Methods of predicting therapeutic benefit of CD19 antibody treatment in a patient

Abstract

The present disclosure relates to a method of identifying a subject having chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), small lymphocytic lymphoma (SLL), or acute lymphoblastic leukemia (ALL) who will respond to treatment with an anti-CD 19 antibody, the method comprising: a) providing a blood sample obtained from the subject prior to treatment with the anti-CD 19 antibody, b) determining the level of at least one biomarker selected from the group consisting of i) peripheral NK cell count and ii) CD 16 expression level on peripheral NK cells in the sample, c) comparing the level of the at least one biomarker in the sample to a predetermined cutoff level, wherein a level of the at least one biomarker that is equal to or higher than the predetermined cutoff level indicates that the subject will benefit from anti-CD 19 antibody treatment. The present disclosure also relates to a method of treating a patient according to the above screening method and use of an anti-CD 19 antibody to treat such a subject.

Description

[0001] This application is a divisional application of application number 201780033793.9, filed on May 30, 2017, entitled "Method for predicting the therapeutic benefit of CD19 antibody therapy in patients", and enjoys priority thereto. Invention Field

[0002] This disclosure relates to the identification of characteristics and biomarkers in patients who benefit from anti-CD19 antibody therapy. Background Technology

[0003] CD19 is a 95-kDa transmembrane glycoprotein of the immunoglobulin superfamily, containing two extracellular immunoglobulin-like domains and an extensive cytoplasmic tail. This protein is a pan-B lymphocyte surface receptor, expressed broadly from the earliest stages of pre-B cell development until it is downregulated during terminal differentiation into plasma cells. It is specific to the B-lymphocyte lineage and is not expressed on hematopoietic stem cells and other immune cells, except for some follicular dendritic cells. CD19 functions as a positive regulator of B cell receptor (BCR) signaling, and is crucial for B cell activation and proliferation, as well as the development of humoral immune responses. It acts as a co-stimulatory molecule in conjunction with CD21 and CD81, which is essential for B cell responses against T-cell-dependent antigens. The cytoplasmic tail of CD19 is physically associated with a family of tyrosine kinases that trigger downstream signaling pathways via src-family protein tyrosine kinases. CD19 is a notable target for lymphoid cancers because it is highly expressed in almost all chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL), as well as many other different types of leukemia, including acute lymphoblastic leukemia (ALL) and hairy cell leukemia (HCL).

[0004] Clinical development of antibodies against CD19 was previously limited by CD19 antigen internalization; however, improvements in antibody modification technology have restored this potential therapeutic target. MOR00208 (formerly known as XmAb5574) is a humanized monoclonal antibody with an Fc-modified structure that binds to CD19. Due to the mutation in the XmAb modification, MOR00208 exhibits increased Fc binding to FcγR compared to the unmodified antibody, significantly enhancing antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and direct cytotoxicity (apoptosis) against tumors in vitro. MOR00208 did not demonstrate complement-dependent cytotoxicity.

[0005] MOR00208 has been investigated in clinical trials for CLL, ALL, and NHL, or is currently under investigation. Specifically, a study titled "..." has been completed. Safety and tolerability of 5574 in chronic lymphocytic leukemia The Phase I trial of Fc-Optimized Anti-CD19 Antibody (MOR208) for the treatment of B-cell Acute Lymphoblastic Leukemia (B-ALL) and the Phase IIa trial of Fc-Optimized Anti-CD19 Antibody (MOR00208) for the treatment of B-cell Acute Lymphoblastic Leukemia (B-ALL) have completed recruitment. A Phase IIa trial of Fc-Optimized Anti-CD19 Antibody (MOR00208) for the treatment of Non-Hodgkin's Lymphoma (NHL) has also completed recruitment.The following trials are planned / ongoing: A Phase II / III trial entitled "A Trial to Evaluate the Efficacy and Safety of MOR00208 With Bendamustine (BEN) and Versus Rituximab (RTX) With BEN in Adult Patients With Relapsed or Refractory Diffuse Large B-cell Lymphoma (DLBCL)" (B-MIND); and a study entitled "A Study to Evaluate Efficacy and Safety of MOR00208 With Idelalisib in R / R CLL / SLL Patients Pre-treated with BTKi" (A Study to Evaluate Efficacy and Safety of MOR00208 With Idelalisib in R / R CLL / SLL). The Phase II trial entitled "A Study to Evaluate the Safety and Efficacy of Lenalidomide With MOR00208 in Patients With RR DLBCL"; and the Phase II trial entitled "MOR00208 in Combination With Lenalidomide for Patients With Relapsed or Refractory CLL, SLL or PLL or Older Patients With Untreated CLL, SLL or PLL".In another current phase II trial (COSMOS), the efficacy and safety of MOR00208 in combination with idelalisib or venetoclax in patients with relapsed or refractory CLL, SLL were investigated.

[0006] The single-agent efficacy of MOR00208 has been reported in CLL and NHL. However, patient response rates to monoclonal antibody therapy are often variable, indicating a need for methods to accurately predict which patients are likely to respond to these antibody treatments, so that treatment can be given to those most likely to benefit. Specific biomarkers or characteristics of patients can be identified, with specific concentrations or ranges of each biomarker correlated with responsiveness to the treatment.

[0007] The impact of natural killer (NK) cell count on survival in DLBCL patients treated with rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate, and prednisone (R-CHOP) has been evaluated. (Kim et al., Blood Research, 49:3, 162-169 (September 2014)). Previously, peripheral NK cell counts have been reported to be associated with clinical outcomes in patients with aaIPI 2-3 DLBCL. (Plonquet et al., Ann Oncol 2007; 18:1209-15).

[0008] Clearly, considerable effort and investment are still needed to discover and identify patient characteristics and biomarkers for these predictive powers. Summary of the Invention

[0009] MOR00208 has been studied in patients with CLL, ALL, NHL, and SLL. Therefore, a detailed analysis of clinical data has been completed to date to identify characteristics or biomarkers of patients most likely to benefit from MOR00208 treatment.

[0010] MOR00208 specifically targets the CD19 surface antigen and mediates direct tumor cell killing via its enhanced ADCC effector function. Preclinical studies have shown efficacy across a wide range of human lymphomas and leukemias (Burkitt's lymphoma, CLL, hairy cell leukemia (HCL), CD19) +In chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), and acute lymphoblastic leukemia (ALL), MOR00208 significantly enhanced ADCC, ADCP, and direct cytotoxicity (apoptosis) in CD19+ tumor cell lines in vitro, with CD19 antigen expression levels ranging from 15,000 to 105,000 molecules / cell. Similar effects have been observed with newly isolated patient CLL or ALL cells and are also expected to transform into primary non-Hodgkin's lymphoma (NHL) cells, as the reported expression range for ALL and CLL B cells encompasses the range observed for NHL B cells (Ginaldi et al., 1998; Olejniczak et al., 2006). Based on the broad and uniform surface expression of CD19 across many types of B-cell tumors, the efficacy of MOR00208 in the current study can be transferred to a wide range of human lymphomas and leukemias, such as CLL, ALL, NHL, and SLL and their subtypes.

[0011] Data from the Phase IIa trial entitled "Study of Fc-Optimized Anti-CD19 Antibody (MOR00208) to Treat Non-Hodgkin's Lymphoma (NHL)" have been meticulously analyzed. As a result of these efforts, the following disclosure provides the characteristics and biomarkers of patients in whom the anti-CD19 antibody was effective.

[0012] Specifically, patients were evaluated for at least the following characteristics: a) age, b) sex, c) whether the patient had received a dose of rituximab within the last 6 months, d) whether the patient was refractory to rituximab, e) whether the patient had a high or low affinity allele for FcγRIIIa, f) whether the patient had a high or low affinity allele for FcγRIIa, g) whether the patient had a response duration of more than 12 months to previous treatment, h) baseline peripheral T cell count (cells / μl), i) baseline peripheral NK cell count (cells / μl), and j) baseline CD16 expression on peripheral NK cells (antibody-ABC bound per cell).

[0013] Both 1) baseline peripheral NK cell count and 2) baseline CD16 expression on peripheral NK cells clearly demonstrated a correlation with patient response to MOR00208 treatment. Specifically, patients with higher baseline peripheral NK cell counts / μl were associated with higher disease control rates (DCR). DCR included patients with complete response (CR) + partial response (PR) + stable disease (SD). Moreover, these patients had significantly better progression-free survival (PFS) compared to patients with lower NK cell counts. Furthermore, patients with baseline CD16 expression on NK cells of at least 60,000 (ABC) were associated with higher disease control rates (DCR).

[0014] Therefore, patients diagnosed with CLL, ALL, NHL, and SLL who have a) high peripheral NK cell count or 2) baseline CD16 expression on peripheral NK cells of at least 60,000 ABC are more likely to benefit from MOR00208 treatment.

[0015] Both 1) baseline peripheral NK cell count and 2) baseline CD16 expression on peripheral NK cells clearly demonstrated a correlation with patient response to MOR00208 treatment. Specifically, patients with a baseline peripheral NK cell count of at least 50 cells / μl were associated with a higher disease control rate (DCR). DCR included patients with a complete response (CR) + partial response (PR) + stable disease (SD). Moreover, patients with a baseline NK cell count of at least 50 cells / μl had significantly better progression-free survival (PFS) compared to patients with lower NK cell counts. Furthermore, patients with a baseline CD16 expression on NK cells of at least 60,000 (ABC) were associated with a higher disease control rate (DCR).

[0016] Therefore, patients diagnosed with CLL, ALL, NHL, and SLL and with a) a baseline peripheral NK cell count of at least 50 cells / μl or 2) baseline CD16 expression on peripheral NK cells of at least 60,000 ABC are more likely to benefit from MOR00208 treatment.

[0017] Both 1) baseline peripheral NK cell count and 2) baseline CD16 expression on peripheral NK cells clearly demonstrated a correlation with patient response to MOR00208 treatment. Specifically, patients with a baseline peripheral NK cell count of at least 100 cells / μl were associated with a higher disease control rate (DCR). DCR included patients with a complete response (CR) + partial response (PR) + stable disease (SD). Moreover, patients with a baseline NK cell count of at least 100 cells / μl had significantly better progression-free survival (PFS) compared to patients with lower NK cell counts. Furthermore, patients with a baseline CD16 expression on NK cells of at least 60,000 (ABC) were associated with a higher disease control rate (DCR).

[0018] Therefore, patients diagnosed with CLL, ALL, NHL, and SLL and with a) a baseline peripheral NK cell count of at least 100 cells / μl or 2) baseline CD16 expression on peripheral NK cells of at least 60,000 ABC are more likely to benefit from MOR00208 treatment. Attached Figure Description

[0019] Figure 1 The amino acid sequences of the MOR00208 variable domain and CDR are shown.

[0020] Figure 2 The complete heavy and light chain amino acid sequences of MOR00208 are shown.

[0021] Figure 3 Receiver operating characteristic (ROC) analysis of peripheral NK cell count as a predictor of DCR is shown.

[0022] Figure 4 ROC analysis of CD16 expression levels (ABC) on peripheral NK cells, which are a predictor of DCR, is shown.

[0023] Figure 5 ROC analysis of peripheral T cell count as a potential predictor of DCR is shown.

[0024] Figure 6 This indicates that peripheral NK cell count and CD16 expression level on peripheral NK cells are independent variables and are not correlated.

[0025] Figure 7 The DCR forest plot is shown for patient subgroups with specific baseline characteristics and biomarkers.

[0026] Figure 8The study showed a difference in progression-free survival between patients with a peripheral NK cell count of at least 100 cells / μl and those with a lower NK cell count.

[0027] Figure 9 This study demonstrated the difference in progression-free survival between patients with CD16 expression of at least 60,000 ABC on peripheral NK cells and patients with lower CD16 expression on NK cells.

[0028] Figure 10 The study showed a difference in progression-free survival between patients with a peripheral T cell count of at least 500 cells / μl and those with a lower T cell count. Detailed Implementation

[0029] The term "antibody" refers to a monoclonal antibody, including any isotype, such as IgG, IgM, IgA, IgD, and IgE. An IgG antibody consists of two identical heavy chains and two identical light chains linked together by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three segments called "complementarity-determining regions" ("CDRs") or "hypervariates," which are primarily responsible for binding to antigenic epitopes. These are called CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The more conserved portion of the variable region outside the CDRs is called the "backbone region." An "antibody fragment" refers to an Fv, scFv, dsFv, Fab, Fab'F(ab')2 fragment, or other fragment containing at least one variable heavy or light chain, each containing both a CDR and a backbone region.

[0030] "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody or antibody fragment. "VL" refers to the variable region of the immunoglobulin light chain of an antibody or antibody fragment.

[0031] The "Fc region" refers to the antibody constant region, which in humans can be IgG1, 2, 3, 4 subclasses or others. The sequence of the human Fc region can be obtained from IMGT, Human IGH C-REGIONs, www.imgt.org / IMGTrepertoire / Proteins / protein / human / IGH / IGHC / Hu_IGHCallgenes.html (searched on May 16, 2011).

[0032] The term "patient" includes people.

[0033] NHL is a heterogeneous malignant tumor originating from lymphocytes. In the United States (US), the estimated incidence is 65,000 per year, with approximately 20,000 deaths (American Cancer Society, 2006; and SEER Cancer Statistics Review). The disease can occur at any age, but typically begins in adults over 40 years of age, with incidence increasing with age. NHL is characterized by the clonal proliferation of lymphocytes accumulating in the lymph nodes, blood, bone marrow, and spleen, although it can involve any major organ. The current classification system used by pathologists and clinicians is the World Health Organization (WHO) Classification of Tumors, which organizes NHL into precursor and mature B-cell or T-cell vegetations. The PDQ currently classifies NHL into indolent or aggressive types for entry into clinical trials. The indolent NHL group primarily includes follicular subtypes, small lymphocytic lymphoma, MALT (mucosa-associated lymphoid tissue), and marginal zone; indolent includes approximately 50% of newly diagnosed B-cell NHL patients. Invasive NHL includes patients histologically diagnosed with primary diffuse large B-cell (DLBL, DLBCL, or DLCL) (diffuse large cells account for 40% of all newly diagnosed patients), Burkitt's, and mantle cell NHL. The clinical course of NHL is highly variable. The primary determinant of the clinical course is the histological subtype. Most indolent NHL is considered an incurable disease. Patients initially respond to chemotherapy or antibody therapy, and most will relapse. Studies to date have not shown that early intervention improves survival. In asymptomatic patients, "observation and waiting" is acceptable until the patient develops symptoms or disease progression appears to accelerate. Over time, the disease can transform into a more aggressive histological type. Median survival is 8–10 years, and patients with indolent NHL often receive 3 or more treatments during their course of treatment. Initial treatment for symptomatic indolent NHL patients has historically been combined with chemotherapy. The most commonly used medications include cyclophosphamide, vincristine, and prednisone (CVP); or cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP). Approximately 70% to 80% of patients will respond to their initial chemotherapy, with remission lasting 2-3 years. Ultimately, most patients relapse. The discovery and clinical use of the anti-CD20 antibody rituximab has provided significant improvements in response and survival. Currently, the standard of care for most patients is rituximab + CHOP (R-CHOP) or rituximab + CVP (R-CVP). Interferon is approved for initial treatment of NHL in combination with alkylating agents, but its use is limited in the United States.Rituximab therapy has shown efficacy in several types of NHL and is currently approved as first-line treatment for both indolent (follicular lymphoma) and aggressive NHL (diffuse large B-cell lymphoma). However, anti-CD20 monoclonal antibodies (mAbs) have significant limitations, including primary resistance (50% response rate in relapsed indolent patients), acquired resistance (50% response rate on retreatment), rare complete responses (2% complete response rate in the relapsed population), and a recurring relapse pattern. Finally, many B cells do not express CD20, therefore many B-cell disorders are untreatable with anti-CD20 antibody therapy.

[0034] Besides NHL, there are several other types of leukemia caused by abnormal B-cell regulation. Chronic lymphocytic leukemia (also known as "chronic lymphocytic leukemia" or "CLL") is an adult-onset leukemia caused by an abnormal accumulation of B lymphocytes. In CLL, malignant lymphocytes may appear normal and mature, but they are unable to effectively fight off infections. CLL is the most common form of leukemia in adults. Men are twice as likely to develop CLL as women. However, a key risk factor is age. More than 75% of new cases are diagnosed in patients over the age of 50. More than 10,000 cases are diagnosed each year, and nearly 5,000 deaths occur annually (American Cancer Society, 2006; and SEER Cancer Statistics Review). CLL is an incurable disease, but in most cases it progresses slowly. Many people with CLL live normal, active lives for many years. Due to its slow onset, early-stage CLL is often left untreated, as early intervention is believed not to improve survival or quality of life. Instead, the disease is monitored over time. Initial treatment for CLL varies depending on the accurate diagnosis and disease progression. Dozens of drugs are available for CLL treatment. Combination chemotherapy regimens such as FCR (fludarabine, cyclophosphamide, and rituximab) and BR (ibrutinib and rituximab) are effective in both newly diagnosed and relapsed CLL. Allogeneic bone marrow (stem cell) transplantation is rarely used as first-line treatment for CLL due to its extremely high risks.

[0035] Another type of leukemia is small lymphocytic lymphoma (SLL), which is considered a variant of CLL that lacks the clonal lymphocytosis required for a CLL diagnosis, but also shares the pathological and immunophenotypic features (Campo et al., 2011). The definition of SLL requires the presence of lymphadenopathy and / or splenomegaly. Furthermore, the number of B lymphocytes in peripheral blood should not exceed 5 x 102. 9 / L. In SLL, the diagnosis should be confirmed by histopathological evaluation of lymph node biopsy whenever possible (Hallek et al., 2008). In the United States, the incidence of SLL is approximately 25% of that of CLL (Dores et al., 2007).

[0036] Another type of leukemia is acute lymphoblastic leukemia (ALL), also known as acute lymphoblastic leukemia. ALL is characterized by the excessive production and continuous proliferation of malignant, immature white blood cells (also called lymphocytes) in the bone marrow. "Acute" refers to the undifferentiated, immature state of circulating lymphocytes ("blasts") and the rapid progression of the disease; without treatment, the life expectancy is weeks to months. ALL is most common in children, with a peak incidence between the ages of 4 and 5. Children aged 12-16 are more likely to die from the disease. Currently, at least 80% of childhood ALL is considered curable. Fewer than 4,000 cases are diagnosed each year, with nearly 1,500 deaths annually (American Cancer Society, 2006; and SEER Cancer Statistics Review).

[0037] The use of CD19 antibodies in nonspecific B-cell lymphoma is discussed in WO2007076950 (US2007154473), both of which are incorporated by reference. The use of CD19 antibodies in CLL, NHL, and ALL is described in Scheuermann et al.'s "CD19 Antigen in Leukemia and Lymphoma Diagnosis and Immunotherapy, Leukemia and Lymphoma, Vol. 18, 385-397 (1995)," which is also incorporated by reference.

[0038] In WO2005012493 (US7109304), WO2010053716 (US12 / 266,999) (Immunomedics); WO2007002223 (US8097703) (Me darex); WO2008022152 (12 / 377,251) and WO2008150494 (Xencor), WO2008031056 (US11 / 852,106) (Medimmune); WO 2007076950 (US11 / 648,505) (Merck Patent GmbH); WO 2009 / 052431 (US12 / 253,895) (Seattle Genetics); and WO2010095031 (12 / 710,442) (Glenmark Other CD19-specific antibodies are described in Pharmaceuticals, WO2012010562 and WO2012010561 (International Drug Development), WO2011147834 (Roche Glycart) and WO2012 / 156455 (Sanofi), all of which are incorporated herein by reference in their entirety.

[0039] The term "CD19" refers to the protein called CD19, which has the following synonyms: B4, B-lymphocyte antigen CD19, B-lymphocyte surface antigen B4, CVID3, differentiation antigen CD19, MGC12802, and T-cell surface antigen Leu-12.

[0040] Human CD19 has the following amino acid sequence:

[0041]

[0042] “MOR00208” is an anti-CD19 antibody. The amino acid sequence of the variable domain is provided by… Figure 1 The amino acid sequences of the heavy and light chain Fc regions of MOR00208 provide information for... Figure 2 In the text, “MOR00208” and “XmAb 5574” are used as synonyms to describe… Figure 1 and 2 The antibody shown is described in U.S. Patent Application No. 12 / 377,251, the entire contents of which are incorporated herein by reference.

[0043] U.S. Patent Application No. 12 / 377,251 describes the following antibody (hereinafter referred to as MOR00208) named 4G7 H1.52 Hybrid S239D / I332E / 4G7L1.155:

[0044] >4G7H1.52S239D / 1332E

[0045]

[0046] >4G7L1.155

[0047]

[0048] The pharmaceutical composition contains an active agent, such as an antibody used for human treatment. The pharmaceutical composition may additionally contain a pharmaceutically acceptable carrier or excipient.

[0049] "Giving" or "administering" means delivering a pharmaceutical composition via an injectable form, such as intravenous, intramuscular, intradermal, or subcutaneous route or mucosal route, for example, as a nasal spray or inhaled aerosol, or as a digestible solution, capsule, or tablet.

[0050] The antibody administered according to this disclosure is given to patients at a therapeutically effective amount. A “therapeutically effective amount” means an amount sufficient to provide a certain improvement in the clinical presentation of a specified disease or condition. For example, patients in the example study received a dose of 12 mg / kg MOR00208 once weekly, maintained every two weeks or once a month.

[0051] The effective dose for a specific therapeutic purpose will depend on the severity of the disease or injury, as well as the subject's weight and general condition. It should be understood that the appropriate dose can be determined using standard trials by constructing a numerical matrix and testing the differences within it, all within the general skill range of trained physicians or clinical scientists.

[0052] The baseline refers to the time before the desired treatment is administered. For example, before the desired anti-CD19 antibody is administered.

[0053] Receiver operating characteristic (ROC) analysis was used to analyze predictability, sensitivity, and specificity, and to identify potential biomarkers such as NK cell count, CD16 expression levels on NK cells, and cut-off values ​​for T cell counts. Additionally, the following methods exist for estimating the cutoff level for optimization: a) "Max.Accuracy" – the cutoff that maximizes accuracy; b) "Max.DOR" – the cutoff that maximizes the diagnostic advantage ratio; c) "Error.rate" – the cutoff that minimizes the error rate; d) "Max.Accuracy.area" – the cutoff that maximizes the area of ​​accuracy; e) "Max.Sens+Spec" – the cutoff that maximizes the total of sensitivity and specificity; f) "Max.Youden" – the cutoff that maximizes the Youden exponent; g) "Se=Sp" – the cutoff that makes sensitivity equal to specificity; h) "Min.ROC.Dist" – the cutoff that minimizes the distance between the curve and the top left corner of the image; i) "Max.Efficiency" – the cutoff that maximizes efficiency; and j) "Min.MCT" – the cutoff that minimizes the misclassification cost term. See Lopez-Raton, M., Rodriguez-Alvarez, MX, Cadarso-Suarez, C., and Gude-Sampedro, F. (2014). Optimal Cutpoints: An RPackage for Selecting Optimal Cutpoints in Diagnostic Tests. Journal of Statistical Software 61(8), 1-36.

[0054] CD19-specific antibodies have been tested in preclinical trials in combination with other drugs. For example, MOR00208 has been combined with nitrogen mustards, purine analogs, thalidomide analogs, and phosphatidylinositol 3-kinase inhibitors. BCL-2 Inhibitors It was tested against Bruton's tyrosine kinase (BTK) inhibitors.

[0055] Nitrogen mustard is a non-specific DNA alkylating agent used in chemotherapy. Alkylating agents add an alkyl group (C0.05) to the nucleic acid bases. n H 2n+1For example, an alkyl group is added to the guanine base of DNA at the 7-nitrogen atom of the imidazole ring. The alkylation step leads to the formation of interchain crosslinks (ICLs). These ICLs are highly cytotoxic because they block essential metabolic processes such as replication and transcription. Nitrogen mustards include cyclophosphamide, chlorambucil, uramustine, ifosfamide, melphalan, and bendamustine.

[0056] Bendamustine is licensed by Mundipharma International Corporation Limited (AstellasPharma GmbH) and Cephalon. and Bendamustine, also known as SDX-105, is sold under this name and is used to treat chronic lymphocytic leukemia (CLL), indolent B-cell non-Hodgkin's lymphoma (NHL), and other lymphomas. Bendamustine has the following structure:

[0057]

[0058] Purine analogues are antimetabolites that mimic the structure of metabolic purines, thereby interfering with nucleic acid synthesis. For example, fludarabine can be incorporated into DNA and RNA by substituting purine nucleotides adenine and guanine. Purine analogues inhibit the growth of rapidly proliferating cells in an individual, such as cancer cells, bone marrow cells, or cells present in the gastrointestinal tract. Purine analogues include mercaptopurine, azathioprine, thioguanine, and fludarabine. Fludarabine or fludarabine phosphate It is a chemotherapy drug used to treat chronic lymphocytic leukemia and indolent non-Hodgkin's lymphoma. Fludarabine is a purine analogue. Fludarabine inhibits DNA synthesis by interfering with ribonucleotide reductase and DNA polymerase, exhibiting S-phase specificity (because these enzymes are highly active during DNA replication). Fludarabine has the following structure:

[0059]

[0060] "Thalidomide analogues" include, but are not limited to, thalidomide itself, lenalidomide (CC-5013, Revlimid) TM Pomalidomide (CC4047, Actimid) TMCompounds disclosed in WO2002068414 and WO2005016326 are incorporated herein by reference in their entirety. The term "thalidomide analogue" refers to a synthetic compound using the thalidomide structure as its backbone (e.g., with the addition of side groups, or with these side groups removed from the parent structure). Analogs differ structurally from thalidomide and its metabolites, for example, in alkyl chain length, molecular fragmentation, one or more functional groups, or ionization variations. The term "thalidomide analogue" also includes metabolites of thalidomide. Thalidomide analogues comprise racemic mixtures of the S- and R-enantiomers of the respective compounds, or separate S- or R-enantiomers. Racemic mixtures are preferred.

[0061] Thalidomide analogues include compounds with the following structures:

[0062] (A) Lenalidomide

[0063]

[0064] "Inositol phosphokinase inhibitors" are a class of drugs that work by inhibiting one or more phosphokinases that are part of the PI3K / AKT / mTOR pathway, which is an important signaling pathway for many cellular functions such as growth control, metabolism, and translation initiation.

[0065] There are several different types and isoforms of PI3K. Class I PI3K has a catalytic subunit called p110, which has four types (isoforms)—p110α, p110β, p110γ, and p110δ. Inhibitors currently under investigation inhibit one or more isoforms of Class I PI3K.

[0066] Phosphoinositol 3-kinase inhibitors include at least Idelalisib, Duvelisib, and Copanlisib. Idelalisib is marketed by Gilead Sciences, Inc. (trade name Zydelig, also known as GS-1101 or CAL-101). Idelalisib is currently marketed for the treatment of relapsed chronic lymphocytic leukemia (CLL) in combination with rituximab in patients for whom rituximab alone is considered an appropriate therapy due to other comorbidities; relapsed follicular B-cell non-Hodgkin's lymphoma (FL) in patients who have received at least two prior systemic therapies; and relapsed small lymphocytic lymphoma (SLL) in patients who have received at least two prior systemic therapies. This substance acts as a phosphoinositol 3-kinase inhibitor; more specifically, it blocks the delta isoform P110δ of phosphoinositol 3-kinase.

[0067] The molecular formula of Idelalisib is:

[0068]

[0069] Bruton's tyrosine kinase (BTK) inhibitors are a class of drugs that exert their effects by inhibiting the tyrosine-protein kinase BTK, which plays a crucial role in B cell development. Specifically, BTK contains a pH domain that binds to (3,4,5) phosphatidylinositol triphosphate (PIP3). PIP3 binding induces BTK to phosphorylate phospholipase C, which in turn hydrolyzes phosphatidylinositol PIP2 into two second messengers, inositol triphosphate (IP3) and diacylglycerol (DAG), which then continue to regulate the activity of downstream proteins in B cell signaling.

[0070] Bruton's tyrosine kinase (BTK) inhibitors include ibrutinib. Ibrutinib is marketed by Pharmacyclics, Inc. and Johnson & Johnson's Janssen Pharmaceutical (brand name Imbruvica, also known as PCI-32765). Ibrutinib is currently marketed for the treatment of patients with mantle cell lymphoma (MCL) who have received at least one prior therapy, patients with chronic lymphocytic leukemia (CLL) who have received at least one prior therapy, and patients with CLL with 17p deletion. Patients with Alzheimer's macroglobulinemia. Ibrutinib has the molecular formula 1-[(3R)-3-[4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]-1-piperidinyl]-2-propen-1-one, and has the following structure:

[0071]

[0072] BCL-2 inhibitors are a class of drugs that work by inhibiting the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein, leading to programmed cell death. One example of a BCL-2 inhibitor is venetoclax, which is sold by AbbVie and Genentech under the brand name VENCLEXTA. TMAlso known as GDC-0199, ABT-199, and RG7601. Venetoclax is currently marketed for the treatment of patients with chronic lymphocytic leukemia (CLL) with a 17p deletion who have received at least one prior therapy, as detected by an FDA-approved test. Venetoclax has the molecular formula 4-(4-{[2-(4-chlorophenyl)-4,4-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide and has the following structure:

[0073]

[0074] “Venetok,” “ABT,” and “ABT-199” are used as synonyms in this article.

[0075] Implementation

[0076] One aspect is a method for identifying subjects with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL) who have responded to anti-CD19 antibody therapy, the method comprising:

[0077] a. Prior to treatment with the anti-CD19 antibody, a sample obtained from the subject shall be provided.

[0078] b. Determine the level of at least one biomarker selected from the following in the sample:

[0079] i. Peripheral NK cell count, and

[0080] ii. CD16 expression level on peripheral NK cells

[0081] c. Compare the level of the at least one biomarker in the sample with a predetermined cut-off level.

[0082] The level of the at least one biomarker is equal to or higher than a predetermined cutoff level as an indication that the subject will benefit from anti-CD19 antibody treatment.

[0083] In one embodiment, the sample is a blood sample. In another embodiment, the sample includes peripheral NK cells.

[0084] In one embodiment, the predetermined cutoff level for the biomarker is a baseline peripheral NK cell count of at least 50 cells / μl, at least 75 cells / μl, at least 100 cells / μl, at least 125 cells / μl, at least 150 cells / μl, at least 175 cells / μl, at least 200 cells / μl, at least 225 cells / μl, or at least 250 cells / μl. In another embodiment, the predetermined cutoff level for the biomarker is a baseline CD16 expression level on peripheral NK cells of at least 45,000 ABC, at least 60,000 ABC, at least 75,000 ABC, or at least 90,000 ABC.

[0085] In this implementation, the predetermined cut-off value for the biomarker is:

[0086] a. Baseline peripheral NK cell count is at least 50 cells / μl, or

[0087] b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 (ABC).

[0088] In this implementation, the predetermined cutoff value for the biomarker is:

[0089] a. Baseline peripheral NK cell count was at least 50 cells / μl, and

[0090] b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 (ABC).

[0091] In one embodiment, the predetermined cutoff level is a baseline peripheral NK cell count of at least 50 cells / μl. In another embodiment, the predetermined cutoff level for the biomarker is a baseline CD16 expression level on peripheral NK cells of at least 60,000 (ABC).

[0092] In this implementation, the predetermined cutoff value for the biomarker is:

[0093] a. Baseline peripheral NK cell count is at least 70 cells / μl, or

[0094] b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 (ABC).

[0095] In this implementation, the predetermined cutoff value for the biomarker is:

[0096] a. Baseline peripheral NK cell count was at least 70 cells / μl, and

[0097] b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 (ABC).

[0098] In one embodiment, the predetermined cutoff level is a baseline peripheral NK cell count of at least 70 cells / μl. In another embodiment, the predetermined cutoff level for the biomarker is a baseline CD16 expression level on peripheral NK cells of at least 60,000 (ABC).

[0099] In this implementation, the predetermined cutoff value for the biomarker is:

[0100] a. Baseline peripheral NK cell count is at least 80 cells / μl, or

[0101] b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 (ABC).

[0102] In this implementation, the predetermined cutoff value for the biomarker is:

[0103] a. Baseline peripheral NK cell count was at least 80 cells / μl, and

[0104] b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 (ABC).

[0105] In one embodiment, the predetermined cutoff level is a baseline peripheral NK cell count of at least 80 cells / μl. In another embodiment, the predetermined cutoff level for the biomarker is a baseline CD16 expression level on peripheral NK cells of at least 60,000 (ABC).

[0106] In this implementation, the predetermined cutoff value for the biomarker is:

[0107] a. Baseline peripheral NK cell count is at least 90 cells / μl, or

[0108] b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 (ABC).

[0109] In this implementation, the predetermined cutoff value for the biomarker is:

[0110] a. Baseline peripheral NK cell count was at least 90 cells / μl, and

[0111] b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 (ABC).

[0112] In one embodiment, the predetermined cutoff level is a baseline peripheral NK cell count of at least 90 cells / μl. In another embodiment, the predetermined cutoff level for the biomarker is a baseline CD16 expression level on peripheral NK cells of at least 60,000 (ABC).

[0113] In this implementation, the predetermined cutoff value for the biomarker is:

[0114] a. Baseline peripheral NK cell count is at least 100 cells / μl, or

[0115] b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 (ABC).

[0116] In this implementation, the predetermined cutoff value for the biomarker is:

[0117] a. Baseline peripheral NK cell count was at least 100 cells / μl, and

[0118] b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 (ABC).

[0119] In one embodiment, the predetermined cutoff level is a baseline peripheral NK cell count of at least 100 cells / μl. In another embodiment, the predetermined cutoff level for the biomarker is a baseline CD16 expression level on peripheral NK cells of at least 60,000 (ABC).

[0120] One aspect is a method for identifying subjects with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL) who have responded to anti-CD19 antibody therapy, the method comprising:

[0121] a. Prior to treatment with the anti-CD19 antibody, a blood sample obtained from the subject shall be provided.

[0122] b. Determine the level of at least one biomarker selected from the following in the sample:

[0123] i. Peripheral NK cell count, and

[0124] ii. CD16 expression level on peripheral NK cells

[0125] c. Compare the level of the at least one biomarker in the sample with a predetermined cutoff level.

[0126] The baseline peripheral NK cell count was at least 50 cells / μl, at least 60 cells / μl, at least 70 cells / μl, at least 80 cells / μl, at least 90 cells / μl, or at least 100 cells / μl, and the baseline CD16 expression level on peripheral NK cells was at least 60,000 (ABC). The anti-CD19 antibody included: the HCDR1 region including the sequence SYVMH (SEQ ID NO:1), the HCDR2 region including the sequence NPYNDG (SEQ ID NO:2), the HCDR3 region including the sequence GTYYYGTRVFDY (SEQ ID NO:3), the LCDR1 region including the sequence RSSKSLQNVNGNTYLY (SEQ ID NO:4), the LCDR2 region including the sequence RMSNLNS (SEQ ID NO:5), and the LCDR3 region including the sequence MQHLEYPIT (SEQ ID NO:6).

[0127] One aspect is a method for identifying subjects with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL) who have responded to anti-CD19 antibody therapy, the method comprising:

[0128] a. Prior to treatment with the anti-CD19 antibody, a blood sample obtained from the subject shall be provided.

[0129] b. Determine the level of at least one biomarker selected from the following in the sample:

[0130] i. Peripheral NK cell count, and

[0131] ii. CD16 expression level on peripheral NK cells

[0132] c. Compare the level of the at least one biomarker in the sample with a predetermined cutoff level.

[0133] The baseline peripheral NK cell count is at least 100 cells / μl, or the baseline CD16 expression level on peripheral NK cells is at least 60,000 (ABC), and the anti-CD19 antibody comprises: an HCDR1 region including the sequence SYVMH (SEQ ID NO:1), an HCDR2 region including the sequence NPYNDG (SEQ ID NO:2), an HCDR3 region including the sequence GTYYYGTRVFDY (SEQ ID NO:3), an LCDR1 region including the sequence RSSKSLQNVNGNTYLY (SEQ ID NO:4), an LCDR2 region including the sequence RMSNLNS (SEQ ID NO:5), and an LCDR3 region including the sequence MQHLEYPIT (SEQ ID NO:6).

[0134] In this implementation, the predetermined cutoff value for the biomarker is:

[0135] a. Baseline peripheral NK cell count was at least 100 cells / μl, and

[0136] b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 (ABC).

[0137] In one embodiment, the predetermined cutoff level for the biomarker is a baseline peripheral NK cell count of at least 50 cells / μl, at least 75 cells / μl, at least 100 cells / μl, at least 125 cells / μl, at least 150 cells / μl, at least 175 cells / μl, at least 200 cells / μl, at least 225 cells / μl, or at least 250 cells / μl. In another embodiment, the predetermined cutoff level for the biomarker is a baseline CD16 expression level on peripheral NK cells of at least 45,000 ABC, at least 60,000 ABC, at least 75,000 ABC, or at least 90,000 ABC.

[0138] One approach is to treat patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL) using anti-CD19 antibodies. This approach includes:

[0139] a. Obtain the patient's baseline peripheral NK cell count, or the patient's baseline CD16 expression level on peripheral NK cells, and

[0140] b. Administer an effective amount of anti-CD19 antibody to patients with a baseline peripheral NK cell count of at least 50 cells / μl, at least 60 cells / μl, at least 70 cells / μl, at least 80 cells / μl, at least 90 cells / μl, or at least 100 cells / μl, or a baseline CD16 expression level on peripheral NK cells of at least 60,000 (ABC).

[0141] One approach is to treat patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL) using anti-CD19 antibodies. This approach includes:

[0142] a. Obtain the patient's baseline peripheral NK cell count, or the patient's baseline CD16 expression level on peripheral NK cells, and

[0143] b. Administer an effective amount of anti-CD19 antibody to patients with a baseline peripheral NK cell count of at least 100 cells / μl or a baseline CD16 expression level on peripheral NK cells of at least 60,000 (ABC).

[0144] In one implementation, the method of treating patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL) with anti-CD19 antibodies includes:

[0145] a. Obtain the patient's baseline peripheral NK cell count, and

[0146] b. Administer an effective amount of anti-CD19 antibody to patients with NK cell counts of at least 50 cells / μl, at least 60 cells / μl, at least 70 cells / μl, at least 80 cells / μl, at least 90 cells / μl, or at least 100 cells / μl.

[0147] In one implementation, the method of treating patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL) with anti-CD19 antibodies includes:

[0148] a. Obtain the patient's baseline peripheral NK cell count, and

[0149] b. Administer an effective amount of anti-CD19 antibody to patients with NK cell counts of at least 50 cells / μl, at least 60 cells / μl, at least 70 cells / μl, at least 80 cells / μl, at least 90 cells / μl, or at least 100 cells / μl.

[0150] In one implementation, the method of treating patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL) with anti-CD19 antibodies includes:

[0151] a. Obtain the patient's baseline peripheral NK cell count, and

[0152] b. Administer an effective amount of anti-CD19 antibody to patients with an NK cell count of at least 100 cells / μl.

[0153] In one implementation, the method of treating patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL) with anti-CD19 antibodies includes:

[0154] a. Obtain the baseline CD16 expression level on the patient's peripheral NK cells, and

[0155] b. Administer an effective amount of anti-CD19 antibody to patients with a CD16 level of at least 60,000 (ABC) on NK cells.

[0156] In one implementation, an anti-CD19 antibody is given to a patient with a baseline peripheral NK cell count of at least 50 cells / μl, at least 60 cells / μl, at least 70 cells / μl, at least 80 cells / μl, at least 90 cells / μl, or at least 100 cells / μl and a baseline CD16 expression level on peripheral NK cells of at least 60,000 (ABC).

[0157] In one implementation, an anti-CD19 antibody is given to a patient with a baseline peripheral NK cell count of at least 100 cells / μl and a baseline CD16 expression level of at least 60,000 (ABC) on peripheral NK cells.

[0158] In one embodiment, anti-CD19 antibody is administered to patients with baseline peripheral NK cell counts of at least 50 cells / μl, at least 75 cells / μl, at least 100 cells / μl, at least 125 cells / μl, at least 150 cells / μl, at least 175 cells / μl, at least 200 cells / μl, at least 225 cells / μl, or at least 250 cells / μl. In another embodiment, anti-CD19 antibody is administered to patients with baseline CD16 expression levels on peripheral NK cells of at least 45,000 ABC, at least 60,000 ABC, at least 75,000 ABC, or at least 90,000 ABC.

[0159] One aspect is a method of treating patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL) using an anti-CD19 antibody, the method comprising:

[0160] a. Prior to treatment with the anti-CD19 antibody, a sample obtained from the subject shall be provided.

[0161] b. Determine the level of at least one biomarker selected from the following in the sample:

[0162] i. Peripheral NK cell count, and

[0163] ii. CD16 expression level on peripheral NK cells

[0164] c. Compare the level of the at least one biomarker in the sample with a predetermined cutoff level.

[0165] d. Administer an effective amount of anti-CD19 antibody to patients with a peripheral NK cell count of at least 100 cells / μl and a CD16 expression level of at least 60,000 on peripheral NK cells (ABC).

[0166] One aspect is a treatment method for patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL), which includes:

[0167] if

[0168] a. The patient's baseline peripheral NK cell count is at least 100 cells / μl, or

[0169] b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 (ABC).

[0170] The patient will then be given an effective amount of anti-CD19 antibody.

[0171] One aspect is a treatment method for patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL), which includes:

[0172] a. Obtain the patient's peripheral NK cell count.

[0173] b. Administer an effective amount of anti-CD19 antibody to patients with a peripheral NK cell count of at least 100 cells / μl.

[0174] One aspect is a treatment method for patients with chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), acute lymphoblastic leukemia (ALL), or small lymphocytic lymphoma (SLL), which includes:

[0175] a. Obtain the CD16 expression level on the patient's peripheral NK cells.

[0176] b. Administer an effective amount of anti-CD19 antibody to patients with a CD16 expression level of at least 60,000 (ABC) on peripheral NK cells.

[0177] In one embodiment, an anti-CD19 antibody is administered to a patient with a baseline peripheral NK cell count of at least 100 cells / μl and a baseline CD16 expression level on peripheral NK cells of at least 60,000 (ABC). In another embodiment, an anti-CD19 antibody is administered to a patient with a baseline peripheral NK cell count of at least 50 cells / μl, at least 75 cells / μl, at least 100 cells / μl, at least 125 cells / μl, at least 150 cells / μl, at least 175 cells / μl, at least 200 cells / μl, at least 225 cells / μl, or at least 250 cells / μl. In yet another embodiment, an anti-CD19 antibody is administered to a patient with a baseline CD16 expression level on peripheral NK cells of at least 45,000 ABC, at least 60,000 ABC, at least 75,000 ABC, or at least 90,000 ABC.

[0178] In this implementation, baseline peripheral NK cell count or baseline CD16 expression level (ABC) on peripheral NK cells is obtained from a blood sample taken from the patient. In this implementation, peripheral NK cell count and / or CD16 expression level (ABC) are measured prior to administration of anti-CD19 antibody.

[0179] In an embodiment, the CD19-specific antibody includes: an HCDR1 region comprising the sequence SYVMH (SEQ ID NO:1), an HCDR2 region comprising the sequence NPYNDG (SEQ ID NO:2), an HCDR3 region comprising the sequence GTYYYGTRVFDY (SEQ ID NO:3), an LCDR1 region comprising the sequence RSSKSLQNVNGNTYLY (SEQ ID NO:4), an LCDR2 region comprising the sequence RMSNLNS (SEQ ID NO:5), and an LCDR3 region comprising the sequence MQHLEYPIT (SEQ ID NO:6).

[0180] In this implementation, baseline peripheral NK cell count or baseline CD16 expression level on peripheral NK cells is obtained from a blood sample taken from the patient.

[0181] In one embodiment, the patient has non-Hodgkin's lymphoma. In another embodiment, the non-Hodgkin's lymphoma is selected from follicular lymphoma, small lymphocytic lymphoma, mucosa-associated lymphoid tissue (MALT), marginal zone lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, and mantle cell lymphoma. In one embodiment, the non-Hodgkin's lymphoma is follicular lymphoma. In another embodiment, the non-Hodgkin's lymphoma is indolent non-Hodgkin's lymphoma. In another embodiment, the non-Hodgkin's lymphoma is small lymphocytic lymphoma. In another embodiment, the non-Hodgkin's lymphoma is MALT. In another embodiment, the non-Hodgkin's lymphoma is marginal zone lymphoma. In another embodiment, the non-Hodgkin's lymphoma is diffuse large B-cell lymphoma. In another embodiment, the non-Hodgkin's lymphoma is Burkitt's lymphoma. In another embodiment, the non-Hodgkin's lymphoma is mantle cell lymphoma. In yet another embodiment, the patient has chronic lymphocytic leukemia. In one implementation, the patient has acute lymphoblastic leukemia. In another implementation, the patient has small lymphocytic lymphoma (SLL).

[0182] In this implementation, the treatment produces therapeutic effects selected from disease control rate (DCR) and longer progression-free survival.

[0183] In one embodiment, the treatment further includes administering an effective amount of nitrogen mustard. In another embodiment, the nitrogen mustard is bendamustine. In another embodiment, the treatment further includes administering an effective amount of a purine analogue. In another embodiment, the purine analogue is fludarabine. In another embodiment, the treatment further includes administering an effective amount of a Bruton's tyrosine kinase (BTK) inhibitor. In another embodiment, the Bruton's tyrosine kinase (BTK) inhibitor is ibrutinib. In another embodiment, the treatment further includes administering an effective amount of a phosphoinositol 3-kinase inhibitor. In another embodiment, the phosphoinositol 3-kinase inhibitor is idelalisib. In another embodiment, the treatment further includes administering an effective amount of a thalidomide analogue. In another embodiment, the thalidomide analogue is lenalidomide. In another embodiment, the treatment further includes administering an effective amount of a BCL-2 inhibitor. In another embodiment, the BCL-2 inhibitor is venetoclax.

[0184] Since the example anti-CD19 antibody and other anti-CD19 antibodies bind to CD19, it is believed that other anti-CD19 antibodies can achieve similar results. Other anti-CD19 antibodies are described in U.S. Patent Application No. 12 / 377,251 (Xencor), WO2005012493, WO2010053716 (Immunomedics); WO2007002223 (Medarex); WO2008022152 (Xencor); WO2008031056 (Medimmune); WO 2007 / 076950 (Merck Patent GmbH); WO 2009 / 052431 (Seattle Genetics); and WO2010095031 (Glenmark Pharmaceuticals), all of which are incorporated herein by reference in their entirety.

[0185] In an embodiment, the CD19-specific antibody includes an antibody that cross-competes with antibodies including: an HCDR1 region comprising the sequence SYVMH (SEQ ID NO:1), an HCDR2 region comprising the sequence NPYNDG (SEQ ID NO:2), an HCDR3 region comprising the sequence GTYYYGTRVFDY (SEQ ID NO:3), an LCDR1 region comprising the sequence RSSKSLQNVNGNTYLY (SEQ ID NO:4), an LCDR2 region comprising the sequence RMSNLNS (SEQ ID NO:5), and an LCDR3 region comprising the sequence MQHLEYPIT (SEQ ID NO:6).

[0186] In an embodiment, the CD19-specific antibody includes an antibody that binds to the same epitope as an antibody including the following: an HCDR1 region including the sequence SYVMH (SEQ ID NO:1), an HCDR2 region including the sequence NPYNDG (SEQ ID NO:2), an HCDR3 region including the sequence GTYYYGTRVFDY (SEQ ID NO:3), an LCDR1 region including the sequence RSSKSLQNVNGNTYLY (SEQ ID NO:4), an LCDR2 region including the sequence RMSNLNS (SEQ ID NO:5), and an LCDR3 region including the sequence MQHLEYPIT (SEQ ID NO:6).

[0187] In this embodiment, the CD19-specific antibody includes the HCDR1 region of the sequence SYVMH (SEQ ID NO:1), the HCDR2 region of the sequence NPYNDG (SEQ ID NO:2), the HCDR3 region of the sequence GTYYYGTRVFDY (SEQ ID NO:3), the LCDR1 region of the sequence RSSKSLQNVNGNTYLY (SEQ ID NO:4), the LCDR2 region of the sequence RMSNLNS (SEQ ID NO:5), and the LCDR3 region of the sequence MQHLEYPIT (SEQ ID NO:6).

[0188] In an embodiment, the CD19-specific antibody comprises a variable heavy chain of the sequence EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWG QGTLVTVSS (SEQ ID NO:10) and a variable light chain of the sequence DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIK (SEQ ID NO:11).

[0189] In an embodiment, the antibody comprises the heavy chain constant region of the sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:12).

[0190] In an embodiment, the CD19-specific antibody includes the light chain constant region of the sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:13).

[0191] In the implementation, the CD19-specific antibody includes the sequence EVQLVESGGGLVKPGGSLKLSCAASGYTFTSYVMHWVRQAPGKGLEWIGYINPYNDGTKYNEKFQGRVTISSDKSISTAYMELSSLRSEDTAMYYCARGTYYYGTRVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKALPAPE EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ The heavy chain of ID NO:8).

[0192] In an embodiment, the CD19-specific antibody comprises a light chain with the sequence DIVMTQSPATLSLSPGERATLSCRSSKSLQNVNGNTYLYWFQQKPGQSPQLLIYRMSNLNSGVPDRFSGSGSGTEFTLTISSLEPEDFAVYYCMQHLEYPITFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9).

[0193] The embodiments include pharmaceutical compositions. In the embodiments, the composition includes an acceptable carrier. In the embodiments, the composition is administered in an effective amount.

[0194] Example

[0195] Example 1: T cell and NK cell count

[0196] The scope of the MOR00208C201 clinical trial includes the evaluation of several exploratory biomarkers. As part of this first-in-class protocol, baseline peripheral T and NK cell counts were performed at the clinical site.

[0197] T cells are a type of lymphocyte (a subtype of leukocyte) that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of T-cell receptors on their cell surface.

[0198] Natural killer (NK) cells are a type of cytotoxic lymphocyte that is crucial for the innate immune system. NK cells provide a rapid response to virus-infected cells, taking effect approximately 3 days after infection and responding to viral formation. Typically, immune cells detect the major histocompatibility complex (MHC) present on the surface of infected cells, triggering the release of cytokines that lead to cell lysis or apoptosis. However, NK cells are unique because they possess the ability to recognize stressed cells in the absence of antibodies and MHC, allowing for a much faster immune response.

[0199] Materials and methods

[0200] TriTest CD3 FITC / CD16+CD56 PE / CD45 PerCP (with TruCOUNT tubes): BD Biosciences, Cat: 340403 (US); 342442 (Europe). Pipettes and pipette tips available in 20μL, 50μL, and 450μL sizes: Gilson Inc. FACS lysis buffer: BD Biosciences, Cat: 349202

[0201] Instruments: Flow cytometer, Vortex mixer

[0202] Flow cytometry background

[0203] Whole blood is stained with an antibody labeled with a fluorescent dye that specifically binds to leukocyte surface antigens (TriTEST reagent). Cells pass through a laser beam and scatter the laser light. The stained cells fluoresce. These scattering and fluorescence signals detected by the instrument provide information about cell size, internal complexity, and relative fluorescence intensity. TriTEST reagent uses fluorescence triggering, allowing direct fluorescence gating of NK- and T-cell lymphocyte populations to reduce contamination from unlysplitted or nucleated red blood cells within the gate.

[0204] dyeing

[0205] For each patient sample, label the TruCOUNT tube with the sample identification number. Pipe 20 μL of TriTEST CD3 / CD16+CD56 / CD45 reagent to the bottom of the tube. Pipe 50 μL of well-mixed anticoagulated whole blood to the bottom of the tube. Anticoagulated blood (EDTA) stored at room temperature (20-25°C) must be stained within 24 hours of extraction and analyzed within 6 hours of staining (keeping at room temperature and protected from light). Gently vortex the tube to mix. Incubate the tube in the dark at room temperature (20-25°C) for 15 minutes. Add 450 μL of 1X FACS lysis buffer to the tube. Vortex the tube and incubate again in the dark at room temperature (20-25°C) for 15 minutes.

[0206] Using TruCOUNT tubes, a known volume of sample is stained directly into the tube. The lyophilized microspheres dissolve in the tube, releasing a known number of fluorescent beads. During analysis, the absolute number of positive cells in the sample (cells / μL) is determined by comparing the number of cell events with the number of bead events.

[0207] Flow cytometry

[0208] Before running the cells on a flow cytometer, vortex them thoroughly (at low speed) to reduce aggregation.

[0209] Data Analysis

[0210] Visually examine the dotted plot of CD45 relative to SSC. Lymphocytes appear as bright, compact cell populations with low to moderate SSC. Monocytes (M) and granulocytes (G) appear as distinct populations. Analysis is completed once monocytes and lymphocytes show clear separation.

[0211] Lymphocytes are first gated to a CD45-positive, low-SSC cell population. CD16 / CD56 is preselected relative to CD3. T cells (T) should appear as compact, bright CD3-positive clusters. NK cells (NK) should appear as compact, bright CD16 / CD56-positive clusters. Gating is then completed, and T and NK cells are counted.

[0212] Bead event counting is performed using a CD16 / CD56 plot relative to CD3 without any pre-selected gates. Beads should appear as PE / FITC double-positive clusters.

[0213] Calculate absolute count

[0214] The absolute number of T cells or NK cells in the sample (cells / μL blood) was determined by comparing the number of cellular events with the number of bead events. Data analysis was performed using MultiSET software or manually (using CellQuest or other software). For manual counting, the number of positive cellular events (#) obtained was divided by the number of bead events obtained (#), and then multiplied by (total TruCOUNT bead count (lot dependent)) divided by the whole blood sample volume of 50 μL). The result is the absolute cell count / µL.

[0215] equation :

[0216]

[0217] Example :

[0218]

[0219] Example 2: Quantification of CD16 on NK cells

[0220] As part of the MOR00208C201 clinical study, CD16 (an exploratory biomarker) on peripheral NK cells was quantified centrally at ICON Central Laboratories (Farmingdale, New York).

[0221] Materials and methods

[0222] Antibodies: CD45 AmCyan (Clone 2D1, BD Biosciences, Cat#339192); CD3 FITC (Clone UCHT1, BioLegend, Cat#300406); mouse IgG FITC (clone MOPC-21, BioLegend, Cat#400110); CD16 PE (clone 3G8, BioLegend, Cat#302008); MOR00208; mouse IgG PE (mouse MOPC-21, BioLegend, Cat#400114); CD56 PerCP-Cy5.5 (clone HCD56, BioLegend, Cat#318322); and mouse IgG PerCP-Cy5.5 (clone MOPC-21, BioLegend, Cat#400150).

[0223] Materials: PharmaTherm heat-insulating packaging (Intelsius, Catalog #PHT014); BD CPT TM Mononuclear Cell Preparation Tube-Sodium Heparin(16x125mm / 8mL)(BD,Catalog#362753); BD Falcon TM 12x75mm round-bottom tubes (BD, Catalog #352052); CS&T beads (BD Biosciences Cat #642212); fetal bovine serum (FBS), heat-inactivated (Sigma F4135, or equivalent); Dulbecco's PBS, Ca-free. ++ and Mg ++ (Gibco, Cat#14190, or equivalent); BD Falcon, cell filter, 100 μm, yellow (BD Bioscience, Cat#352360); FACS buffer, 3% heat-inactivated FBS in 1X DPBS; deionized water, laboratory stock; crushed (wet) ice; ice bucket; aluminum foil; conical tube, 50 mL; conical tube, 15 mL; sterile, filter pipette tip; BD Pharm Lyse lysis buffer (BD Biosciences, Cat#555899); ViViD LIVE / A kit for fixing purple dead cells for 405nm excitation (Life Technologies, Cat#L34955); ArC amine reactive beads (Life Technologies, Cat#A10346); BD QuantiBRITE beads (BD Biosciences, Cat#340495); and 52μm nylon mesh (Miami Aqua Culture, for Cat:Nylon 52μm, with 32% open region woven into the material).

[0224] Equipment: Centrifuge (refrigeration capacity); Lab quake (tube shaker); vortex mixer; laminar flow hood; incubator (set to 37°C, 5% CO2); Advia (cell counter); BD FACSCANTO II flow cytometer; Desi-Vac TM Container, 1.5 liters (VWR, Cat#62344-930); Humidity Sponge TM Indicating(VWR,Cat#61161-319); and Traceable Humidity-On-A-Card(VWR,Cat#15551-012).

[0225] Table 1. Quantitative analysis of CD16 in PBMCs (peripheral blood mononuclear cells)

[0226]

[0227]

[0228] PBMC Preparation and Labeling Methods

[0229] Peripheral blood from patients was collected in CPT tubes and transported overnight from the clinical site to the central laboratory in insulated packaging. The CPT tubes were centrifuged at 1800 x g for 25 minutes at RT with the brakes engaged. Immediately after centrifugation, the CPT tubes were inverted and placed on a labquake for 10 minutes to resuspend the PBMC layer in autologous plasma and dissolve most of the formed cell aggregates. Under aseptic conditions, the homogenized PBMC / plasma suspension was slowly decanted into the center of a 100 μm cell strainer placed atop a 50 mL sterile conical tube. An equal volume of 1X DPBS was added to the PBMC / plasma suspension (approximately 4 mL) in the 15 mL conical tube. The tube was centrifuged at 300 x g for 10 minutes at 4°C, and the supernatant was removed. The tube was vortexed to resuspend the cell clumps. The cell clumps were washed with DBPS, centrifuged, the supernatant removed, and resuspended by vortexing. Add the washed PBMC suspension to an Eppendorf microcentrifuge tube containing 1 μL of ViViD stock solution; incubate on ice for 15 minutes in the dark (covered with aluminum foil). Transfect the stained ViViD PBMCs into newly labeled conical tubes, then add ice-cold FACS buffer. Centrifuge the cells and vortex again for resuspension. Label polystyrene centrifuge tubes for each sample (Table 1). Add the antibody or allotype control antibody to the appropriate tube. Add an aliquot of ViViD-stained PBMCs (Table 1) to each tube. Vortex the tubes and incubate. Add FACS buffer, centrifuge the cells, and vortex again for resuspension. Add BD Pharm Lyse lysis buffer, vortex the cells, centrifuge, and aspirate to remove the supernatant, then vortex again for resuspension. Add FACS buffer again, centrifuge the cells, and vortex again for resuspension. The samples were then fed into a FACSCanto II hematology counter, and the ABC (Antibodies Bound Per Cell) was inferred from the normalized MFI, as described in the following literature: Iyer S et al., Expression of CD69 on activated T cells using R-phycoerythrinlabeled beads, Cytometry, 1996; #AC78(Suppl.8):113 and Iyer S et al., QuantiBRITE: A New Standard for Fluorescence Quantitation, Becton Dickinson Immunocytometry Systems, San Jose, CA. 1997. White Paper.

[0230] Example 3: NHL test

[0231] The study of Fc-Optimized Anti-CD19 Antibody (MOR00208) to Treat Non-Hodgkin's Lymphoma (NHL) (ClinicalTrials.gov ID NCT01685008) is no longer recruiting.

[0232] The selection criteria are as follows: :

[0233] 1. Male or female patients aged ≥18 years.

[0234] 2. Histologically confirmed according to REAL / WHO classification, with the following B-cell lymphomas: a. FL, b. MCL, c. DLBCL, d. other indolent NHL (e.g., MZL / MALT).

[0235] 3. The patient's NHL must have progressed after at least one prior regimen containing rituximab.

[0236] 4. A measurable disease site as measured by magnetic resonance imaging (MRI) or computed tomography (CT) is defined as at least one lesion measuring at least 1.5 × 1.5 cm, with the following exceptions: for patients with only MCL, patients with unmeasurable disease but with evaluable sites (bone marrow, spleen, peripheral blood, gastrointestinal tract) may be recruited.

[0237] 5. Patients who have previously received autologous stem cell transplantation must be at least 4 weeks post-transplantation before receiving the study drug and must have demonstrated complete hematologic recovery.

[0238] 6. The investigational drug may be administered only after at least 60 days of discontinuous prior monoclonal antibody therapy (except rituximab) or radioimmunotherapy.

[0239] 7. Rituximab should be discontinued for at least 14 days before screening visits can be conducted to confirm no response or disease progression after rituximab treatment.

[0240] 8. DLBCL patients have a positive [18F] fluorodeoxyglucose-positron emission tomography (FDG-PET) scan at baseline (Cheson response criteria).

[0241] 9. Predicted survival > 3 months.

[0242] 10. ECOG performance status < 3.

[0243] 11. Laboratory criteria for screening: a) Absolute neutrophil count (ANC) ≥ 1.0 (1000 / mm²) 3 b) Platelet count ≥75×10 9 c) Hemoglobin ≥ 8.0 g / dL (can be administered via transfusion). d) Serum creatinine < 2.0 x Upper Limit of Normal (ULN). e) Total bilirubin ≤ 2.0 x ULN. f) Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) ≤ 2.5 x ULN.

[0244] 12. If a woman is likely to become pregnant, she must confirm a negative pregnancy test before registration, use dual barrier contraception (oral contraceptive plus barrier device), or confirm that she has undergone clinically proven total hysterectomy and / or oophorectomy, or tubal ligation.

[0245] 13. If the patient is male and has been sexually active with a woman who may become pregnant, he must use an effective barrier method of contraception during the study period and for 3 months after the last dose.

[0246] 14. Able to comply with all research-related processes, drug use, and evaluations.

[0247] 15. Able to understand and provide written informed consent and comply with the research protocol.

[0248] Exclusion criteria are as follows :

[0249] 1. Screening patients who have received prior treatment with cytotoxic chemotherapy, immunotherapy, radiotherapy or other lymphoma-specific therapies within 14 days prior to the screening visit, or who have not yet recovered from the side effects of prior lymphoma-specific treatment.

[0250] 2. Systemic experimental drug treatment within 28 days prior to the screening visit.

[0251] 3. Prior treatment with anti-CD19 antibodies or fragments.

[0252] 4. Prior allogeneic stem cell transplantation.

[0253] 5. Known or suspected hypersensitivity to excipients contained in the study drug formulation.

[0254] 6. Clinically significant cardiovascular disease or heart failure, cardiomyopathy, pre-existing clinically significant arrhythmia, acute myocardial infarction within the past 3 months, or angina pectoris within the past 3 months.

[0255] 7. Clinical or laboratory evidence of active hepatitis B or hepatitis C.

[0256] 8. History of HIV infection.

[0257] 9. Any active systemic infection (viral, fungal, or bacterial) requiring aggressive parenteral antibiotic treatment within 4 weeks of administration of the study drug.

[0258] 10. Currently treated with immunosuppressants other than prescribed corticosteroids (not exceeding 10 mg prednisone equivalent).

[0259] 11. Major surgery or radiation therapy within 4 weeks prior to the first administration of the investigational drug.

[0260] 12. In the researchers' view, this would prevent the research and treatment of systemic diseases (cardiovascular, renal, hepatic, etc.).

[0261] 13. Central nervous system (CNS), meningeal, or epidural diseases, including a history of brain metastases or clinical evidence.

[0262] 14. Aggressive treatment / chemotherapy for other primary malignant tumors within the past 5 years.

[0263] 15. Pregnant or breastfeeding women, and women who are likely to become pregnant and are not using an acceptable method of contraception.

[0264] 16. Patients who do not adhere to their medical history or are considered potentially uncooperative or unreliable.

[0265] Patients were treated with MOR00208 as follows: Patients were treated in two 28-day cycles, with MOR00208 administered at a dose of 12 mg / kg on days 1, 8, 15, and 22. At the end of the two cycles, patients with stable or better condition were treated in a third 28-day cycle using the same dose and regimen as the first two cycles. At the end of the third cycle, patients with a partial response or better entered the maintenance phase. During the maintenance phase, MOR00208 was administered at a dose of 12 mg / kg every 14 or 28 days until disease progression.

[0266] At the end of the study, the patient characteristics were as follows:

[0267] Table 2

[0268]

[0269]

[0270] DLBCL, diffuse large B-cell lymphoma; ECOG PS, Eastern Cooperative Oncology Group performance status; iNHL, indolent non-Hodgkin's lymphoma (including follicular lymphoma and other iNHL); MCL, mantle cell lymphoma; mos, month. (%)

[0271] Other iNHL refers to indolent, non-invasive NHL types in the heterologous group that are not further specified, such as limbic cell lymphoma, marginal zone lymphoma, and mucus-associated lymphoid tissue (MALT) lymphoma.

[0272] The key primary and secondary endpoints are as follows:

[0273] • First and foremost: Overall Response Rate (ORR) = CR + PR

[0274] ·second:

[0275] Disease control rate (DCR) = CR + PR + SD

[0276] Progression-free survival (PFS)

[0277] Table 3:

[0278]

[0279] Data is n (%). *Researcher evaluation. This includes follicular lymphoma and other indolent NHL. No baseline response assessment / data unavailable. § n = 25, 40, 11, and 76, respectively. DCR, Disease Control Rate; DLBCL, Diffuse Large B-Cell Lymphoma; iNHL, Indolent Non-Hodgkin's Lymphoma; MCL, Mantle Cell Lymphoma; ORR, Overall Response Rate.

[0280] The response criteria in this study are defined in Table 4. They are all based on the International Working Group Response Criteria (2007).

[0281] Table 4: Reaction Standards

[0282]

[0283] Abbreviations: CR, complete remission; FDG, [ 18 F] Fluorodeoxyglucose; PET, Positron Emission Tomography; CT, Computed Tomography; PR, Partial Remission; SPD, Sum of the Products of Diameters; SD, Stable Disease; PD, Disease Progression.

[0284] In this trial, DCR (CR+PR+SD) was considered the most relevant power endpoint in the patient characteristics and biomarker analysis because most SD patients had significant reduction in the target lesion, but the study design itself did not include treatment beyond the third cycle. Therefore, patients with SD were included in the analysis.

[0285] Patients were evaluated for at least the following characteristics to determine if there was a correlation between characteristics of patients treated with anti-CD19 antibodies and observed disease control rate (DCR): a) age, b) sex, c) whether the patient had received rituximab doses within the last 6 months, d) whether the patient was rituximab refractory, e) whether the patient had high or low affinity alleles of FCγRIIIa, f) whether the patient had high or low affinity alleles of FCγRIIa, g) whether the patient had a duration of response to previous treatment longer than 12 months, h) baseline peripheral T cell count (cells / μl), i) baseline peripheral NK cell count (cells / μl), and j) baseline CD16 expression on peripheral NK cells (antibody-ABC bound per cell).

[0286] Baseline peripheral NK cell counts, T cell counts, and baseline CD16 expression on peripheral NK cells were evaluated using Examples 1 and 2 above. The data are shown in Table 2.

[0287] Predictability, sensitivity, and specificity were analyzed using receiver operating characteristic (ROC) analysis, and cutoff values ​​were determined for potential biomarkers NK cell count, T cell count, and CD16 expression levels on peripheral NK cells (ABC). ROC plots show the performance of binary classifiers with sequential or disjoint outputs. As the output thresholds are shifted across all possible values, they indicate sensitivity (the proportion of correctly classified positive observations) and specificity (the proportion of correctly classified negative observations). See Swets JA: The Relative Operating Characteristic in Psychology. Science 1973, 182: 990–1000; and Pepe MS: The statistical evaluation of medical tests for classification and prediction. Oxford: Oxford University Press; 2003. In the ROC context, the area under the curve (AUC) measures the performance of the classifier and is therefore frequently used for comparing methods. A higher AUC indicates better classification. The AUCs for peripheral NK / T cell count and CD16 expression on NK cells were 0.66, 0.53, and 0.61, respectively. Figure 3 , 4 and 5).

[0288] Typically, the determination of the cutoff depends on the objectives involved in each method. Various criteria, such as maximum precision, maximum diagnostic advantage ratio, minimum error rate, maximum sensitivity, and / or maximum specificity, will lead to the determination of different cutoffs. Furthermore, further criteria, such as the balance between sensitivity and specificity, will also lead to the determination of a specific cutoff.

[0289] Therefore, there are several methods or criteria for selecting the optimal cutoff, including those that maximize precision, sensitivity plus specificity, predictive value, diagnostic probability ratio, or prevalence. Due to the asymmetry of the CD16 expression-ROC curve (see...),... Figure 4 Most methods yield a cutoff of 60,000ABC (the point where the distance between the ROC curve and the bisection line is greatest), however, the symmetry of the NK cell count ROC curve (see...) Figure 3This explains why different optimal cutoff values ​​can be obtained when different methods are applied. In this specific study, more weight was allocated to sensitivity for the two biomarkers; therefore, CD16 expression levels of 100 NK cells / μl and 60,000 ABC were selected as cutoff values ​​to analyze DCR and PFS within subgroups. For peripheral T cell counts, the AUC was 0.53, and the ROC curve was close to the bisection line at any specificity and sensitivity values. Therefore, even choosing different cutoff values ​​greater than 500 cells / μl did not affect the negative results of DCR and PFS subgroup analyses.

[0290] The determination of the cutoff value can also be balanced to favor sensitivity or specificity. Even if more weight is allocated to sensitivity in determining the optimal cutoff value, the methodology will differ, and a lower cutoff value for NK cell counts is expected. In this case, the cutoff value is determined to be at least 50 NK cells / μl. Alternatively, the cutoff value can be determined to be at least 60 NK cells / μl, at least 70 NK cells / μl, at least 80 NK cells / μl, at least 90 NK cells / μl, or at least 100 NK cells / μl.

[0291] To maximize the specificity of the disclosed method, the cutoff value for NK cell counting is increased and determined to be between at least 100 NK cells / μl and at least 150 NK cells / μl. Therefore, for maximizing specificity, the cutoff value is selected to be at least 100 NK cells / μl, at least 110 NK cells / μl, at least 120 NK cells / μl, at least 130 NK cells / μl, at least 140 NK cells / μl, or at least 150 NK cells / μl.

[0292] The cutoff values ​​determined in this specific study (CD16 expression level of 100 NK cells / μl and 60,000 ABC) were used for the following statistical analysis.

[0293] Forest plot analysis was used to analyze all patient characteristics and biomarkers to determine the correlation between each characteristic and DCR. Results are shown in... Figure 7 Based on forest plot analysis of different patient characteristics and their correlation with DCR, the following characteristics showed statistically significant differences between DLBCL and iNHL patients: 1) baseline peripheral NK cell count of approximately 100 cells / μl and baseline CD16 expression on peripheral NK cells of approximately 60,000 ABC(χ²). 2 Unadjusted p-value = 0.029 / 0.003 Figure 7 ).

[0294] To ensure that CD16 expression and NK cell count were independent properties and did not influence each other, parametric and nonparametric correlation analyses were performed. Data on CD16 expression and NK cell count from 51 patients were available. The Pearson correlation coefficient (Pearson's r) was 0.019 with a two-tailed p-value of 0.9, and the Spearman correlation coefficient (Spearman's r) was 0.036 with a two-tailed p-value of 0.8. The results are illustrated in [Figure number missing]. Figure 6 In summary, CD16 expression and NK cell count were not correlated at the defined threshold, and therefore could be considered as a completely independent indicator of the likelihood that a patient would benefit from MOR00208 treatment.

[0295] The following characteristics were found not to predict DCR: a) age, b) sex, c) whether the patient received rituximab doses within the last 6 months, d) whether the patient was rituximab-refractory, e) whether the patient possessed high or low affinity alleles of FcγRIIIa, f) whether the patient possessed high or low affinity alleles of FcγRIIa, g) whether the patient had a duration of response to previous treatment longer than 12 months, and h) baseline peripheral T cell count. See also Figure 7 .

[0296] Both 1) baseline peripheral NK cell count and 2) baseline CD16 expression on peripheral NK cells clearly demonstrated a correlation with patient response to MOR00208 treatment. Specifically, patients with a baseline NK cell count of at least 100 cells / μl were associated with a higher disease control rate (DCR). DCR included patients with a complete response (CR) + partial response (PR) + stable disease (SD). Furthermore, patients with baseline CD16 expression on peripheral NK cells of at least 60,000 (ABC) were associated with a higher disease control rate (DCR).

[0297] Progression-free survival (PFS) refers to the length of time a patient lives with the disease without its progression during and after treatment. This is another important endpoint in clinical trials and an indicator of efficacy in patients. PFS was compared across the following patient characteristics: a) baseline peripheral NK cell count of at least 100 cells / μl or less, b) baseline CD16 expression on peripheral NK cells of at least 60,000 ABC or less, and c) baseline peripheral T cell count of at least 500 cells / μl or less. Results were displayed on... Figure 8-10 In comparison, patients with NK cell counts of at least 100 cells / μl showed a statistically significant difference in PFS compared to patients with lower NK cell counts (HR = 0.1561, unadjusted log-rank p = 0.0003). This further confirms the predictive power of NK cell count in the response to MOR00208 treatment in patients with CLL, NHL, ALL, or SLL.

[0298] It should be understood that while the specification, specific embodiments, and data point out exemplary implementations, they are merely illustrative and not intended to limit the invention. Various changes and modifications within the scope of this invention will be apparent to those skilled in the art from the discussion, disclosure, and data contained herein, and are therefore considered part of this invention.

Claims

1. Use of an anti-CD19 antibody in the preparation of a medicament for treating a subject with chronic lymphocytic leukemia or acute lymphoblastic leukemia, wherein the anti-CD19 antibody comprises a heavy chain having the sequence shown in SEQ ID NO:8 and a light chain having the sequence shown in SEQ ID NO:9, and wherein the subject satisfies: a. Baseline peripheral NK cell count at least 50 cells / μl, or b. Baseline CD16 expression levels on peripheral NK cells are at least 60,000 ABC.

2. The use according to claim 1, wherein the baseline peripheral NK cell count of the subject is at least 60 cells / μl.

3. The use according to claim 1, wherein the baseline peripheral NK cell count of the subject is at least 70 cells / μl.

4. The use according to claim 1, wherein the baseline peripheral NK cell count of the subject is at least 80 cells / μl.

5. The use according to claim 1, wherein the baseline peripheral NK cell count of the subject is at least 100 cells / μl.

6. The use according to claim 1, wherein the baseline CD16 expression level on the peripheral NK cells of the subject is at least 60,000 ABC.

7. The use according to any one of claims 1-6, wherein the subject suffers from chronic lymphocytic leukemia.

8. The use according to any one of claims 1-6, wherein the subject suffers from acute lymphoblastic leukemia.

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