SCLC patients had tumor samples that stained 3 homo or more. Two of these three achieved a partial response. This data suggests that, in the patient subset in the Phase 2 trial (i.e., chemonaive SCLC patients), a correlation between elevated CD56 expression and treatment response may exist.

Example 10

ABC Determination

Characterization of test reagents

] PE-labeled anti-CD56 antibodies (either humanized or murine N901) were used to quantify CD56 levels on tumor cell lines. PE-conjugated murine anti-CD56 clone N901 (muN901-PE) was obtained commercially. PE-conjugated humanized anti-CD56 clone N901 (huN901-PE) was prepared and shown to have a molar ratio of N901 to PE of 1 : 1 and was greater than 90% monomer. The ability of muN901-PE and huN901-PE to reach comparable saturable binding was determined by flow cytometry using CD56-positive H929 cells. All samples were tested in duplicate. Cells were washed with cold sterile PBS and resuspended in FACS buffer (1 % BSA in PBS). The cells were counted by trypan blue and diluted to a final volume of 4.0 x 10 ⁶ cells/mL. Antibody concentrations were prepared in a 96-well plate. The stock concentration (2x) of antibody-PE (12 x 10 ^" M) was prepared in FACS buffer and serially diluted 1 :3 over 1 1 dilutions. Each dilution of antibody-PE ( 100 μί) was incubated with cells ( 100 μί; 4.0 x 10 ^? cells/well of a 96 well plate). Control samples were also included where FACS buffer (100 μί,) was incubated with cells (100 μί). After incubation at 4 ^° C for 2 h, plates were centrifuged at 4 ^° C for 5 min at 2000 g. The supernatant was rapidly flicked off, and the wells were washed (gentle agitation followed by 4 C centrifugation, and flicking) twice with 200 μίΛνεΙΙ FACS buffer. Cells were resuspended in PBS containing a 1% formaldehyde solution (175 μΕΛνεΙΙ). and the plates were kept at 4 C for approximately 24 h in the dark. Fixed cells were resuspended prior to FACS analysis. Using a flow cytometer (FACS Calibur BD Biosciences), 2000 events were collected from each well, and a single population of cells was selected based on the forward and side scatters (FSC and SSC). The geometric means of PE fluorescence (FL2) from the antigen-positive population was plotted against the log of the concentration of either huN901 -PE or muN901-PE. Sigmoidal dose-response (variable slope) curves were generated by GraphPad PrismVersion 4.03 software. Estimation of ABC values for cell pellet controls

[00262] The quantification of CD56 on tumor cells was studied using murine or humanized N901 -PE and the BD Quantibrite™ beads kit. Each step of the assay was performed at 4'C unless otherwise stated. Cells rinsed in PBS were diluted to approximately 5 x 10 ⁵ cells/mL in FACS buffer (PBS containing 2% BSA). Two samples were prepared (test and control) for each cell line by the addition of 100 μΕ cells into a 12 x 75 mm Falcon tube. To the test samples, N901 -PE was added (muN901 -PE for H526 and H929 and huN901 -PE for OPM2 at a final concentration of 1 .0 and 6.7 nM, respectively). FACS buffer was added to control samples. All samples were allowed to incubate for approximately 1 h. Each sample was quenched and washed by the addition of 5 mL of FACS buffer. All samples were centrifuged at 300 g for 5 min and the supernatant from each sample was decanted. To each sample was added 200 \xL of

FACS sheath buffer containing 2% formaldehyde. Each sample was incubated overnight prior to flow cytometry acquisition. The levels of bound fluorescent antibody were determined by flow cytometry using a FACS Calibur. Instrument settings were adjusted and evaluated for cells from the control sample. Readings from test samples were acquired using established instrument settings ( 10,000 events were collected for H929 and H526 cells and 2000 events were collected for OPM2 cells), and the mean FL2 values were obtained. The Quantibrite kit, containing four populations of beads with varying quantities of PEs, was prepared and the data was acquired according manufacturer ^' s instructions using the same instrument settings as the test samples. The number of N901 -

PE antibodies bound per cell (ABC) was determined as described in the product specification sheet. Briefly, the log of the mean FL2 value of each bead population was plotted against the log of the PE number (provided in the product specifications) to create a standard curve that depicts the linear relationship of log (PE) against log (FL2). The log of the mean FL2 value was determined for each cell line test sample and was used to calculate the number of cell surface PE molecules.

Confirmation of muN901 -PE and huN901 -PE as test reauents for antigen quantification

[00263] Saturable binding was determined for both muN901 -PE and huN901 -PE by flow cytometry. Binding curves obtained by incubating cells with varying concentrations of

N901 -PE, indicate that both muN901 -PE and huN901 -PE exhibit saturable binding. The upper asymptotes of each binding curve give comparable FL2 values and indicate that the respective ABC values from each clone are nearly equivalent. The data suggests that that either clone can provide a similar estimation of CD56 antigen level for a given cell line.

ABC values of turner cell lines

[00264] ABC values were determined by measuring the amount of bound N901-PE using flow cytometry. Estimated ABC values for each cell line (provided in Example 1 and

T able 4. above) were determined by comparison of the mean fluorescence ( FL2) values to the PE standard curve. The ABC values ranged from about 70,000 for H929 cells to over

300,000 for H526 cells. OPM2 cells had an intermediate ABC value (about 160,000). As

H526 cells predominantly grow in multicellular clusters, quantification was limited to a subset of cells that could be disaggregated by extensive pipetting. The high level of homogeneous staining by IHC of cell pellets generated without disaggregation suggests thai the single cell population analyzed by flow cytometry is representative of the cell line.

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[00265] All publications, patents, patent applications, internet sites, and accession numbers/database sequences (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.