Specific Anti-CD38 antibodies for treating human cancers

Abstract

To provide use of an anti-CD38 antibody as an agent for treating cancers such as multiple myeloma or in the manufacture of such an agent.SOLUTION: The present invention relates to an antibody that specifically binds CD38 and is capable of killing a CD38+ cell by induction of apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity. The antibody of the present disclosure may be used as a preparation or in the manufacture of a preparation, wherein the antibody is to be administered to a human subject in a safe therapeutic dose of about 20 mg / kg or below. In one embodiment, the preparation is for treating CD38+ multiple myeloma in humans.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 61 / 898,309, filed October 31, 2013, and European Patent Application No. EP14306220.6, filed July 31, 2014, which are incorporated herein by reference in their entireties for all purposes.

[0002] Sequence Listing This application is accompanied by a sequence listing in computer readable form that reproduces exactly the sequences set forth herein.

[0003] This disclosure relates to the treatment of diseases using antibodies. More specifically, this disclosure relates to the use of anti-CD38 antibodies as or in the manufacture of a medicament for treating cancer, such as multiple myeloma. [Background technology]

[0004] CD38 is a 45 kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. The CD38 protein is a bifunctional extracellular enzyme that can catalyze the conversion of NAD+ to cyclic ADP-ribose (cADPR) and also hydrolyze cADPR to ADP-ribose.

[0005] CD38 is upregulated in many hematological malignancies and in cell lines derived from various hematological malignancies. Furthermore, most early pluripotent stem cells in the blood system express CD38. - The expression of CD38 in hematological malignancies and its correlation with disease progression in chronic lymphocytic leukemia (CLL) makes it an attractive target for antibody therapy.

[0006] Anti-CD38 antibodies that specifically recognize CD38 have been previously described, for example, in Patent Document 1. However, these antibodies have not been used as single agents to inhibit CD38. +When incubated with expressing cells, it is unable to induce apoptosis.

[0007] Specific monoclonal anti-CD38 antibodies such as 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 have been previously described in U.S. Patent No. 5,629,999, which describes three cytotoxic activities of these specific anti-CD38 antibodies: apoptosis, ADCC and CDC.

[0008] Furthermore, these specific anti-CD38 antibodies in combination with cytotoxic drugs such as cytarabine, vincristine, cyclophosphamide, and melphalan + The use of antibodies has been reported in US Patent Nos. 5,629,299, 5,729,302, 5,729,310, 5,729,320, and 5,729,330. However, the use of anti-CD38 antibodies as single agents has not been reported. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Patent Application No. WO2006 / 099875 [Patent Document 2] International Patent Application No. WO2008 / 047242 [Patent Document 3] International Patent Application No. WO2010 / 061357 [Patent Document 4] International Patent Application No. WO2010 / 061358 [Patent Document 5] International Patent Application No. WO2010 / 061359 [Patent Document 6] International Patent Application No. WO2010 / 061360 Summary of the Invention [Means for solving the problem]

[0010] In one embodiment, the present disclosure relates to an antibody that specifically binds to CD38 for use as a medicament, wherein the antibody is administered to a human subject at a safe therapeutic dose of 20 mg / kg or less.

[0011] In another embodiment, a safe therapeutic dose of an antibody of the disclosure is about 5 mg / kg, or about 10 mg / kg, or about 20 mg / kg.

[0012] In another embodiment, the present disclosure relates to an antibody that specifically binds to CD38 or a pharmaceutical composition comprising an antibody that specifically binds to CD38, wherein the antibody or pharmaceutical composition can be used as a medicament in the treatment of relapsed and / or refractory multiple myeloma, and wherein the antibody is administered to a human subject at a safe therapeutic dose of about 20 mg / kg or less.

[0013] In another embodiment, the present disclosure relates to the use of anti-CD38 antibodies as single agents to reduce the patient's chemotherapy burden.

[0014] In another embodiment, the present disclosure further relates to a safe therapeutic dose of an antibody that specifically binds CD38 in a human subject, said safe therapeutic dose being less than or equal to about 20 mg / kg.

[0015] In another embodiment, the present disclosure provides a method for the treatment of CD38 in a human subject in need thereof. + The present invention relates to a method of treating a hematological malignancy, comprising administering to the human subject an antibody that specifically binds to CD38 in a safe, therapeutically effective amount of less than or equal to about 20 mg / kg.

[0016] In another embodiment, the present disclosure provides a method for the treatment of CD38 in a human subject in need thereof. + The present invention relates to a method of treating multiple myeloma, comprising administering to said human subject an effective amount of an antibody that specifically binds to CD38.

[0017] In another embodiment, the present disclosure relates to the use of an antibody that specifically binds CD38 for the manufacture of a medicament, wherein the antibody is administered to a human subject at a safe therapeutic dose of 20 mg / kg or less, or about 20 mg / kg or less.

[0018] In one embodiment, the agent inhibits CD38 + Hematological malignancies, particularly multiple myeloma, most particularly relapsed and / or refractory CD38 + For the treatment of multiple myeloma.

[0019] In another embodiment, the present disclosure further provides a method for the detection of CD38 + Multiple myeloma, specifically relapsed and / or refractory CD38 + The present invention relates to the use of an antibody that specifically binds to CD38 for the manufacture of a medicament for the treatment of multiple myeloma.

[0020] In one embodiment, disclosed is an antibody or epitope-binding fragment thereof capable of specifically binding to CD38 for use as a medicament, wherein the antibody or epitope-binding fragment does not induce the production of autoantibodies against the antibody or epitope-binding fragment by a human subject when the antibody or epitope-binding fragment is administered to the human subject at a dose of about 20 mg / kg or less.

[0021] In another embodiment, a compound capable of specifically binding to CD38 for use as a medicament is provided. Disclosed is an antibody or epitope-binding fragment thereof that is capable of demonstrating detectable CD38 receptor occupancy in a human subject when administered to the human subject at a dose level of about 1 mg / kg every two weeks.

[0022] In another embodiment, an antibody or epitope-binding fragment thereof capable of specifically binding to CD38 is disclosed for use as a medicament, wherein when administered to a human subject at a dose level of about 10 mg / kg every two weeks, the antibody or epitope-binding fragment thereof is capable of exhibiting at least about 84.1% CD38 receptor occupancy in the human subject.

[0023] In another embodiment, an antibody or epitope-binding fragment thereof capable of specifically binding to CD38 is disclosed for use as a medicament, wherein when administered to a human subject at a dose level of about 10 mg / kg every two weeks, the antibody or epitope-binding fragment thereof is capable of exhibiting at least about 97.7% CD38 receptor occupancy in the human subject.

[0024] In another embodiment, disclosed is an antibody or epitope-binding fragment thereof capable of specifically binding to CD38 for use as a medicament, wherein when administered to a human subject at a dose level ranging from about 5 mg / kg to about 20 mg / kg every two weeks or every week, the antibody or epitope-binding fragment thereof is capable of inhibiting tumor growth in the human subject.

[0025] In another embodiment, disclosed is an antibody or epitope-binding fragment thereof capable of specifically binding to CD38 for use as a medicament, wherein when administered to a human subject at a dose level ranging from about 10 mg / kg to about 20 mg / kg every two weeks or every week, the antibody or epitope-binding fragment thereof is capable of inhibiting tumor growth in a human subject.

[0026] In another embodiment, a pharmaceutical composition comprising any of the antibodies of the present disclosure and a pharmaceutically acceptable carrier or excipient is also disclosed.

[0027] In another embodiment, the present disclosure provides a unit dosage form comprising the pharmaceutical composition disclosed herein.

[0028] In another embodiment, the present disclosure also provides an article of manufacture comprising a pharmaceutical composition disclosed herein and a container.

[0029] In another embodiment, the methods of the present disclosure may be suitable for treating a human subject having a disease or disorder in which CD38 is abnormally upregulated. Such methods may include, among other steps, administering to the human subject an antibody or epitope-binding fragment thereof capable of specifically binding to CD38, wherein the antibody is capable of inhibiting tumor growth in the human subject when administered to the human subject at a dose level ranging from about 1 mg / kg to about 20 mg / kg every two weeks or once a week. In one aspect, such a disease or disorder is characterized by an abnormal upregulation of CD38. + In another embodiment, such a disease or disorder is a hematological malignancy. + It is multiple myeloma. [Brief explanation of the drawings]

[0030] [Figure 1] 1 is a graph showing response over time in patients with multiple myeloma treated with the specific anti-CD38 antibody hu38SB19 at different doses (1, 3, 5, and 10 mg / kg) (N=17). (Several patients were excluded after short-term treatment due to progressive disease. However, several patients remained stable during treatment, and others showed partial responses at the 1 mg / kg and 10 mg / kg dose levels. PR=partial response, MR=minimal response, SD=stable disease, or PD=progressive disease.) DETAILED DESCRIPTION OF THE INVENTION

[0031] In the context of this disclosure, the term "CD38" refers to the CD38 protein, a 45 kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. The CD38 protein is a bifunctional extracellular enzyme that can catalyze the conversion of NAD+ to cyclic ADP-ribose (cADPR) and also hydrolyze cADPR to ADP-ribose. During ontogeny, CD38 appears on CD34+-associated stem cells and lineage-committed progenitor cells of lymphoid, erythroid, and myeloid cells. CD38 expression mostly persists in the lymphoid lineage, with varying expression levels at different stages of T cell and B cell differentiation.

[0032] CD38 in signal transduction + The role of is further described in International Patent Application WO2008 / 047242. In particular, CD38 refers to a type II transmembrane protein comprising an amino acid sequence similar to, for example, Genbank Accession No. NP_001766.2 (accessed October 7, 2013).

[0033] CD38 is upregulated in many hematological malignancies and in cell lines derived from various hematological malignancies.

[0034] "Hematologic malignancies" are types of cancer that affect the blood, bone marrow, and lymph nodes. Because the three are closely related in the immune system, diseases that affect one of the three may also affect the other two. Hematologic malignancies include non-Hodgkin's lymphoma (NHL) (including, for example, Burkitt's lymphoma (BL) and T-cell lymphoma (TCL)), multiple myeloma (MM), chronic lymphocytic leukemia (CLL) (such as, for example, B-cell chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL)), B- and T-cell acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML).

[0035] On the other hand, most early pluripotent stem cells in the blood system express CD38 -The expression of CD38 in hematological malignancies and its correlation with disease progression makes it an attractive target for antibody therapy.

[0036] "CD38 + "Cells" are cells that express the CD38 protein. + The cell is a mammalian cell. In one embodiment, the cell is a CD38 + The cells are non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), B and T cell acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma (HL), or chronic myeloid leukemia (CML) cells that express the CD38 protein.

[0037] Therefore, "CD38 + Hematologic malignancies are characterized by tumors in which cancer cells express CD38 + cells or CD38 + The previously described hematologic malignancies include cells.

[0038] In the context of the present disclosure, CD38 + Specifically, hematological malignancies are characterized by the expression of CD38 + cells or CD38 + Non-Hodgkin's lymphoma (NHL) (including, for example, Burkitt's lymphoma (BL) and T-cell lymphoma (TCL)), multiple myeloma (MM), chronic lymphocytic leukemia (CLL) (such as, for example, B-cell chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia (HCL)), B- and T-cell acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML).

[0039] For details, see CD38 +The hematological malignancies are B-cell non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (B-cell ALL) and / or chronic lymphocytic leukemia (CLL), more particularly multiple myeloma (MM), most particularly relapsed and / or refractory multiple myeloma.

[0040] Methods for identifying hematologic malignancies are known to those skilled in the art and include, as a first step, a complete blood count (CBC) and examination of a peripheral blood smear. Final diagnosis usually requires a full bone marrow aspirate and / or biopsy for morphological testing, ultimately complemented by flow cytometric analysis, cytogenetics, and additional molecular techniques.

[0041] Cells derived from this hematologic malignancy express CD38 + Techniques for determining identity are known to those skilled in the art and include, for example, standard molecular biology techniques such as polymerase chain reaction (PCR) and / or immunochemical methods such as Western blot analysis.

[0042] In the context of this disclosure, "subject" refers to a human.

[0043] In particular, the "subject" and / or "subject in need thereof" may be a CD38 + The term "subject" as used herein refers to an individual suffering from a hematological malignancy and under medical care or treatment. Accordingly, the term "subject" as used herein can refer to a patient.

[0044] Subjects according to the present disclosure may be male or female.

[0045] In some embodiments, the subject has been previously treated with an anti-cancer therapy. In particular, the prior anti-cancer therapy can be selected from the group consisting of chemotherapy, targeted cancer therapy, radiation therapy, bone marrow and / or stem cell transplantation, and immunotherapy.

[0046] "Chemotherapy" refers to the treatment of cancer with one or more cytotoxic antitumor drugs (chemotherapeutic agents) as part of a standard regimen. Chemotherapy agents are usually given in cycles, with periods of treatment followed by rest periods to allow the body to recover.

[0047] For example, "chemotherapeutic agents" used to treat hematological malignancies include, but are not limited to, cytarabine (cytosine arabinoside or ara-C) and anthracyclines (such as daunorubicin and / or daunomycin, doxorubicin and liposomal doxorubicin, idarubicin, and mitoxantrone), gemtuzumab, clofarabine, cladribine, hydroxyurea (Hydrea®), etoposide, amsacrine, FLT3-inhibitors, and demethylating agents (5-azacytidine and decitabine), melphalan, cyclophosphamide, vincristine, proteasome inhibitors such as bortezomib, lenalidomide, thalidomide, and / or pomalidomide, in particular bortezomib and / or lenalidomide.

[0048] "Targeted cancer therapy" refers to drugs or other substances that block the growth and spread of cancer by interfering with specific molecules involved in tumor growth and progression.

[0049] "Radiation therapy" or "irradiation" uses high-energy radiation to eliminate cancer cells. Radiation therapy can be used before bone marrow or peripheral blood stem cell transplantation.

[0050] "Bone marrow and / or stem cell transplant" refers to a cell transplant aimed at restoring stem cells destroyed by high-dose chemotherapy and / or radiation therapy. Sources of stem cells include bone marrow, peripheral blood, and erythrocytes. This includes peripheral blood and umbilical cord blood. Depending on the source of the stem cells to be transplanted, the procedure can be classified as bone marrow transplantation (BMT), peripheral blood stem cell transplantation (PBSCT), or umbilical cord blood transplantation (UCBT). Furthermore, bone marrow and / or stem cell transplantation can refer to autologous stem cell transplantation and / or allogeneic stem cell transplantation.

[0051] In an "autologous transplant," a subject's own stem cells are removed from their bone marrow or peripheral blood. The stem cells are frozen and stored while the person undergoes treatment (high-dose chemotherapy and / or radiation). A process called "purging" can be used to attempt to remove any leukemia cells in the sample. The stem cells are then reinfused into the subject's blood after treatment.

[0052] An "allogeneic transplant" is a transplant from a matched donor. The advantage of allogeneic bone marrow transplantation is that the cells transplanted from the donor can establish a new immune system that can detect leukemia cells as foreign and eliminate them. The disadvantages of allogeneic transplantation are the restriction of a matched donor and side effects.

[0053] "Immunotherapy" refers to stimulating a subject's immune system to attack disease-causing malignant tumor cells. This may be done by immunizing the subject, for example by administering a cancer vaccine, in which the subject's own immune system is trained to recognize tumor cells as targets for destruction, or by administering a therapeutic antibody as a drug, in which the therapeutic antibody recruits the subject's immune system to destroy tumor cells.

[0054] In the context of the present disclosure, the subject may have been previously treated with standard anti-cancer therapy as defined above, but for relapsed and / or refractory hematological malignancies.

[0055] Thus, in certain embodiments, the subject is + Multiple myeloma, specifically relapsed and / or refractory CD38 + I have multiple myeloma.

[0056] "Relapsed" refers to a subject whose hematological malignancy has been treated and improved, but whose hematological malignancy has recurred.

[0057] "Refractory" refers to a subject whose hematological malignancy has been treated without any improvement and thus the hematological malignancy has progressed.

[0058] In one embodiment, the subject has been previously treated with bortezomib and / or lenalidomide.

[0059] In one embodiment, the subject has previously undergone autologous stem cell transplantation (ASCT).

[0060] In one embodiment, the subject relapses within six months of autologous transplant.

[0061] It is known in the art that subjects with multiple myeloma and certain genetic features, such as chromosome 17p deletions, translocations such as t(4,14), t(14,16), t(14,20), or amplifications such as more than three copies of 1q21, are associated with poor outcomes.

[0062] Thus, in one embodiment, the subject has a 17p deletion, a t(4,14), a t(14,16), a t(14,20), and / or more than three copies of 1q21.

[0063] Recently, researchers have developed genomic profiling studies for subjects with multiple myeloma. This test allows doctors to classify subjects with multiple myeloma based on their genomic expression profile as well as a few chromosomal abnormalities.

[0064] In one embodiment, the subject may therefore have a high-risk gene expression profiling (GEP) signature (Shaughnessy JD Jr., Zhan F, Burington BE et al. (2007) Blood 109:2276-2284).

[0065] A subject may exhibit any combination of the above characteristics.

[0066] Multiple myeloma can be detected by the presence of a monoclonal protein (M protein).

[0067] "M-protein" refers to a paraprotein (monoclonal protein, or M protein). This paraprotein is an immunoglobulin or immunoglobulin light chain that is overproduced by clonal proliferation of plasma cells. Amounts above a certain threshold indicate multiple myeloma. M-protein is usually quantified in serum and urine.

[0068] M-protein levels in serum are typically measured by serum electrophoresis or, for example, by specific immunoglobulin assays; however, quantification of specific immunoglobulins always overestimates M-protein because normal immunoglobulins are included in the results. For this reason, baseline and follow-up measurements of M-protein must be performed by the same method (Riches PG et al., 1991).

[0069] In one embodiment, M-protein in serum greater than 0.5 g / dL is indicative of multiple myeloma. In another embodiment, M-protein in serum greater than about 0.5 g / dL is indicative of multiple myeloma.

[0070] Urinary M-protein refers to the M-protein excreted in urine measured over a 24-hour period, typically by measuring the total amount of protein excreted over a 24-hour period and multiplying it by the percentage of urinary M-protein determined by electrophoresis of concentrated urinary protein.

[0071] In one embodiment, greater than 200 mg of urinary M-protein in a 24 hour urine period is indicative of multiple myeloma. In another embodiment, greater than about 200 mg of urinary M-protein in a 24 hour urine period is indicative of multiple myeloma.

[0072] Multiple myeloma can be further identified by the presence of immunoglobulin light chains in urine. This paraprotein, called Bence Jones protein, is a urinary paraprotein composed of lambda (λ) and / or kappa (κ) free light chains. These free light chains (FLC) can be measured by commercial tests. Measurement of free light chains refers to the measurement of FLC kappa and FLC lambda free light chains, which gives the FLC kappa to FLC lambda free light chain ratio (FLC κ / λ ratio). A normal FLC κ / λ ratio ranges from 0.26 to 1.65.

[0073] In patients with multiple myeloma, light chains, either kappa or lambda, are predominantly produced, which results in an altered FLC κ / λ ratio.

[0074] Thus, an abnormal FLC κ / λ ratio indicative of multiple myeloma is an FLC κ / λ ratio less than 0.26 or greater than 1.65.

[0075] In one embodiment, high levels of serum free light chains (FLC) with an abnormal FLC ratio of greater than 10 mg / dL are indicative of multiple myeloma. In another embodiment, high levels of serum free light chains (FLC) with an abnormal FLC ratio of greater than about 10 mg / dL are indicative of multiple myeloma.

[0076] Thus, in one embodiment, a subject with multiple myeloma has a) measurable serum M-protein greater than about 0.5 g / dL, and / or b) urinary M-protein greater than about 200 mg (24-hour urine), and / or c) high levels of serum free light chains (FLC) with an abnormal FLC ratio, with FLC greater than about 10 mg / dL.

[0077] In one embodiment, the subject is tolerant to leached protein products.

[0078] Antibodies in the context of the present disclosure specifically bind to CD38. According to one embodiment, anti-CD38 antibodies used within the scope of the present disclosure inhibit CD38 by inducing apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). + It can kill cells.

[0079] As used herein, "apoptosis" refers to the process of programmed cell death.

[0080] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a mechanism of cell-mediated immune defense whereby effector cells of the immune system actively lyse target cells whose membrane surface antigens have been bound by specific antibodies.

[0081] "Complement dependent cytotoxicity" or "CDC," in the context of this disclosure, refers to the lysis of target cells in the presence of complement system proteins.

[0082] As used herein, the terms "conjugate," "immunoconjugate," "antibody-drug conjugate," or "ADC" have the same meaning and are interchangeable.

[0083] An "antibody" may be a natural or conventional antibody consisting of two heavy chains linked to each other by disulfide bonds, and each heavy chain linked to a light chain by a disulfide bond. Two types of light chains exist: lambda (λ) and kappa (κ). Five major heavy chain classes (or isotypes) determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. Light chains contain two domains or regions: a variable domain (VL) and a constant domain (CL). Heavy chains contain four domains: one variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions (VL) and (VH) of both the light and heavy chains determine binding recognition and specificity to antigens. The constant region domains (CL) and (CH) of the light and heavy chains confer important biological properties, such as antibody chain association, secretion, placental transfer, complement fixation, and Fc receptor (FcR) binding. The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portion of one light chain and one heavy chain. Antibody specificity resides in the structural complementarity between the antibody-combining site and an antigenic determinant. The antibody-combining site is composed of residues primarily from the hypervariable or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) influence the overall domain structure and therefore the combining site.

[0084] "Complementarity-determining region" or "CDR" refers to the amino acid sequences that collectively define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. Each light and heavy chain of an immunoglobulin has three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Thus, the antigen-binding site of a conventional antibody consists of six CDRs, each containing a set of CDRs from the V region of each of the heavy and light chains. Including DR.

[0085] The term "framework region" (FR) refers to the amino acid sequence inserted between the CDRs, i.e., the amino acid sequence portion of the immunoglobulin light and heavy chain variable regions that is relatively conserved among different immunoglobulins of a single species. Each immunoglobulin light and heavy chain has four FRs, designated FR1-L, FR2-L, FR3-L, and FR4-L, and FR1-H, FR2-H, FR3-H, and FR4-H, respectively.

[0086] As used herein, a "human framework region" is a framework region that is substantially (about 85% or more, particularly 90%, 95%, 97%, 99% or 100%) identical to the framework region of a naturally occurring human antibody.

[0087] The definition of CDR / FR for an immunoglobulin light or heavy chain is given based on the definition of Kabat (http: / / www.bioinf.org.uk / abs / ).

[0088] The term "antibody" as used herein refers to conventional antibodies and fragments thereof, as well as single domain antibodies and fragments thereof, in particular the variable heavy chains of single domain antibodies, and chimeric, humanized, bispecific or multispecific antibodies.

[0089] As used herein, antibody or immunoglobulin is a more recently described term that also includes "single domain antibodies," which are antibodies whose complementarity-determining regions are part of a single domain polypeptide. Examples of single domain antibodies include heavy chain antibodies, antibodies naturally lacking light chains, single domain antibodies derived from traditional four-chain antibodies, and engineered single domain antibodies. Single domain antibodies can be derived from any species, including but not limited to mice, humans, camels, llamas, goats, rabbits, and cattle. Single domain antibodies can be naturally occurring single domain antibodies known as heavy chain antibodies lacking light chains. In particular, camelid species, such as camels, dromedaries, llamas, alpacas, and guanacos, produce heavy chain antibodies naturally lacking light chains. Camelid heavy chain antibodies also lack the CH1 domain.

[0090] The variable heavy chains of these single domain antibodies, which lack light chains, are known in the art as "VHH" or "nanobodies". Like conventional VH domains, VHHs contain four FRs and three CDRs. Nanobodies have advantages over conventional antibodies: they are approximately 10 times smaller than IgG molecules, and as a result, correctly folded, functional nanobodies can be produced by in vitro expression with high yields. Furthermore, nanobodies are very stable and resistant to the action of proteases. The properties and production of nanobodies have been reviewed by Harmsen and De Haard (Harmsen and De Haard (2007) Appl.Microbiol.Biotechnol.77:13-22).

[0091] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody molecule of a single amino acid composition that is directed against one specific antigen and should not be construed as requiring production of the antibody by any particular method. A monoclonal antibody can be produced by a single clone of B cells or hybridoma, but can also be recombinant, i.e., produced by protein engineering.

[0092] The term "chimeric antibody" refers to an engineered antibody in which the constant region or a portion thereof has been modified, replaced, or exchanged, and thus the variable region is combined with a constant region of a different species, or a species belonging to another antibody class or subclass. Chimeric antibodies also refer to antibodies in which the variable region or a portion thereof has been modified, replaced, or exchanged, and thus the constant region is combined with a variable region of a different species, or a species belonging to another antibody class or subclass. In particular, chimeric antibodies are The chimeric antibody comprises the VH and VL domains of an antibody derived from a non-human animal associated with the CH and CL domains of another antibody, particularly a human antibody. The non-human animal can be any animal, such as a mouse, rat, hamster, or rabbit. The chimeric antibody can also exhibit multispecificity, having specificity for at least two different antigens. In one embodiment, the chimeric antibody has variable domains of mouse origin and constant domains of human origin.

[0093] The term "humanized antibody" refers to an antibody that is originally of wholly or partially non-human origin and that has been modified, particularly by replacing certain amino acids in the framework regions of the heavy and light chains, to avoid or minimize an immune response in humans. The constant domains of a humanized antibody are often human CH and CL domains. In one embodiment, a humanized antibody has constant domains of human origin.

[0094] For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences.

[0095] While amino acid residues that are part of CDRs are typically not modified for humanization, in certain cases it may be desirable to modify individual CDR amino acid residues to remove, for example, glycosylation sites, deamidation sites, unwanted cysteine ​​residues, or, in the case of ADCs, lysine residues. N-linked glycosylation occurs through the attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except Pro. Removal of N-glycosylation sites can be achieved by mutation of either Asn or Ser and / or Thr residues to different residues, particularly by conservative amino acid substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, primarily when present in the sequence Asn-Gly, and to a lesser extent, in other dipeptide sequences such as Asn-Ala. When such deamidation sites, particularly Asn-Gly, exist in the CDR sequence, it may be desirable to remove the site, typically by conservative substitution to remove one of the relevant residues. In the case of ADCs, the binding of a cytotoxic drug to a mAb may be created by covalent bonding to a lysine side chain residue. This steric hindrance may interfere with the binding of the mAb to the antigen. Therefore, it may be desirable to remove the lysine residue, typically by conservative substitution with arginine. Substitution in the CDR sequence to remove one of the relevant residues is also intended to be encompassed by the present disclosure.

[0096] The goal of humanization is to reduce the immunogenicity of xenogeneic antibodies, such as murine antibodies, for introduction into humans while maintaining the full antigen-binding affinity and specificity of the antibody. Humanized antibodies, or antibodies adapted for non-rejection by other mammals, can be produced using several techniques, including antibody resurfacing and CDR grafting. As used herein, resurfacing technology uses a combination of molecular modeling, statistical analysis, and mutagenesis to modify the non-CDR surfaces of antibody variable regions to resemble known antibody surfaces of the target host.

[0097] Strategies and methods for antibody resurfacing, as well as other methods for reducing the immunogenicity of antibodies in different hosts, are disclosed in U.S. Patent No. 5,639,641. Briefly, a preferred method involves: (1) generating a positional alignment of a group of antibody heavy and light chain variable regions to obtain a set of heavy and light chain variable region framework surface-exposed positions, where the aligned positions for all variable regions are at least about 98% identical; (2) defining a set of heavy and light chain variable region framework surface-exposed amino acid residues for a rodent antibody (or fragment thereof); (3) identifying a set of heavy and light chain variable region framework surface-exposed amino acid residues that most closely resemble the rodent set of surface-exposed amino acid residues; (4) replacing the set of heavy and light chain variable region framework surface-exposed amino acid residues defined in step (2) with the set of heavy and light chain variable region framework surface-exposed amino acid residues identified in step (3), except for amino acid residues that are within 5 Å of any atom of any residue in the complementarity-determining regions of the rodent antibody; and (5) producing a humanized rodent antibody having the binding specificity. Thus, in one embodiment, the humanized antibody can also be referred to as a "resurfaced" antibody.

[0098] Antibodies can be further humanized using a variety of other techniques, including CDR-grafting (EP 0239400; WO 91 / 09967; U.S. Patent Nos. 5,530,101 and 5,585,089), veneering or resurfacing (EP 0592106; EP 0519596; Padlan (1991) Molecular Immunology 28 (4 / 5): 489-498; Studnicka et al. (1994) Protein Engineering 7 (6): 805-814; Roguska et al. (1994) Proc. Natl. Acsd. Sci USA 91: 969-973), and chain shuffling (U.S. Patent No. 5,565,332). Human antibodies can be produced by a variety of methods known in the art, including phage display methods. See also U.S. Patent Nos. 4,444,887, 4,716,111, 5,545,806 and 5,814,318; and International Patent Applications WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741.

[0099] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functionality of a protein. Examples of amino acid groups having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine-tryptophan, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.

[0100] A "fragment" of a (traditional) antibody comprises a portion of a complete antibody, in particular the antigen-binding or variable region of the complete antibody. Examples of antibody fragments include Fv, Fab, F(ab')2, Fab', dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies formed from antibody fragments, bispecific and multispecific antibodies. A fragment of a conventional antibody may be a heavy chain antibody or a single domain antibody such as a VHH.

[0101] The term "Fab" refers to an antibody fragment having a molecular weight of approximately 50,000 Da and antigen-binding activity, in which approximately half of the N-terminal heavy chain and the entire light chain are bound together via disulfide bonds between fragments obtained by treating IgG with the protease papain.

[0102] The term "F(ab')2" refers to an antibody fragment having a molecular weight of approximately 100,000 Da and antigen-binding activity, which is slightly larger than Fab and is formed by treating IgG with the protease pepsin and linking fragments via disulfide bonds in the hinge region.

[0103] The term "Fab" refers to the F(ab')2 fragment obtained by cleavage of the disulfide bond in the hinge region. It refers to an antibody fragment obtained by the above procedure, which has a molecular weight of approximately 50,000 Da and antigen-binding activity.

[0104] Single-chain Fv ("scFv") polypeptides are covalently linked VH::VL heterodimers that are typically expressed from gene fusions containing VH- and VL-encoding genes linked by a peptide-encoding linker. Human scFv fragments of the present disclosure specifically contain the CDRs held in the appropriate conformation by the use of genetic engineering techniques. Bivalent and multivalent antibody fragments may form spontaneously by association of monovalent scFvs or can be generated by linking monovalent scFvs with peptide linkers, such as bivalent sc(Fv)2.

[0105] "dsFv" is a VH::VL heterodimer stabilized by a disulfide bond.

[0106] "(dsFv)2" indicates two dsFvs linked by a peptide linker.

[0107] The term "bispecific antibody" or "BsAb" refers to an antibody that combines the antigen-binding sites of two antibodies in a single molecule. BsAbs are therefore capable of simultaneously binding to two different antigens. Genetic engineering is frequently used to design, modify, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions, as described, for example, in EP 2050764 A1.

[0108] The term "multispecific antibody" refers to an antibody that combines the antigen-binding sites of two or more antibodies in a single molecule.

[0109] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains can pair with the complementary domains of another chain and create two antigen-binding sites.

[0110] The antibody used within the scope of the present disclosure may be one of the anti-CD38 monoclonal antibodies designated 38SB13, 38SB18, 38SB19, 38SB30, 38SB31 and 38SB39 described in WO2008 / 047242, or a derivative thereof obtained by resurfacing technology.

[0111] In particular, an anti-CD38 antibody in the context of the present disclosure comprises at least one heavy chain comprising CDR-H1 of sequence SEQ ID NO: 1, CDR-H2 of sequence SEQ ID NO: 2, and CDR-H3 of sequence SEQ ID NO: 3, and at least one light chain comprising three consecutive CDRs with amino acid sequences consisting of SEQ ID NOs: 4, 5, and 6. In particular, said antibody has a heavy chain variable domain comprising SEQ ID NO: 44 and a light chain variable domain comprising SEQ ID NO: 38.

[0112] In particular, an anti-CD38 antibody in the context of the present disclosure comprises at least one heavy chain comprising CDR-H1 of sequence SEQ ID NO: 7, CDR-H2 of sequence SEQ ID NO: 8, and CDR-H3 of sequence SEQ ID NO: 9, and at least one light chain comprising three consecutive CDRs with amino acid sequences consisting of SEQ ID NOs: 10, 11, and 12. In particular, said antibody has a heavy chain variable domain comprising SEQ ID NO: 45 and a light chain variable domain comprising SEQ ID NO: 39.

[0113] In particular, an anti-CD38 antibody in the context of the present disclosure comprises at least one heavy chain comprising CDR-H1 of the sequence SEQ ID NO: 13, CDR-H2 of the sequence SEQ ID NO: 14, and CDR-H3 of the sequence SEQ ID NO: 15, and at least one light chain comprising three consecutive CDRs having the amino acid sequences consisting of SEQ ID NOs: 16, 17, and 18. In particular, said antibody comprises at least one heavy chain comprising CDR-H1 of the sequence SEQ ID NO: 13, CDR-H2 of the sequence SEQ ID NO: 14, and CDR-H3 of the sequence SEQ ID NO: 15, and at least one light chain comprising three consecutive CDRs having the amino acid sequences consisting of SEQ ID NOs: 16, 17, and 18. 46 and a light chain variable domain comprising SEQ ID NO:40.

[0114] In particular, an anti-CD38 antibody in the context of the present disclosure comprises at least one heavy chain comprising CDR-H1 of sequence SEQ ID NO: 19, CDR-H2 of sequence SEQ ID NO: 20, and CDR-H3 of sequence SEQ ID NO: 21, and at least one light chain comprising three consecutive CDRs with amino acid sequences consisting of SEQ ID NOs: 22, 23, and 24. In particular, said antibody has a heavy chain variable domain comprising SEQ ID NO: 47 and a light chain variable domain comprising SEQ ID NO: 41.

[0115] In particular, an anti-CD38 antibody in the context of the present disclosure comprises at least one heavy chain comprising CDR-H1 of sequence SEQ ID NO: 25, CDR-H2 of sequence SEQ ID NO: 26, and CDR-H3 of sequence SEQ ID NO: 27, and at least one light chain comprising three consecutive CDRs with amino acid sequences consisting of SEQ ID NOs: 28, 29, and 30. In particular, said antibody has a heavy chain variable domain comprising SEQ ID NO: 48 and a light chain variable domain comprising SEQ ID NO: 42.

[0116] In particular, an anti-CD38 antibody in the context of the present disclosure comprises at least one heavy chain comprising CDR-H1 of sequence SEQ ID NO: 31, CDR-H2 of sequence SEQ ID NO: 32, and CDR-H3 of sequence SEQ ID NO: 33, and at least one light chain comprising three consecutive CDRs with amino acid sequences consisting of SEQ ID NOs: 34, 35, and 36. In particular, said antibody has a heavy chain variable domain comprising SEQ ID NO: 49 and a light chain variable domain comprising SEQ ID NO: 43.

[0117] Further included within the context of the present disclosure are antibodies comprising sequences that are at least 85%, more particularly at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequences disclosed herein.

[0118] A sequence that is "at least 85% identical to a reference sequence" is a sequence that has 85% or more, more particularly 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity over its entire length to the entire length of the reference sequence.

[0119] The percentage of "sequence identity" can be determined by comparing two sequences that are optimally aligned over a comparison window, where a portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of the comparison sequences is performed by global pairwise alignment, for example, using the algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970). The percentage of sequence identity can be easily determined, for example, using the program Needle with the BLOSUM62 matrix and the following parameters: gap-open=10, gap-extend=0.5.

[0120] In one embodiment, the anti-CD38 antibody is a humanized anti-CD38 antibody obtained by resurfacing technology. Such antibodies may also be referred to as "resurfaced" antibodies.

[0121] In one embodiment, the resurfaced and / or humanized antibody comprises at least one heavy chain and at least one light chain, wherein the heavy chain comprises three contiguous complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 1, 2, and 3, and the light chain comprises three contiguous complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 4, 5, and 6.

[0122] In another embodiment, the resurfaced and / or humanized antibody comprises at least one heavy chain and at least one light chain, wherein the heavy chain comprises three contiguous complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 7, 8, and 9, and the light chain comprises three contiguous complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 10, 11, and 12.

[0123] In another embodiment, the resurfaced and / or humanized antibody comprises at least one heavy chain and at least one light chain, wherein the heavy chain comprises three contiguous complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 13, 37, and 15, and the light chain comprises three contiguous complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 16, 17, and 18.

[0124] In another embodiment, the resurfaced and / or humanized antibody comprises at least one heavy chain and at least one light chain, wherein the heavy chain comprises three contiguous complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 19, 20, and 21, and the light chain comprises three contiguous complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 22, 23, and 24.

[0125] In another embodiment, the resurfaced and / or humanized antibody comprises at least one heavy chain and at least one light chain, wherein the heavy chain comprises three contiguous complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 25, 26, and 27, and the light chain comprises three contiguous complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 28, 29, and 30.

[0126] In a further embodiment, the resurfaced and / or humanized antibody comprises at least one heavy chain and at least one light chain, wherein the heavy chain comprises three contiguous complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 31, 32, and 33, and the light chain comprises three contiguous complementarity determining regions having the amino acid sequences represented by SEQ ID NOs: 34, 35, and 36.

[0127] In one embodiment, the present disclosure provides a V having an amino acid sequence selected from the group of SEQ ID NOs: 50 and 51. H In a further embodiment, the resurfaced and / or humanized antibody comprises a V having the amino acid sequence represented by SEQ ID NO: 50. H In another embodiment, the humanized antibody comprises a V having the amino acid sequence represented by SEQ ID NO: 51. H Includes:

[0128] In another embodiment, the present disclosure provides a V having an amino acid sequence selected from the group of SEQ ID NOs: 52, 53, 54 and 55. L In another embodiment, the humanized antibody has an amino acid sequence selected from the group consisting of SEQ ID NOs: 52 and 53. L In another embodiment, the humanized antibody comprises a V having an amino acid sequence selected from the group of SEQ ID NOs: 54 and 55. L Includes:

[0129] In a specific embodiment, the antibody according to the present disclosure comprises a V H V having the amino acid sequence represented by SEQ ID NO: 52 L and a humanized and / or resurfaced antibody designated hu38SB19, comprising:

[0130] In another embodiment, the antibody according to the present disclosure is daratumumab.

[0131] In specific embodiments, the antibodies for use in accordance with the present disclosure are naked antibodies, i.e., That is, the antibody is not conjugated with any drug to form an antibody-drug conjugate.

[0132] Antibodies for use in accordance with the present disclosure specifically bind to CD38 and inhibit CD38 by inducing apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and + In a specific embodiment, the antibodies for use in accordance with the present disclosure are capable of killing CD38 cells by inducing apoptosis in vitro, even when the cells are treated with an antibody that is not cross-linked with an anti-human IgG secondary antibody. + It can kill cells.

[0133] Also included within the context of the present disclosure are humanized antibodies comprising a sequence that is at least 85%, more particularly at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence previously disclosed herein.

[0134] Antibodies according to the present disclosure can be obtained by standard techniques of animal immunization, hybridoma formation, and selection of antibodies with specific properties. In particular, the CD38 antibody 38SB19 was obtained as described in Example 4 of International Patent Application WO2008 / 047242, i.e., according to the following protocol for cloning and sequencing the light and heavy chains of the anti-CD38 antibody:

[0135] (i) RNA preparation from hybridoma cells producing the 38SB19 antibody as follows: Total RNA was prepared from 5 x 10 38SB19 antibody-producing hybridoma cells using the RNeasy miniprep kit from Qiagen. 6 Briefly, 5 × 10 hybridoma cells were obtained. 6The cells were pelleted and resuspended in 350 μL of RLT buffer (containing 1% β-mercaptoethanol). The suspension was homogenized by passing it through a 21.5-gauge needle and syringe approximately 10–20 times, or until it was no longer viscous. Ethanol (350 μL of 70% aqueous ethanol) was added to the homogenate and mixed thoroughly. The solution was transferred to a spin column, placed in a 2 mL collection tube, and spun at >8000 × g for 15 seconds. The column was washed twice with 500 μL of RPE buffer, then transferred to a new tube and eluted with 30 μL of RNase-free water and spun for 1 minute. The eluate (30 μL) was then transferred back to the column for a second 1-minute elution spin. An aliquot of the 30 μL eluate was diluted with water and used to measure UV absorbance at 260 nm for RNA quantification.

[0136] (ii) cDNA preparation using a reverse transcriptase (RT) reaction as follows: Variable region 38SB19 antibody cDNA was generated from total RNA using Invitrogen's Superscript II kit. Up to 5 μg of total RNA from Qianeasy mini prep was used, strictly following the kit's protocol. Briefly, RNA, 1 μL of random primer, and 1 μL of dNTP mix were brought up to 12 μL with RNase-free sterile distilled water and incubated at 65°C for 5 minutes. The mix was then placed on ice for at least 1 minute. Next, 4 μL of 5x reaction buffer, 2 μL of 0.1 M DTT, and 1 μL of RNaseOUT were added, and the mix was incubated at 25°C for 2 minutes in an MJ Research thermal cycler. The thermal cycler was stopped to allow for the addition of 1 μL of Superscript II enzyme and then resumed at 25°C for an additional 10 minutes before shifting to 55°C for 50 minutes. The reaction was heat inactivated by heating to 70°C for 15 min, RNA was removed by adding 1 μL of RNase H, and incubated at 37°C for 20 min.

[0137] (iii) Degenerate PCR reaction: The procedure for the first round degenerate PCR reaction on the cDNA from hybridoma cells was based on the method described in Wang et al. (2000) and Co et al. (1992). The primers for this round (Table 1) were: pBlue Contains restriction sites to facilitate cloning into the scriptII plasmid.

[0138] [Table 1]

[0139] PCR reaction components (Table 2) were mixed on ice in thin-walled PCR tubes and then transferred to a preheated MJ research thermal cycler and terminated at 94°C. Reactions were run using a program based on Wang et al. (2000) as follows: Name:Wang45 94°C for 3:00 min 94°C for 0:15 seconds 45℃ 1:00 min 72°C for 2:00 minutes Return to 2 and repeat 29 times 72°C for 6:00 minutes Long time at 4℃ end.

[0140] The PCR reaction mixture was then run on a 1% low-melting agarose gel, and a 300-400 bp band was excised, purified using Zymo DNA minicolumns, and sent to Agencourt biosciences for sequencing. The 5' and 3' PCR primers, respectively, were used as sequencing primers to generate 38SB19 variable region cDNA from both directions.

[0141] (iv) Cloning of 5'-end sequences as follows: The degenerate primers used to clone the 38SB19 variable region light and heavy chain cDNA sequences altered the 5'-end sequences, so additional sequencing effort was required to decipher the complete sequence. The preliminary cDNA sequence from the method was used to search the NCBI IgBlast site for the murine germline sequence from which the 38SB19 sequence was derived (http: / / www.ncbi.nlm.nih.gov / igblast / ). PCR primers were designed (Table 3) to anneal to the leader sequence of the murine antibody, so that a new PCR reaction could generate the complete variable region cDNA unmodified by the PCR primers. PCR reactions, band purification, and sequencing were performed as previously described.

[0142] [Table 2]

[0143] (v) Peptide analysis for sequence confirmation as follows: The cDNA sequence information for the variable regions was combined with the germline constant region sequence to obtain the full-length antibody cDNA sequence. The molecular weights of the heavy and light chains were then calculated and compared to the molecular weight of the murine 38SB19 antibody obtained by LC / MS analysis. Table 5 of U.S. Patent No. 8,153,765 provides the masses calculated from the cDNA sequences for the LC and HC of 38SB19, along with the values ​​measured by LC / MS. The molecular weight measurements are consistent with the cDNA sequences for both the light and heavy chains of 38SB19.

[0144] [Table 3]

[0145] Table 3 provides the 5'-terminal murine leader sequence primers used in the 38SB19 second round PCR reactions. The 3'-terminal primers are identical to those used in the first round reactions, as they prime with the respective constant region sequences.

[0146] More specifically, the humanized 38SB19 antibody can be produced as described in Example 5 of International Patent Application WO 2008 / 047242, i.e., according to the following protocol: The variable region sequences for hu38SB19 were codon-optimized and synthesized by Blue Heron Biotechnology. These sequences are flanked by restriction enzyme sites for cloning in-frame with the respective constant sequences within both single-chain and tandem double-chain mammalian expression plasmids. The light chain variable region is cloned into the EcoRI and BsiWI sites in both the ps38SB19LCZv1.0 and ps38SB19v1.00 plasmids (Figures 2A and 2C of WO 2008 / 047242). The heavy chain variable region is cloned into the HindIII and Apa1 sites in both the ps38SB19HCNv1.0 and ps38SB19v1.00 plasmids (Figures 2B and 2C of WO 2008 / 047242). These plasmids can be used to express hu38SB19 in mammalian cells either transiently or by stable transfection. Similar expression vector constructs have been used to produce other chimeric and humanized antibodies. Transient transfection to express hu38SB19 in HEK-293T cells was performed using CaPO4 reagent from BD biosciences. The supplied protocol was slightly modified for high expression yields. Briefly, 2 x 10 6 HEK-293T cells were plated onto 10 cm tissue culture plates coated with polyethyleneimine (PEI) 24 h before transfection. Transfection was initiated by washing the cells with PBS and replacing the medium with 10 mL of DMEM (Invitrogen) containing 1% Ultra Low IgG FBS (Hyclone). Solution A (10 μg DNA, 86.8 μL Ca) was added. 2+Solution B, and up to 500 μL of H2O were added dropwise to Solution B while vortexing. The mixture was incubated at room temperature for 1 minute, and 1 mL of the mixture was added dropwise to each 10 cm plate. Approximately 16 hours after transfection, the medium was replaced with 10 mL of fresh DMEM containing 1% Ultra Low IgG FBS. Approximately 24 hours later, 2 mM sodium butyrate was added to each 10 cm plate. Transfectants were harvested 4 days later. Protein A antigen was isolated by adding 1 / 10 volume of 1 M Tris / HCl buffer, pH 8.0. The supernatant was prepared for affinity chromatography. The pH-adjusted supernatant was filtered through a 0.22 μm filter membrane and loaded onto a Protein A Sepharose column (HiTrap Protein A HP, 1 mL, Amersham Biosciences) equilibrated with binding buffer (PBS, pH 7.3). A Q-Sepharose precolumn (10 mL) was connected upstream of the Protein A column during sample loading to reduce contamination from cellular materials such as DNA. After sample loading, the precolumn was removed, and the Protein A column direction was reversed for washing and elution. The column was washed with binding buffer until a stable baseline was obtained with no absorbance at 280 nm. The antibody was eluted with 0.1 M acetate buffer, pH 2.8, containing 0.15 M NaCl at a flow rate of 0.5 mL / min. Approximately 0.25 mL fractions were collected and neutralized by adding 1 / 10 volume of 1 M Tris / HCl buffer, pH 8.0. The peak fraction was dialyzed twice overnight against PBS and OD 280 Purified antibodies were quantified by absorbance at 1000 Hz. Humanized and chimeric antibodies can also be purified using a Protein G column using a slightly different procedure.

[0147] All exemplary chimeric, humanized and / or resurfaced anti-CD38 antibodies were expressed and purified using procedures similar to those previously described.

[0148] The inventors have determined appropriate administration doses and regimens for the antibodies of the present disclosure and have demonstrated that CD38 + Hematological malignancies, specifically CD38 +Multiple myeloma, more particularly relapsed and / or refractory CD38 + A well-tolerated anti-cancer therapy has been achieved that can treat subjects with multiple myeloma.

[0149] Therefore, an accelerated dose escalation schedule was used for the first five dose levels ranging from 0.0001 mg / kg to 0.1 mg / kg, administered every two weeks, with one evaluable subject per dose level. All subsequent dose levels, including 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, and 20 mg / kg, were administered every two weeks, with a classical 3 + 3 dose escalation design based on dose-level toxicity, including weekly administration of 10 mg / kg, with a median cycle of administration ranging from 2.0 to 50.0.

[0150] Within this study, the inventors demonstrated that the antibody used had a manageable safety profile at a maximum tolerated dose of over 10 mg / kg.

[0151] Immunogenicity studies performed by the present inventors showed that human subjects did not produce antibodies against the anti-CD38 antibody.

[0152] Furthermore, the present inventors have demonstrated that the antibodies of the present disclosure have high binding efficiency in humans, as evidenced by high receptor occupancy at low doses, which was detected at dose levels of, for example, 1 mg / kg every two weeks and reached a range of 84.1-97.7% at 10 mg / kg.

[0153] Receptor occupancy is typically assessed using two monoclonal antibodies (mAbs) that bind to two different epitopes on the CD38 antigen. MAb1 is specific for the same epitope as, for example, hu38SB19 and is therefore a reporter of free CD38 sites (e.g., not occupied by hu38SB19). A second monoclonal antibody, mAb2, directed against a different epitope from that of hu38SB19, binds to CD38. +This provides a positive control for the presence of CD38 antigen on the cells and an assessment of the amount of CD38 antigen remaining on the cell surface after in vitro drug stimulation. The use of the beads as a calibrator and indirect detection allows for a quantitative approach without altering the binding capacity of hu38SB19. The combined results provide receptor density and occupancy per cell.

[0154] C in Cycle 1 for the dose range of 0.03 to 3 mg / kg max Compared with cycle 2, C max There was no significant increase in

[0155] The inventors have demonstrated that the antibody is effective in treating C18+ / C28+ / C38+ / C48+ / C58+ / C68+ / C78+ / C88+ / C9 ... max This further demonstrates that threshold tumor growth inhibition was achieved in

[0156] The present inventors have investigated the CD38 + Hematological malignancies, specifically CD38 + Multiple myeloma, most particularly relapsed and / or refractory CD38 + It is specifically demonstrated that subjects suffering from multiple myeloma showed a detailed response when administered anti-CD38 antibodies of the present disclosure at doses of 1 mg / kg, 5 mg / kg, and 10 mg / kg.

[0157] We evaluated C at the 5 mg / kg dose level for one patient and at the 10 mg / kg dose level for five patients. max It was observed in detail that the tumor growth inhibition threshold was reached at .

[0158] According to the present disclosure, the antibody is for use as a pharmaceutical, and the antibody is administered to a human subject at a safe therapeutic dose of 20 mg / kg or less.

[0159] In one embodiment, the antibody is CD38 +It is for use in treating hematological malignancies, particularly in treating multiple myeloma, most particularly in treating relapsed and / or refractory multiple myeloma.

[0160] CD38 treated in the context of the present disclosure + Hematologic malignancies are described in the section "CD38 + Hematologic malignancies are defined in the "Hematologic malignancies" section.

[0161] Human subjects are defined above in the section "Subjects."

[0162] In the context of the present disclosure, the term "treat" or "treatment" as used herein means reversing, alleviating, inhibiting, or preventing the progression of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

[0163] As used herein, the term "CD38 + Treating Hematologic Malignancies by targeting tumor CD38 + Malignant cell proliferation and / or CD38 + This means inhibiting the progression of metastases from a tumor. Such treatment may also lead to regression of tumor growth, i.e., a reduction in the size of a measurable tumor. In particular, such treatment involves inhibiting the growth of CD38 + Tumor or CD38 + leading to complete regression of metastases.

[0164] By "therapeutically effective amount" of an antibody in the context of this disclosure is meant the CD38 + It refers to an amount of antibody sufficient to treat a hematological malignancy.

[0165] In certain embodiments, the therapeutically effective amount of antibody administered to a subject is a dose in the range of 0.0001 mg / kg to 20 mg / kg, particularly a dose in the range of 1 mg / kg to 20 mg / kg, and more particularly 0.1 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 7.5 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg or 20 mg / kg.

[0166] In one embodiment, the antibodies of the disclosure can be administered once a week or once every two weeks.

[0167] In another embodiment, the therapeutically effective amount of antibody administered to a subject is, for example, once per week or once every two weeks, at a dose ranging from 1 mg / kg to 20 mg / kg, 3 mg / kg to 20 mg / kg, 5 mg / kg to 20 mg / kg, or 10 mg / kg to 20 mg / kg, which have indeed been shown to be safe while demonstrating efficacy in some individuals.

[0168] In yet another embodiment, the therapeutically effective amount of antibody administered to a subject is a dose of 10 mg / kg once a week, or 20 mg / kg, for example, once a week or once every two weeks.

[0169] In some embodiments, the antibodies of the present disclosure can be administered according to an intermittent regimen with one or two week intervals between each dose, which can be extended by one to two weeks depending on the tolerance to the previous dose, although the inventors have observed that in most cases the side effects are not severe.

[0170] Thus, in certain embodiments, the administration of the antibody is repeated in a new cycle immediately following the previous cycle.

[0171] As used herein, a "cycle" refers to one week in the case of "weekly" administration, and one cycle corresponds to two weeks in the case of "biweekly" administration.

[0172] In one embodiment, the number of cycles of administration may be 2 to 50, particularly 2, 3, 4, 5, 6, 7, 8, 9, 0, 12, 14, 16, 18, 20, 25, 30, 35, 45, 50 cycles.

[0173] Thus, the number of cycles of administration of the antibodies of the disclosure can be selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 0, 12, 14, 16, 18, 20, 25, 30, 35, 45, 50 cycles.

[0174] The antibodies of the present disclosure are administered intravenously.

[0175] In some embodiments, the dose can be increased during treatment after disease response assessment.

[0176] The minimum dose in the context of this disclosure corresponds to 0.0001 mg / kg, a dose level of 0.0001 mg / kg representing a theoretical 10% CD38 receptor occupancy on normal B and T cells.

[0177] In some embodiments, the administration is intravenous at a specific infusion rate.

[0178] Specifically, the antibody is administered at an initial infusion rate of 0.042 mg / hour to 250 mg / hour, particularly an initial infusion rate of 0.042 mg / hour.

[0179] In some embodiments, the initial infusion rate depends on the dose being administered.

[0180] Thus, in one example, an antibody of the disclosure may be administered at a dose of 0.0001 mg / kg at an initial rate of 0.042 mg / hour for a total of 3 ml, at a dose of 0.001 mg / kg at an initial rate of 0.42 mg / hour for a total of 3 ml, or at a dose of 1.4 mg / hour for a total of 3 ml. for example, at a dose of 0.01 mg / kg at an initial rate of 4.2 mg / hour, for example, at a dose of 0.03 mg / kg at an initial rate of 4.2 mg / hour, for example, at a dose of 0.1 mg / kg at an initial rate of 7 mg / hour, for example, at a dose of 0.01 mg / kg at an initial rate of 1.4 mg / hour, for example, at a dose of 0.3 mg / kg at an initial rate of 10.5 mg / hour, for example, at a dose of 1 mg / kg at an initial rate of 17.5 mg / hour, for example, at a dose of 3 mg / kg at an initial rate of 52.5 mg / hour, for example, at a dose of 5 mg / kg at an initial rate of 87.5 mg / hour, for example, at a dose of 10 mg / kg at an initial rate of 175 mg / hour, and for example, at a dose of 20 mg / kg at an initial rate of 250 mg / hour.

[0181] During administration, subjects are typically observed for signs of a hypersensitivity reaction, and administration is continued only in the absence of a hypersensitivity reaction.

[0182] However, the exact time of administration (weekly or biweekly), number of cycles, and initial infusion rate will fall within the ranges disclosed herein, however the exact values ​​will vary depending on the CD38 concentration being treated for any particular subject. + It will be understood that the treatment will be determined by the attending physician depending on factors including the severity of the hematological malignancy or disorder; the activity of the specific antibody used; the specific composition used, the age, weight, general health, sex, and diet of the subject.

[0183] It is understood that the attending physician can modify and adapt the administration regimen based on disease response.

[0184] "Disease response" can be determined according to standard criteria for hematological malignancies and staging.

[0185] Hematological malignancies, specifically CD38 + Methods for assessing disease response of hematological malignancies are known to those of skill in the art.

[0186] Typically, methods for assessing disease response can be selected from the group consisting of Karnofsky performance status assessment, quantification of specific markers, bone marrow biopsy and / or bone marrow aspirate, radiological imaging of plasmacytoma, skeletal survey, M-protein quantification (serum and / or 24-hour urine) and serum free light chain levels or urinary light chain levels, serum beta2-microglobulin, lymph node biopsy and / or radiological tumor assessment (by X-ray, computed tomography [CT] scan, PET scan, or magnetic resonance imaging [MRI]), blood count including blast count.

[0187] CD38 is the best method to assess disease response in hematological malignancies + Depending on the type of hematological malignancy, these methods are known to those skilled in the art.

[0188] Based on the results obtained from the assessment of disease response, disease response can be stratified according to standard criteria for underlying disease and can be classified as complete response or complete remission (CR), partial response (PR), stable disease (SD) or progressive disease (PD).

[0189] The "Karnofsky score" refers to a score from 100 to 0, where 100 is "perfect" health and 0 is death.

[0190] "Markers" as used in the context of responsiveness assessment may typically include serum and / or plasma markers such as, but not limited to, Hs-CRP, tumor necrosis factor alpha (TNF-α), IL-6, IL-1-β, IFN-λ, and CD38 receptor density and occupancy markers.

[0191] In one example, techniques for assessing disease response in subjects with multiple myeloma are bone marrow biopsy and / or bone marrow aspirate, radiological imaging of plasmacytoma, skeletal examination, quantification of M-protein as previously described, and measurement of serum β2-microglobulin.

[0192] Assessment of disease response can further include receptor density and receptor occupancy on circulating tumor cells (peripheral blood), receptor density and receptor occupancy on blasts and plasma cells in the bone marrow, and levels of human anti-drug antibodies (ADA).

[0193] The antibodies of the present disclosure can be administered in the form of a pharmaceutical composition containing pharmaceutically acceptable excipients and, optionally, a slow-release matrix, such as a biodegradable polymer, to form a therapeutic composition.

[0194] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecules and compositions that do not produce adverse, allergic or other untoward reactions when administered to a mammal, especially a human, in a timely manner. A pharmaceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating substance or formulation auxiliary.

[0195] The type of pharmaceutical composition comprising the antibodies of the present disclosure will, of course, depend on the condition being treated, the severity of the disease, the age, weight, and sex of the subject, and the like.

[0196] The antibodies of the present disclosure are formulated for intravenous administration.

[0197] In particular, pharmaceutical compositions comprising antibodies of the present disclosure can comprise pharmaceutically acceptable vehicles for injectable formulations, which may in particular be isotonic, sterile, saline solutions (such as mono- or di-sodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, or mixtures of such salts), or dried, in particular freeze-dried compositions that can constitute an injectable solution upon addition of sterile water or saline, as the case may be.

[0198] To prepare pharmaceutical compositions, an effective amount of the antibodies of the present disclosure can be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0199] The pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.In all cases, the pharmaceutical form must be sterile and fluid enough to allow easy injection.It must be stable under the conditions of manufacture and storage, and must be protected against the contaminating action of microorganisms such as bacteria and fungi.

[0200] The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerin, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, stabilizers, antifreeze agents, or antioxidants. Prevention of the action of microorganisms can be achieved by antibacterial and antifungal agents. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride.

[0201] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with several other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating various sterile active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients other than those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any other desired ingredient from a previously sterile-filtered solution thereof.

[0202] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the injectable solutions described above, although drug release capsules and the like are also available.

[0203] For intravenous administration of aqueous solutions, for example, the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous administration. In this regard, sterile aqueous vehicles that can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dose can be dissolved in 1 mL of isotonic NaCl solution and added to 1000 mL of subcutaneous infusion fluid or injected intravenously at a designated infusion site (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some dosage variations may be necessary depending on the condition of the subject being treated. The individual administering the solution will, in any event, determine the appropriate dose for the individual subject.

[0204] In one example, the antibody is formulated for intravenous administration and is therefore presented as a concentrate of infusion solution in a vial containing, for example, 5 mg / mL (100 mg / 20 mL) of an antibody of the present disclosure.

[0205] For administration to a subject, an appropriate volume of the antibody formulation is typically diluted in an infusion bag, for example, with 0.9% sodium chloride solution. The final infusion volume corresponding to the dose of the antibody of the present disclosure is administered over a period of time depending on the dose administered, and thus the amount of protein given per hour.

[0206] In a specific embodiment, an anti-CD38 antibody for use in accordance with the present disclosure is administered as a naked antibody. The naked anti-CD38 antibody can be administered alone, with dexamethasone, or in combination with a chemotherapeutic agent selected from the group consisting of lenalidomide, carfilzomib, bortezomib, melphalan, vincristine, cytarabine, and cyclophosphamide, optionally with dexamethasone. If not administered alone, the anti-CD38 antibody and the other agent can be administered either simultaneously or separately (e.g., sequentially over a period of time).

[0207] The antibodies of the disclosure can be administered in combination with drug therapies to prevent or control fatigue, nausea, fever, cough, vomiting, hypercalcemia, headache, constipation, bone pain, chills, diarrhea, pneumonia, anemia, dysgeusia, hypokalemia, fever, and hyperglycemia.

[0208] In another embodiment, medications to prevent or control fatigue, nausea, fever, cough, vomiting, hypercalcemia, headache, constipation, bone pain, chills, diarrhea, pneumonia, anemia, dysgeusia, hypokalemia, fever, and hyperglycemia can be administered prior to antibody treatment.

[0209] In the context of this disclosure, a physician can assess disease response and adapt the dosing regimen accordingly.

[0210] Throughout this application, the term "comprising" should be interpreted as encompassing all specifically mentioned features, as well as optional additional, unspecified features. As used herein, use of the term "comprising" also discloses embodiments in which no features are present (i.e., "consisting of") other than those specifically mentioned.

[0211] Some embodiments of the present disclosure are further illustrated below for illustrative purposes without limiting the disclosure:

[0212] Item 1: In one embodiment, a method of treating a patient with relapsed and / or refractory multiple myeloma is disclosed, comprising administering to the patient an antibody that specifically binds to CD38, wherein the antibody is administered to the patient at a safe therapeutic dose of 20 mg / kg or less.

[0213] Item 2: In another embodiment, a pharmaceutical composition comprising an antibody that specifically binds CD38 for use as a medicament in the treatment of relapsed and / or refractory multiple myeloma is disclosed, wherein the antibody is administered to a human subject at a safe therapeutic dose of about 20 mg / kg or less.

[0214] Item 3: In another embodiment, the antibody inhibits CD38 in a human subject by inducing apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). + Disclosed herein is a method or pharmaceutical composition according to item 1 or 2, which is capable of killing cells.

[0215] Item 4: Also disclosed herein is a method or pharmaceutical composition according to any one of items 1 to 3, wherein in another embodiment, the patient has at least one condition selected from the group consisting of: (a) a measurable serum M-protein level of greater than about 0.5 g / dL; (b) a urinary M-protein level (24-hour urine) of greater than about 200 mg; (c) a high level of serum free light chain (FLC) of greater than about 10 mg / dL with an abnormal FLC ratio; and combinations thereof.

[0216] Item 5: Also disclosed herein is a method or pharmaceutical composition according to any one of Items 1 to 4, wherein in another embodiment, the antibody comprises at least one heavy chain and at least one light chain, wherein the heavy chain comprises three contiguous complementarity-determining regions (CDRs) having amino acid sequences represented by SEQ ID NOs: 13, 37, and 15, and the light chain comprises three contiguous complementarity-determining regions (CDRs) having amino acid sequences represented by SEQ ID NOs: 16, 17, and 18.

[0217] Item 6: Also disclosed herein is a method or pharmaceutical composition according to any one of Items 1 to 5, wherein in another embodiment, the antibody comprises at least one heavy chain comprising the amino acid sequence represented by SEQ ID NO: 50 and at least one light chain comprising the amino acid sequence represented by SEQ ID NO: 52.

[0218] Item 7: Also disclosed herein is a method or pharmaceutical composition according to any one of Items 1 to 6, wherein in another embodiment, the safe therapeutic dose is from about 1 mg / kg to about 20 mg / kg.

[0219] Item 8: Also disclosed herein is a method or pharmaceutical composition according to any one of items 1 to 6, wherein in another embodiment, the safe therapeutic dose is about 5 mg / kg, or about 10 mg / kg, or about 20 mg / kg.

[0220] Item 9: In another embodiment, the present invention also discloses a method or pharmaceutical composition according to any one of items 1 to 8, wherein the safe therapeutic dose of the antibody is administered intravenously.

[0221] Item 10: Also disclosed herein is a method or pharmaceutical composition according to any one of Items 1 to 9, wherein in another embodiment, a safe therapeutic dose of the antibody is administered once a week or once every two weeks.

[0222] Item 11: Also disclosed herein is a method or pharmaceutical composition according to any one of items 1 to 10, wherein in another embodiment, the safe therapeutic dose is about 10 mg / kg or about 20 mg / kg administered once every two weeks.

[0223] Item 12: Also disclosed herein is a method or pharmaceutical composition according to any one of items 1 to 10, wherein in another embodiment, the safe therapeutic dose is about 10 mg / kg or about 20 mg / kg administered once weekly.

[0224] Item 13: Also disclosed herein is a method or pharmaceutical composition according to any one of Items 1 to 12, wherein in another embodiment, a safe therapeutic dose of the antibody is administered at an initial infusion rate ranging from about 0.042 mg / hour to about 250 mg / hour.

[0225] Item 14: Also disclosed herein is a method or pharmaceutical composition according to any one of Items 1 to 13, wherein in another embodiment, the antibody is administered in combination with dexamethasone.

[0226] Item 15: In another embodiment, the present disclosure also discloses a method or pharmaceutical composition according to any one of Items 1 to 14, wherein the antibody, when administered to a human subject at a dose of about 20 mg / kg or less, does not produce autoantibodies against the antibody.

[0227] Item 16: Also disclosed herein is a method or pharmaceutical composition according to any one of items 1 to 10, wherein in another embodiment, the antibody is capable of exhibiting detectable CD38 receptor occupancy in a human subject when administered to the human subject at a dose level of about 1 mg / kg every two weeks.

[0228] Item 17: Also disclosed herein is the method or pharmaceutical composition of any one of Items 1 to 10, wherein in another embodiment, the antibody, when administered to a human subject at a dose level of about 10 mg / kg or about 20 mg / kg every two weeks, is capable of exhibiting a CD38 receptor occupancy of at least about 84.1% in the human subject.

[0229] Item 18: Also disclosed herein is a method or pharmaceutical composition according to any one of items 1 to 10, wherein in another embodiment, the antibody, when administered to a human subject at a dose level of about 10 mg / kg or about 20 mg / kg every two weeks, is capable of exhibiting a CD38 receptor occupancy of at least about 97.7% in the human subject.

[0230] Item 19: Also disclosed herein is a method or pharmaceutical composition according to any one of Items 1 to 10, wherein in another embodiment, the antibody is capable of inhibiting tumor growth in a human subject when administered to the human subject at a dose level ranging from about 5 mg / kg to about 20 mg / kg every two weeks.

[0231] Item 20: Also disclosed herein is a method or pharmaceutical composition according to any one of Items 1 to 10, wherein in another embodiment, the antibody is capable of inhibiting tumor growth in a human subject when administered to the human subject at a dose level ranging from about 5 mg / kg to about 20 mg / kg once a week.

[0232] Item 21: Also disclosed herein is a method or pharmaceutical composition according to any one of Items 1 to 10, wherein in another embodiment, the antibody is capable of inhibiting tumor growth in a human subject when administered to the human subject at a dose level ranging from about 10 mg / kg to about 20 mg / kg every two weeks.

[0233] Item 22: In another embodiment, the antibody is administered at a dose of about 10 mg / kg to about 20 mg / kg weekly. Also disclosed herein is the method or pharmaceutical composition according to any one of items 1 to 10, which, when administered to a human subject at a dose level ranging from 100 mg / kg, is capable of inhibiting tumor growth in said human subject.

[0234] Item 23: In another embodiment, a unit dosage form comprising the pharmaceutical composition according to any one of Items 1 to 22 is also disclosed herein.

[0235] Item 24: In another embodiment, an article of manufacture is also disclosed herein, comprising the pharmaceutical composition of any one of Items 1 to 22 and a container.

[0236] A brief description of arrays SEQ ID NOs: 1 to 3 show the sequences of CDR1-H, CDR2-H, and CDR3-H of the "38SB13" antibody. SEQ ID NOs: 4 to 6 show the sequences of CDR1-L, CDR2-L, and CDR3-L of the "38SB13" antibody. SEQ ID NOs: 7 to 9 show the sequences of CDR1-H, CDR2-H, and CDR3-H of the "38SB18" antibody. SEQ ID NOs: 10 to 12 show the sequences of CDR1-L, CDR2-L, and CDR3-L of the "38SB18" antibody. SEQ ID NOs: 13 to 15 show the sequences of CDR1-H, CDR2-H, and CDR3-H of the "38SB19" antibody. SEQ ID NOs: 16 to 18 show the sequences of CDR1-L, CDR2-L, and CDR3-L of the "38SB19" antibody. SEQ ID NOs: 19 to 21 show the sequences of CDR1-H, CDR2-H, and CDR3-H of the "38SB30" antibody. SEQ ID NOs: 22 to 24 show the sequences of CDR1-L, CDR2-L, and CDR3-L of the "38SB30" antibody. SEQ ID NOs: 25 to 27 show the sequences of CDR1-H, CDR2-H, and CDR3-H of the "38SB31" antibody. SEQ ID NOs: 28 to 30 show the sequences of CDR1-L, CDR2-L, and CDR3-L of the "38SB31" antibody. SEQ ID NOs: 31 to 33 show the sequences of CDR1-H, CDR2-H, and CDR3-H of the "38SB39" antibody. SEQ ID NOs: 34 to 36 show the sequences of CDR1-L, CDR2-L, and CDR3-L of the "38SB39" antibody. SEQ ID NO: 37 shows the sequence of CDR2-H of the humanized "38SB19" antibody. SEQ ID NOs: 38 to 43 show the VL sequences of the antibodies. SEQ ID NOs: 44 to 49 show the VH sequences of the antibodies. SEQ ID NOs: 50 to 51 show the VH sequences of the humanized antibodies. SEQ ID NOs: 52 to 55 show the VL sequences of the humanized antibodies. SEQ ID NOs: 56 to 61 show the sequences of primers used in the first round degenerate PCR reactions for cloning and sequencing the light and heavy chains of anti-CD38 antibodies. SEQ ID NOs: 62-63 show the 5'-end murine leader sequence primers used in the 38SB19 second round PCR reactions for cloning and sequencing the light and heavy chains of the anti-CD38 antibody.

[0237] The present disclosure is further illustrated by the following examples. [Example]

[0238] The present inventors have determined the appropriate administration dose and regimen for antibody hu38SB19. , CD38 + Well-tolerated anti-cancer therapies have been achieved that can treat patients with hematological malignancies, particularly multiple myeloma, and more particularly relapsed and / or refractory multiple myeloma.

[0239] Thus, the following examples demonstrate the effectiveness of CD38 in multiple myeloma, such as in humans. + We disclose the results of a phase 1 clinical trial that demonstrated a safe therapeutic dose of this specific anti-CD38 antibody, hu38SB19, for the effective treatment of hematological malignancies.

[0240] patient A total of 32 patients were treated in this study. 40.6% of the population were women; the mean age was 64.8 (±8.8) years. Kornofsky status was greater than 60% in all patients.

[0241] The 32 patients included three patients with B-cell non-Hodgkin's lymphoma (NHL), two patients with chronic lymphocytic leukemia (CLL), and 27 patients with multiple myeloma (MM).

[0242] 80.0% of multiple myeloma patients were women, and the mean age was 63.6 (±8.0) years. Multiple myeloma patients received prior anticancer therapy with bortezomib in 100% of cases, lenalidomide in 92.6%, and autologous stem cell transplantation (ASCT) in 81.5%.

[0243] method The naked humanized IgG1 monoclonal antibody (mAb) hu38SB19 was used to select CD38 + It was administered as a single agent as an IV infusion every week (QW) or every two weeks (Q2W) to adult patients with ongoing hematologic malignancies, or after standard therapy, or for whom no effective standard therapy exists.

[0244] An accelerated dose escalation schedule was used for the first five dose levels (0.0001 mg / kg to 0.1 mg / kg every 2 weeks), with one patient evaluable per dose level unless a toxic dose level (DLT) was experienced. All subsequent dose levels (0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg every 2 weeks, and 10 mg / kg every week) followed a classical 3 + 3 design for dose escalation based on dose-level toxicity.

[0245] Thirty-two patients were evaluable for dose-level toxicity endpoint assessments and 32 were evaluable for tumor response assessments.

[0246] The median number of cycles ranged from 2.0 to 50.0.

[0247] result Thirty-two patients were treated across all dose levels, including three patients with B-cell non-Hodgkin's lymphoma (NHL), two patients with chronic lymphocytic leukemia (CLL), and 27 patients with multiple myeloma (MM).

[0248] Studies involving dose levels of 20 mg / kg every 2 weeks and 10 mg / kg once weekly are currently being evaluated, and the maximum tolerated dose has not been reached.

[0249] Dose-limiting toxicity was limited to a grade 2 infusion reaction during cycle 1 at a DL of 0.3 mg / kg and a DL of 3.0 mg / kg, which was mitigated by routine pre-treatment with methylprednisone, diphenhydramine, ranitidine, and acetaminophen. did.

[0250] The most frequent adverse events (≥10%) across all dose levels, regardless of cause, were fatigue (46.9%), nausea (31.3%), fever (28.1%), cough (25%), vomiting (21.9%), and hypercalcemia (18.8%), with headache, constipation, bone pain, chills, and diarrhea each occurring in 15.6% of patients. Additionally, pneumonia, anemia, dysgeusia, and hypokalemia each occurred in 12.5% ​​of patients.

[0251] Serious adverse events related to therapy included grade 3 pneumonia (6.3%), fever-related (3.1%), hyperglycemia (3.1%), and one grade 2 infusion reaction (3.1%).

[0252] Of the 19 patients treated at dose levels of 1.0 mg / kg to 10 mg / kg every 2 weeks, 1 had chronic lymphocytic leukemia (CLL), 1 had non-Hodgkin's lymphoma (NHL), and 17 had multiple myeloma (MM).

[0253] The 17 multiple myeloma patients were elderly and heavily pretreated; median age was 64 years (range, 55-74 years); and median number of prior regimens was seven (range, 2-14). All MM patients had received prior lenalidomide and bortezomib. The median time from diagnosis to first dose of hu38SB19 was 6.8 years (range, 1.8-16.8 years).

[0254] Responses in this group (Figure 1) according to the European Group for Bone Marrow and Transplant (EBMT) multiple myeloma criteria included one partial response (PR) at 1 mg / kg (n=3) and 5 mg / kg (n=3) dose levels, and one minimal response (MR) at the 3 mg / kg dose level (n=6). The 10 mg / kg dose level demonstrated three partial responses (PR) and two stable disease (SD) among the six multiple myeloma patients treated.

[0255] For the 19 patients treated with DL of 1 mg / kg or more, the mean duration of treatment was 8 weeks (range 2–50 weeks).

[0256] Immunogenicity studies show no anti-hu38SB19 antibodies.

[0257] Receptor occupancy was detectable from the dose level of 1 mg / kg, reaching a range of 84.1-97.7% at 10 mg / kg.

[0258] Pharmacokinetic analysis (PK) shows a more dose-proportional increase in exposure over the dose range of 0.03 to 10 mg / kg, and clearance in a similar range from 5 mg / kg to 10 mg / kg.

[0259] C in Cycle 2 over a dose range of 0.03 to 3 mg / kg max No accumulation was observed based on

[0260] The tumor growth inhibition threshold was C at DL 5 mg / kg for one patient and DL 10 mg / kg for five patients. max reached.

[0261] The anti-CD38 antibody hu38SB19 demonstrates promising single-agent activity in patients with heavily pretreated relapsed and / or refractory multiple myeloma.

[0262] The following references are cited throughout this specification or listed below (see, for example, references to patents, patent applications, and publications): The contents of all cited references (including cross-references) are expressly incorporated by reference in their entirety into this disclosure for all purposes. This disclosure may employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, and cell biology that are well known in the art.

Claims

1. A method for treating a human subject with relapsed and / or refractory multiple myeloma, comprising administering to the human subject an antibody that specifically binds to CD38, wherein the antibody is administered to the human subject at a safe therapeutic dose of 20 mg / kg or less.

2. A pharmaceutical composition comprising an antibody that specifically binds to CD38 for use as a drug in the treatment of relapsed and / or refractory multiple myeloma, wherein the antibody is administered to a human subject at a safe therapeutic dose of about 20 mg / kg or less.

3. The antibody inhibits CD38 in human subjects by inducing apoptosis, antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). ＋ 3. The method or pharmaceutical composition of claim 1 or 2, which is capable of killing cells.

4. Human subjects are a) a measurable serum M-protein level greater than about 0.5 g / dL; b) urinary M-protein levels greater than about 200 mg (24-hour urine); c) high levels of serum free light chains (FLC) greater than about 10 mg / dL with an abnormal FLC ratio; and combinations thereof The method or pharmaceutical composition of any one of claims 1 to 3, wherein the patient has at least one condition selected from the group consisting of:

5. 5. The method or pharmaceutical composition of any one of claims 1 to 4, wherein the antibody comprises at least one heavy chain and at least one light chain, the heavy chain comprising three contiguous complementarity determining regions (CDRs) having amino acid sequences represented by SEQ ID NOs: 13, 37, and 15, and the light chain comprising three contiguous complementarity determining regions (CDRs) having amino acid sequences represented by SEQ ID NOs: 16, 17, and 18.

6. 6. The method or pharmaceutical composition of any one of claims 1 to 5, wherein the antibody comprises at least one heavy chain comprising the amino acid sequence represented by SEQ ID NO: 50 and at least one light chain comprising the amino acid sequence represented by SEQ ID NO:

52.

7. The method or pharmaceutical composition of any one of claims 1 to 6, wherein a safe therapeutic dose is from about 1 mg / kg to about 20 mg / kg.

8. The method or pharmaceutical composition of any one of claims 1 to 6, wherein a safe therapeutic dose is about 5 mg / kg, or about 10 mg / kg, or about 20 mg / kg.

9. 9. The method or pharmaceutical composition of any one of claims 1 to 8, wherein a safe therapeutic dose of the antibody is administered intravenously.

10. 10. The method or pharmaceutical composition of any one of claims 1 to 9, wherein a safe therapeutic dose of the antibody is administered once a week or once every two weeks.

11. The method or pharmaceutical composition of any one of claims 1 to 10, wherein a safe therapeutic dose is about 10 mg / kg or about 20 mg / kg administered once every two weeks.

12. 11. The method or pharmaceutical composition of any one of claims 1 to 10, wherein a safe therapeutic dose is about 10 mg / kg or about 20 mg / kg administered once weekly.

13. 13. The method or pharmaceutical composition of any one of claims 1 to 12, wherein a safe therapeutic dose of the antibody is administered at an initial infusion rate ranging from about 0.042 mg / hour to about 250 mg / hour.

14. The method or pharmaceutical composition of any one of claims 1 to 13, wherein the antibody is administered in combination with dexamethasone.

15. 15. The method or pharmaceutical composition of any one of claims 1 to 14, wherein the antibody does not produce autoantibodies against the antibody when administered to a human subject at a dose of about 20 mg / kg or less.

16. 11. The method or pharmaceutical composition of any one of claims 1 to 10, wherein the antibody is capable of demonstrating detectable CD38 receptor occupancy in a human subject when administered to the human subject at a dose level of about 1 mg / kg every two weeks.

17. 11. The method or pharmaceutical composition of any one of claims 1 to 10, wherein the antibody is capable of demonstrating at least about 84.1% CD38 receptor occupancy in a human subject when administered to the human subject at a dose level of about 10 mg / kg or about 20 mg / kg every two weeks.

18. 11. The method or pharmaceutical composition of any one of claims 1 to 10, wherein the antibody is capable of demonstrating at least about 97.7% CD38 receptor occupancy in a human subject when administered to the human subject at a dose level of about 10 mg / kg or about 20 mg / kg every two weeks.

19. 11. The method or pharmaceutical composition of any one of claims 1 to 10, wherein the antibody is capable of inhibiting tumor growth in a human subject when administered to the human subject at a dose level ranging from about 5 mg / kg to about 20 mg / kg every two weeks.

20. 11. The method or pharmaceutical composition of any one of claims 1 to 10, wherein the antibody is capable of inhibiting tumor growth in a human subject when administered to the human subject at a dose level ranging from about 5 mg / kg to about 20 mg / kg weekly.

21. 11. The method or pharmaceutical composition of any one of claims 1 to 10, wherein the antibody is capable of inhibiting tumor growth in a human subject when administered to the human subject at a dose level ranging from about 10 mg / kg to about 20 mg / kg every two weeks.

22. 11. The method or pharmaceutical composition of any one of claims 1 to 10, wherein the antibody is capable of inhibiting tumor growth in a human subject when administered to the human subject at a dose level ranging from about 10 mg / kg to about 20 mg / kg weekly.

23. A unit dosage form comprising the pharmaceutical composition of any one of claims 1 to 22.

24. An article of manufacture comprising the pharmaceutical composition of any one of claims 1 to 22 and a container.

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