TW201729832A - Immune modulation and treatment of solid tumors with antibodies that specifically bind CD38 - Google Patents

Abstract

The present invention relates to methods of immunomodulation and treating patients having solid tumors with antibodies that specifically bind CD38.

Description

Immunomodulation and treatment of solid tumors using antibodies that specifically bind to CD38 [Reciprocal Reference of Related Applications]

The present application claims US Application No. 15/191808, filed on Jun. 24, 2016, and International Application No. US/J. U.S. Provisional Application No. 62/263,307, filed on December 4, 2015, and U.S. Provisional Application No. 62/250,566, filed on November 4, 2015, and November 2, 2015 The benefit of U.S. Provisional Application Serial No. 62/249,546, the entire contents of which is incorporated herein by reference.

The present invention relates to a method for immunomodulating and treating solid tumors with antibodies that specifically bind to CD38.

The immune system is tightly controlled by the network of co-stimulation and co-suppression of ligands and receptors. These molecules provide a secondary signal for T cell activation and provide a balanced network of positive and negative signals to maximize immune responses to infections and tumors while limiting immunity to themselves (Wang et al., (Epub Mar.). 7, 2011) J Exp Med 208(3): 577-92; Lepenies et al. (2008) Endocr Metab Immune Disord Drug Targets 8: 279-288 ).

Immunological checkpoint therapy for solid tumors, which targets the co-suppressor pathway in T cells to promote anti-tumor immune response, is approved for anti-CTLA-4 and anti-PD-1 antibody YERVOY ^® (ipilimumab), The case of KEYTRUDA ^® (pembrolizumab) and OPDIVO ^® (nivolumab) has led to advances in clinical care for cancer patients. Although the anti-PD-1/PD-L1 antibody demonstrates a clinical response that promotes patients with multiple solid tumors, the response rate is still quite low, ranging from 15% to 20% in pre-treated patients (Swaika et al. Person, (2015) Mol Immunol doi: 10.1016/j.molimm.2015.02.009).

Although natural killer cells (NK), dendritic cells (DC), and effector T cells can drive potent anti-tumor responses, tumor cells often induce an immunosuppressive microenvironment that favors the development of immunosuppressive immune cell populations. For example, bone marrow-derived suppressor cells (MDSCs), regulatory T cells (Tregs), or regulatory B cells (Breg), which cause tumor immunotolerance and immunotherapy regimens in cancer patients and experimental tumor models.

Therefore, there is still a need to develop new cancer immunotherapies that induce adaptive immune responses against tumors or target immunosuppressive immune cells.

The present invention provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38.

The invention also provides a method for treating a disease having a regulatory T cell (Treg) vector comprising administering to a patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38.

The invention also provides a method for treating a disease having a bone marrow-derived suppressor cell (MDSC) vector comprising administering to a patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38.

The invention also provides a method for treating a disease having a regulatory B cell (Breg) vector comprising administering to a patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38.

The invention also provides a method of inhibiting the activity of a regulatory T cell (Treg) comprising contacting the Treg with an antibody that specifically binds to CD38.

The invention also provides a method of inhibiting the activity of a bone marrow-derived suppressor cell (MDSC) comprising contacting the MDSC with an antibody that specifically binds to CD38.

The invention also provides a method of inhibiting the activity of a regulatory B cell (Breg) comprising contacting the Breg with an antibody that specifically binds to CD38.

The invention also provides a method of enhancing an immune response in a patient comprising administering to the patient an antibody that specifically binds to CD38.

The invention also provides a method of treating a patient having a solid tumor comprising reducing the number of Treg cells in the patient by administering to the patient an antibody that specifically binds to CD38.

The present invention also provides a method of treating a patient having a solid tumor comprising reducing the number of bone marrow-derived suppressor cells (MDSCs) in the patient by administering to the patient an antibody that specifically binds to CD38.

The present invention also provides a method of inhibiting the activity of an immunosuppressive cell comprising contacting the immunosuppressive cell with an antibody that specifically binds to CD38.

The invention also provides a method of treating a patient suffering from a viral infection comprising administering to a patient in need thereof an antibody that specifically binds to CD38.

Figure 1 shows that the median number of lymphocytes in patients increased the response to DARZALEXTM (dalacil ⁾ at 8 mg/kg (upper line) or 16 mg/kg (lower line) over time. And the number of lymphocytes returned to the base period after the end of treatment. Research: SIRIUS. The X-axis indicates time, which is expressed in terms of the treatment period and the number of days of administration in each treatment cycle (C1D1: Day 1 of the first cycle; C1D4: Day 4 of the first cycle, etc.). SCR: base period; EOT: end of treatment; WK: week; POST-WK: designated weeks after treatment; post-PD FU: follow-up after progression. The range highlighted by the shades of gray indicates that the interquartile range (IQR) of the data points of each visitor's visit is 25 to 27%.

Figure 2 shows the percentage change (%) from the base period of absolute counts of CD3 ⁺ T cells in peripheral blood of patients treated with DARZALEX ^(TM) (darlazumab ⁾ for each individual patient (light gray line). Research: SIRIUS (MMY 2002). The X-axis indicates time, which is expressed in terms of the treatment period and the number of days of administration in each treatment cycle (C1D1: Day 1 of the first cycle; C1D4: Day 4 of the first cycle, etc.). WK: Week; POST-WK: designated weeks after treatment; POST-PD FU: Tracking after progression. The black line shows the % change in the median of all patients.

Figure 3 shows the percentage change (%) of absolute counts of CD4 ⁺ T cells from peripheral blood in peripheral blood of patients treated with DARZALEX ^(TM) (darlazumab ⁾ for each individual patient (light gray line). Research: SIRIUS. The X-axis indicates time, which is expressed in terms of the treatment period and the number of days of administration in each treatment cycle (C1D1: Day 1 of the first cycle; C1D4: Day 4 of the first cycle, etc.). WK: Week; POST-TMT: After treatment. The black line shows the % change in the median of all patients.

Figure 4 shows the percentage change (%) of absolute counts of CD8 ⁺ T cells from the peripheral phase of peripheral blood of patients treated with DARZALEX ^(TM) (darlazumab ⁾ for each individual patient (light gray line). Research: SIRIUS. The X-axis indicates time, which is expressed in terms of the treatment period and the number of days of administration in each treatment cycle (C1D1: first cycle, day 1; C1D4: day 4 of the first cycle, etc.). WK: Week; Pre-PD FU: Tracking before progress; Post-PD FU: Tracking after progress. The black line shows the % change in the median of all patients.

Figure 5 shows that the number of CD45 ⁺ CD3 ⁺ cells in bone marrow extracts (measured as a percentage of lymphocytes) increased over time during treatment with DARZALEX ^(TM) (dalabizumab) at a dose of 8 mg/kg or 16 mg/kg. The chart includes responders and non-responders as indicated. Research: SIRIUS. The X-axis indicates time, expressed as the treatment cycle and the number of days of dosing in each treatment cycle (C2D22: Day 22, Day 22; etc.). SCR: Base period; Post-PD FU1: Tracking after progress. The range highlighted by shades of gray indicates four points for data points for each visit to non-responders administered at 8 mg/kg, responders administered at 16 mg/kg, or non-responders administered at 16 mg/kg. The number of bits (IQR) is 25 to 27%, respectively. NR: no responder; R: responder.

Figure 6 shows that the number of CD45 ⁺ CD3 ⁺ CD8 ⁺ cells (measured as a percentage of lymphocytes) in bone marrow aspirate increased over time during treatment with DARZALEX ^TM (dalacil) at a dose of 8 mg/kg or 16 mg/kg. . The chart includes responders and non-responders as indicated. Research: SIRIUS. The X-axis indicates time, expressed as the treatment cycle and the number of days of dosing in each treatment cycle (C2D22: Day 22, Day 22, etc.). SCR: Base period; Post-PD FU1: Tracking after progress. The range highlighted by shades of gray indicates four points for data points for each visit to non-responders administered at 8 mg/kg, responders administered at 16 mg/kg, or non-responders administered at 16 mg/kg. The number of bits (IQR) is 25 to 27%, respectively. NR: no responder; R: responder.

Figure 7A shows an increase in the ratio of CD8 ⁺ /Treg and CD8 ⁺ /CD4 ⁺ cells in peripheral blood as indicated by the median value of all treated patients during DARZALEX ^(TM) (duracil) treatment. Time point: C1D1: Day 1 of the first cycle; C3D1: Day 1 of the 3rd cycle; C4D1: Day 1 of the 4th cycle. Research: SIRIUS. SRC: Base period.

Figure 7B shows that the ratio of CD8 ⁺ /Treg cells in bone marrow aspirate expressed as a median value for all treated patients increased over time during DARZALEX ^(TM) (darabizumab) treatment. Time point: C1D1: Day 1 of the first cycle; C3D1: Day 1 of the 3rd cycle; C4D1: Day 1 of the 4th cycle. Research: SIRIUS.

Figure 8A shows that the responder's CD8 ⁺ T cells are purely increased when compared to non-responders, as measured by % abundance change (CIA) of specific pure lineage cells. Study: GEN501 17 subset of patients.

Figure 8B shows the fold change in CD8 ⁺ T cell pureness in individual patients after DARZALEX ^(TM) (dalabizumab) treatment. Responders are indicated by an asterisk. The pure lineage is measured as a multiple of the abundance change (CIA) of a particular lineage of cells. Study: GEN501 17 subset of patients.

Figure 8C shows that when compared to non-responders (Group B), the responders (Group A) had a larger total amplification in the TCR depot, which was measured using CIA (abundance change). P=0.037. Study: GEN501 17 subset of patients.

Figure 8D shows the sum of absolute abundance changes (CIA) of each expanded T cell colony in the responder and non-responder. P = 0.035 between the responder (group A) and the non-responder (group B). Study: GEN501 17 subset of patients.

Figure 8E shows the maximum CIA for individual cell lines in the responders (Group A) and non-responders (Group B). Study: GEN501 17 subset of patients.

Figure 8F shows that the maximum amplification of a single colony of responders (Group A) was greater when compared to non-responders (Group B), which was measured using the maximum CIA%. P = 0.0477. Study: GEN501 17 subset of patients.

Figure 9A shows non-responders (NR, black squares) and patients with at least minimal response to DARZALEX ^(TM) (dalabizumab ⁾ at the base period, or after 2 weeks, 4 weeks, or 8 weeks of treatment, or after relapse ( MR8, white square) peripheral blood CD8 ⁺ initial cells (na The percentage (%) of ve cell). Study: GEN501 17 subset of patients. **p=1.82×10 ^-4 .

Figure 9B shows non-responders (NR, black squares) and patients with at least minimal response to DARZALEX ^(TM) (darazumab ⁾ at the base period, or after 2 weeks, 4 weeks, or 8 weeks of treatment, or after relapse ( MR, white square) Percentage of CD8 ⁺ central memory cells (Tem) in peripheral blood. Study: GEN501 17 subset of patients. *p=4.88×10 ^-2 .

Figure 9C shows the percent increase in HLA class I-restricted CD8 ⁺ T cells in peripheral blood at the base period, or at 1, 4, or 8 weeks of treatment, or after relapse. Study: GEN501 17 subset of patients.

Figure 9D shows that CD38 is expressed at low levels in peripheral blood CD8 ⁺ naive T cells and CD8 ⁺ central memory cells (Tem) during the base phase or treatment. Study: GEN501 17 subset of patients. MFI: Average fluorescence intensity.

Figure 10A shows a FACS analysis showing the frequency of Treg (CD3 ⁺ CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim ) in multiple myeloma patients (upper profile, P4 cell population) and CD38 ⁺ Treg in the basal phase Frequency within the Treg group (lower profile, P5 cell population). Study: GEN501 17 subset of patients.

Figure 10B shows a profile of FACS analysis showing the frequency of Treg (CD3 ⁺ CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim ) in multiple myeloma patients after DARZALEX ^TM (duracil) treatment (upper profile, P4 cell population) and the frequency of CD38 ⁺ Treg in the Treg population (lower profile, P5 cell population). DARZALEX ^TM (Dara mAb) therapy to CD38 ⁺ Treg loss. Study: GEN501 17 subset of patients.

Figure 10C shows CD38 ^high CD3 ⁺ in patients treated with DARZALEX ^(TM) (dalacil ⁾ at the base period, or at 1 week, 4 weeks, 8 weeks, after relapse, or at the end of 6 months of treatment (EOT). Frequency of CD4 ⁺ CD25 ⁺ CD127 ^dim Treg. As the frequency of the CD38 ^high Treg DARZALEX ^TM (Dara mAb) treatment reduces, and returns to the base of at EOT. Y-axis: % of CD38 ^high CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim Treg of CD3 ⁺ T cells. Study: GEN501 17 subset of patients.

Figure 10D shows CD8 ⁺ /Treg cell ratios in responders and non-responders at baseline, at 1 week, 4 weeks, and 8 weeks of treatment. At the 8th week of treatment, the CD8 ⁺ /Treg cell ratio of the responders to non-responders was significantly higher (p = 0.00955). Study: GEN501 17 subset of patients.

Figure 10E shows that effector cell proliferation is more effectively inhibited in the presence of CD38 ⁺ Treg when compared to CD38 ^- Treg or a negative control. Error bars represent standard errors. The asterisk indicates a significant change. Samples were obtained from multiple healthy donors. Cell proliferation was assessed by dilution of carboxyfluorescein amber imidate (CFSE).

Figure 11 shows the presence of bone marrow-derived suppressor cells (MDSCs) in multiple myeloma patients (top panel , boxed cells), and about half of the cells exhibit CD38 (middle panel, boxed cells). CD38 high population of MDSC in the primarily DARZALEX ^TM (Dara mAb) infusion therapy in patients loss (lower graph, boxed cells). Study: GEN501 17 subset of patients.

Figure 12 shows CD38 high MDSC (CD11b ⁺ HLADR ^- CD14 ^- CD33 ⁺ CD15 ⁺ ) in patients after 1 week, 4 weeks, or 8 weeks of treatment with DARZALEX ^TM (dalabiza ⁾ compared to the base phase. The number is reduced and returns to near the base period after the end of treatment (EOT). Relapsed patients still showed a reduction in CD38 high MDSC. Black squares: no response; open square: patients having at least a minimum of reaction DARZALEX ^TM (Dara mAb) therapy. The vertical line indicates the median value in each group. Patients 2, 4, 15, 16, and 17 exhibited a high initial CD38 high MDSC population. Study: GEN501 17 subset of patients.

Figure 13 shows that patients with the highest CD38 high MDSC (patients 2, 4, 15, 16, and 17) had the highest progression free survival (PFS). These patients had a partial response (PR) or minimum reaction (MR) of DARZALEX ^TM (Dara mAb) therapy. SD: stable disease; PD: disease progression. The X-axis shows the PFS for each patient with a different number.

Figure 14 shows MDSC sensitivity to DARZALEX ^(TM) (dalabizumab) induced ADCC. Daudi cells were used as positive controls for the target cells in the assay. The % cell lysis was measured.

Figure 15A shows that CD38 ⁺ Breg was depleted in patients treated with DARZALEX ^(TM) (dalacil ⁾ at weeks 1st, 4th, and 8th week of treatment.

Figure 15B shows that CD38 ⁺ Breg secretes IL-10 after stimulation.

Figure 16A shows the production of CMV, EBV, and influenza virus specific (CEF) IFN-γ in PBMCs of patients treated with DARZALEX ^(TM) (Dalabiza ⁾ with VGPR at the base phase and at specified times during the treatment period. The antiviral response measured. OD: optical density. White bars: negative control; black bars: added CEF; short bars: only allogeneic PBMC. Asterisks indicate statistically significant changes. Pre 4, 8, 10 = 4, 8, or 10 weeks of treatment.

Figure 16B shows the production of CMV, EBV, and influenza virus specific (CEF) IFN-γ in PBMCs of patients treated with DARZALEXTM (Dalabiza ⁾ with CR at the base phase and at specified times during the treatment period. The antiviral response measured. OD: optical density. White bars: negative control; black bars: added CEF; short bars: only allogeneic PBMC. Asterisks indicate statistically significant changes. Pre 4, 8, 10 = 4, 8, or 10 weeks of treatment.

Figure 16C shows the production of CMV, EBV, and influenza virus specific (CEF) IFN-γ in PBMCs of patients treated with DARZALEXTM (dalabizumab ⁾ with PD at the base phase and at specified times during treatment. The antiviral response measured. OD: optical density. White bars: negative control; black bars: added CEF; short bars: only allogeneic PBMC. Ns: Not significant. Pre 4, 8 = 4 or 8 weeks of treatment.

Figure 16D shows the production of CMV, EBV, and influenza virus specific (CEF) IFN-[gamma] in PBMCs treated with DARZALEX ^(TM) (Dalabizumab ⁾ with MR at baseline and at specified times during treatment. The antiviral response measured. OD: optical density. White bars: negative control; black bars: added CEF; short bars: only allogeneic PBMC. Ns: Not significant. Pre 4, 8 = 4 or 8 weeks of treatment.

Figure 16E shows the percentage (%) of proliferating virus-reactive T cells in PBMC of patients treated with DARZALEX ^(TM) (Dalabiza ⁾ with VGPR at the base period and at the time indicated during treatment. White bars: negative control; black bars: add CEF. Asterisks indicate statistically significant changes. Pre 4, 8, 10 = 4, 8, or 10 weeks of treatment.

Figure 16F shows the percentage (%) of proliferating virus-reactive T cells in PBMCs of patients treated with DARZALEX ^(TM) (darabumab ⁾ with CR at the base period and at the time indicated during treatment. White bars: negative control; black bars: add CEF. Asterisks indicate statistically significant changes. Pre 4, 8, 10 = 4, 8, or 10 weeks of treatment.

Figure 17A shows a profile of FACS analysis showing the level of performance of CD38 on natural donor killer cells (NK), monocytes, B cells, and T cells.

Figure 17B shows a profile of FACS analysis showing the level of expression of CD38 on plasma cells, natural killer cells (NK), monocytes, B cells, and T cells in multiple myeloma patients.

Figure 17C shows a comparison of mean fluorescence intensity (MFI) of CD38 in CD38+ Treg, Breg, NK, B cells, and T cells in patients with relapsed and refractory multiple myeloma. When compared to CD38+Treg, Bregs, and NK cells, CD38 is expressed at lower levels in B cells and T cells.

Figure 18 shows that PD-L1 protein is down-regulated in the PBMC sample of responder (R) over time and upregulated in non-responder (NR). SD: The disease is stable. C1D1: Day 1 of the first cycle; C3D1: Day 1 of the third cycle. The Y axis shows the log2 protein concentration value.

In the specification and the appended claims, the singular forms "a" and "the" Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

"CD38" refers to human CD38 protein (synonym: ADP ribosyl cyclase 1, cADPr hydrolase 1, ring ADP ribose hydrolase 1). Human CD38 has the amino acid sequence shown in GenBank Accession No. NP_001766 and SEQ ID NO: 1. It is well known that CD38 is a type II single-transmembrane protein having: amino acid residues 1 to 21, which represent the cytoplasmic domain; amino acid residues 22 to 42, which represent the transmembrane domain; And residues 43 to 300, which represent the extracellular domain of CD38.

SEQ ID NO: 1

As used herein, "antibody" means, in a broad sense, and includes immunoglobulin molecules, including monoclonal antibodies (including murine, human, humanized, and chimeric individuals). Antibody, antibody fragment, bispecific or multispecific antibody, dimer, tetramer, or multimeric antibody, single chain antibody, domain antibody, and any other antigen binding site comprising the desired specificity ( The antigen binding site is a modified configuration of immunoglobulin.

Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be assigned to one of two distinct types (ie, kappa (κ) and lambda (λ)), depending on the amino acid sequence of the constant domains.

"Antibody fragment" refers to a portion of an immunoglobulin molecule that retains heavy and/or light chain antigen binding sites, such as heavy chain complementarity determining regions (HCDRs) 1, 2, and 3, and light chain complementation. Decision regions (LCDR) 1, 2, and 3, heavy chain variable region (VH), or light chain variable region (VL). Antibody fragments include Fab fragments, ie, monovalent fragments consisting of VL, VH, CL, and CH1 domains; F(ab) ₂ fragments, ie, bivalent fragments, contained in the hinge region linked by a disulfide bridge Two Fab fragments; the Fd fragment consists of the VH and CH1 domains; the Fv fragment consists of the VL and VH domains of the antibody's one-arm; the domain antibody (dAb) fragment (Ward et al, Nature 341:544-6, 1989), which consists of the VH domain. The VH and VL domains can be engineered and linked together via synthetic linkers to form various types of single-chain antibody designs in which the VH/VL domain is subjected to intramolecular pairing, or in the VH and VL domains by separate single-chain antibodies In the case of constructs, intermolecular pairing is performed to form a monovalent antigen binding site, such as a single chain Fv (scFv) or a diabody; for example, as described in PCT International Publication No. WO 1998/44001, WO 1988 No. /01649, WO1994/13804, and WO1992/01047. These antibody fragments are obtained using well-known techniques known to those of ordinary skill in the art, and these fragments are screened for utility in the same manner as full length antibodies.

By "isolated antibody" is meant an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (eg, an isolated antibody that specifically binds to CD38 is substantially free of specific binding) An antibody to an antigen other than human CD38). However, isolated antibodies that specifically bind to CD38 may cross-react with other antigens, such as heterologs of human CD38, such as macaca fascicularis (cynomolgus) CD38. In the case of a bispecific antibody, the bispecific antibody specifically binds to two antigens of interest and is substantially free of antibodies that specifically bind to antigens that are not two antigens of interest. Furthermore, the isolated antibodies can be substantially free of other cellular materials and/or chemicals. "Isolated antibodies" encompass antibodies that are isolated to higher purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure antibody.

"Specific binding (specific binding)" or "bind" refers to an antibody that binds to an antigen or an epitope within the antigen with greater affinity than other antigens. Typically, the antibody is at about 1 x 10 ^-8 M or less (e.g., about 1 x 10 ^-9 M or less, about 1 x 10 ^-10 M or less, about 1 x 10 ^-11 M or less, or An equilibrium dissociation constant (K _D ) of about 1 × 10 ^-12 M or less, bound to an epitope within an antigen or antigen, usually with its binding to a non-specific antigen (eg, BSA, casein) _{D is} at least a hundred times more K _D combined. Dissociation constants can be measured using standard procedures. However, an antibody that specifically binds to an epitope within an antigen or antigen may be cross-reactive with other related antigens, such as for the same antigen (homolog) from other species, such as humans or monkeys, the monkey. For example, Crab Macaque ( Macaca fascicularis , cynomolgus, cyno), chimpanzee ( Pan troglodytes , chimpanzee, chimp), or 狨 ( Calithrix jacchus , common marmoset, marmoset). Although a monospecific antibody specifically binds to one antigen or one epitope, a bispecific antibody specifically binds two different antigens or two different epitopes.

The antibody variable region consists of a "framework" region interrupted by three "antigen binding sites". These antigen binding sites are defined in various terms: complementarity determining regions (CDRs) (three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3)) based on sequence variability (Wu and Kabat (1970) J Exp Med 132:211-50; Kabat et al. Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991); "Hypervariable region" ), "HVR", or "HV" (three in VH (H1, H2, H3) and three in VL (L1, L2, L3)) are structural heights as defined by Chothia and Lesk The region of the variable variable domain of the hypervariable (Chothia and Lesk (1987) Mol Biol 196:901-17). Other terms include "IMGT-CDR" (Lefranc et al. (2003) Dev Comparat Immunol 27: 55-77) and "Specificity Determining Residue Usage" (SDRU) (Almagro (2004) Mol Recognit 17:132-43). The International ImMunoGeneTics (IMGT) database (http://www_imgt_org) provides definitions of standardized numbers and antigen binding sites. Correspondence between CDR, HV, and IMGT delineation is described in Lefranc et al. (2003) Dev Comparat Immunol 27: 55-77.

As used herein, "Chothia residue" is an antibody VL and VH residue numbered according to Al-Lazikani (Al-Lazikani et al., (1997) J Mol Biol 273: 927-48).

A "framwork" or "framwork sequence" is defined as a sequence other than an antigen binding site in a variable region. Since the antigen binding site can be defined by various terms as described above, the exact amino acid sequence of the architecture depends on how the antigen binding site is defined.

"Humanized antibody" refers to an antibody whose antigen binding site is derived from a non-human species and whose variable region architecture is derived from a human immunoglobulin sequence. Humanized antibodies may include substitutions in the framework regions, so the framework may not be an exact replica of the human immunoglobulin or germline gene sequences.

"Human antibody" refers to an antibody having a heavy chain and a light chain variable region, wherein both the framework and the antigen binding site are derived from a human sequence. If the antibody contains a constant region, the constant region is also derived from a human sequence.

Human antibodies comprise a heavy or light chain variable region derived from a human derived sequence, wherein the variable region of the antibody is obtained from the use of a human germline immunoglobulin or a rearranged immunoglobulin gene. system. Such systems include human immunoglobulin gene libraries presented on phage, and gene transfer non-human animals (such as mice bearing human immunoglobulin loci). "Human antibodies" may contain amino acid differences when compared to human germline immunoglobulin or rearranged immunoglobulin genes due to, for example, naturally occurring somatic mutations, or deliberate introduction in architecture or antigen binding sites. Replace, or both. Typically, a "human antibody" has at least about 80%, 81%, 82%, 83% of the amino acid sequence encoded by an amino acid sequence encoded by a human germline immunoglobulin or a rearranged immunoglobulin gene. 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % identity. In some cases, a "human antibody" may contain a consensus framework sequence derived from human architecture sequence analysis, as described, for example, by Knappik et al., (2000) J Mol Biol 296: 57-86, or incorporated into a phage. The synthetic HCDR3 in the human immunoglobulin gene library is described, for example, in Shi et al., (2010) J Mol Biol 397:385-96 and International Patent Publication No. WO 2009/085462.

Human antibodies derived from human immunoglobulin sequences can be produced using systems such as phage displays incorporating synthetic CDRs and/or synthetic frameworks, or can be induced in vitro to improve antibody properties, thereby obtaining in vivo An antibody that does not naturally occur in the human antibody reproductive system repertoire.

Antibodies whose antigen binding sites are derived from non-human species are not included in the definition of human antibodies.

"Recombinant antibody" includes all antibodies produced, expressed, created, or isolated by recombinant means. An isolated animal, such as a mouse or a rat having a human immunoglobulin gene (such as a mouse or a rat), or a fusion tumor prepared therefrom (described further below); a self-transformed transform An antibody that is isolated from a host cell expressing the antibody; an antibody that is isolated from the recombinant antibody pool; and prepared, expressed, created, or singulated by any other means involved in splicing the human immunoglobulin gene sequence to other DNA sequences An antibody that is isolated; or an antibody produced by Fab arm exchange in vitro, such as a bispecific antibody.

A "monoclonal antibody" refers to a preparation of an antibody molecule consisting of a single molecule. The monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope, or dual binding specificity for two different epitopes in the case of a bispecific monoclonal antibody. "Single antibody" thus refers to a population of antibodies having a single amino acid composition in each heavy chain and in each light chain, except for changes that may be well known, such as removal of the C-terminal amide acid from the antibody heavy chain. Within the antibody population, monoclonal antibodies can have heterogeneous glycosylation. Individual antibodies can be monospecific or multispecific, or monovalent, bivalent, or multivalent. Bispecific anti-systems are included in the term monoclonal antibodies.

"Epitope" means an antigenic moiety that specifically binds to an antibody. Epitopes often consist of chemically active (such as polar, non-polar, or hydrophobic) surface grouping of molecular moieties such as amino acids or polysaccharide side chains, and may have specific three dimensional structural properties, as well as specific charges. characteristic. An epitope can be composed of contiguous and/or noncontiguous amino acids that form a conformational spatial unit. Against non Adjacent epitopes, amino acids from distinct portions of the linear sequence of antigens are in close proximity in a 3-dimensional space by folding of the protein molecules.

"Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications (eg, substitutions, insertions, or deletions).

"In combination with" means that two or more therapeutic agents are administered together in a mixture to a subject, while being administered in a single dose or sequentially in a single dose in any order. Generally, each agent will be administered in a single dose and/or in accordance with a schedule determined for the agent.

"treat or treatment" means a therapeutic treatment in which the purpose is to slow (reduce) undesired physiological changes or diseases, such as the development or spread of tumors or tumor cells, or to provide benefits or benefits during treatment. The clinical outcome of the desire. Beneficial or desirable clinical outcomes include a reduction in symptoms, a reduction in the extent of the disease, stabilization of the disease state (ie, no deterioration), delay or slowing of the progression of the disease, no metastasis, improvement or mitigation of the disease state, and Mitigation (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to the expected survival of a subject who has not received treatment. Those in need of treatment include those who have had unwanted physiological changes or diseases, as well as those who are susceptible to the physiological changes or diseases.

"therapeutically effective amount" means an effective amount of the dose and time required to achieve the desired therapeutic result. The therapeutically effective amount can vary depending on factors such as the disease state of the subject, age, sex, and weight, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the subject. Exemplary indicators of an effective therapeutic or combination of therapeutic agents include, for example, an improvement in patient well-being, a reduction in tumor burden, a slowing or cessation of tumor growth, and/or other locations where cancer cells have not metastasized to the body.

"Inhibit growth" (for example, when referring to a tumor cell) means when the growth of the same tumor cell or tumor tissue is reduced or delayed as compared to the absence of a combination of a therapeutic agent or a therapeutic drug, When contacted with a therapeutic or therapeutic agent or combination of drugs, there is a measurable decrease or delay in tumor cell growth or tumor tissue in vitro or in vivo. Inhibition of growth of tumor cells or tumor tissues in vitro or in vivo may At least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

"regulatory T cell", or "Tregs", or "Treg" refers to the regulation of the activity of one or more other T cells and/or other immune cells (often by inhibiting their activity) Lymphocytes. Treg can be a CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim T cell. It should be understood that Treg may not be completely limited to this phenotype and may exhibit Foxp3.

"Effector T cell", or "Teffs_", or "Teff" refers to a T lymphocyte that performs the function of an immune response, such as killing tumor cells and/or activating an anti-tumor immune response, which can cause tumors. The cells are cleared by themselves. Teff can be CD3 ⁺ with CD4 ⁺ or CD8 ⁺ . Teff can secrete, contain, or exhibit markers such as IFN-[gamma], granzyme B, and ICOS. It should be understood that Teffs may not be completely limited to these phenotypes.

"Function of Tregs" or "Treg function" refers to the inhibitory function of Treg, which is related to the regulation of host immune response and/or prevention of autoimmunity. The function of Treg can inhibit the anti-tumor response elicited by CD8 ⁺ T cells, natural killer (NK) cells, MØ cells, B cells, or dendritic cells (DC), or inhibit the proliferation of effector T cells.

"Inhibit function of Tregs" or "inhibit Treg function" refers to reducing the level of Treg function in vitro or in an animal or human subject, which can be used in the art. Known prior art decisions. The level of function of the Treg can be reduced, for example, by at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%. "Inhibiting the function of Treg" includes reducing the number of Tregs, for example by killing Treg via antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC).

"myeloid-derived suppressor cell", or "MDSCs", or "MDSC" refers to the hematopoietic lineage and exhibits macrophage/mononuclear marker CD11b and particle marker Gr-1/Ly-6G. Specialized cell population. The phenotype of MDSC can be, for example, CD11b ⁺ HLA-DR ^- CD14 ^- CD33 ⁺ CD15 ⁺ . MDSC exhibits low or undetectable expression of mature antigen presenting cell markers MHC class II and F480. MDSC is an immature cell of the myeloid lineage and can be further differentiated into several cell types, including macrophages, neutrophils, dendritic cells, mononuclear spheres, or granules. MDSC can be found naturally in normal adult bone marrow of humans and animals or in areas of normal hematopoiesis such as the spleen.

"Inhibit function of MDSCs" or "inhibit MDSC function" refers to a level of reducing the function of MDSC in vitro or in an animal or human subject, which can be used in the art. Known prior art decisions. The level of functionality of the MDSC can be reduced, for example, by at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%. "Suppressing the function of MDSC" includes reducing the number of MDSCs, for example by killing MDSC via antibody effector functions such as ADCC. MDSC can inhibit T cell responses by various mechanisms such as the production of active oxide peroxynitrite, an increase in arginase metabolism due to high levels of arginase, and an increase in nitrous oxide synthase. Such as hyperplasia, pure lineage amplification, or interleukin production. MDSC can react with IFN-γ and several interleukins such as IL-4 and IL-13. IFN-γ activates MDSC, which induces the activity of nitric oxide synthase 2 (NOS2). Alternatively, Th2 interleukins such as interleukin-4 (IL-4) and IL-13 activate MDSC, which can induce the activity of arginase-1 (ARG1). Metabolism of L-arginine by NOS2 or ARG1 can cause inhibition of T cell proliferation, and the activity of the two enzymes together can cause apoptosis of T cells by producing reactive nitrogen oxides.

"Treg related disease" refers to a disease or condition associated with T regulatory cells (Treg). Treg-associated diseases can be caused by Treg function (eg, inhibition of anti-tumor response or inhibition of effector T cell proliferation). The disease of the Treg medium can be cancer. "Treg-related diseases" and "Treg mediated diseases" are used interchangeably herein.

"Enhance response of effector T cells" or "enhanced T cell responses" means enhancing or stimulating effector T cells in vitro or in an animal or human subject to have persistence Or amplify biological functions, or renew or reactivate depleted or inactive T cells. Exemplary T cell response is proliferative, gamma-interferon secretion from CD8 ⁺ T cells, antigenic reactivity, or pure lineage amplification. The manner in which this enhancement is measured is known to those of ordinary skill in the art.

"MDSC related disease" refers to a disease or condition associated with bone marrow-derived suppressor cells (MDSC). MDSC-associated diseases can be caused by MDSC function (eg, inhibition of anti-tumor response or effector T cell proliferation). The disease of the MDSC vector can be cancer. "MDSC-related diseases" and "MDG mediated diseases" are used interchangeably herein.

"Regulatory B cell", or "Breg" or "Bregs" refers to a B lymphocyte that suppresses an immune response. Breg can be a CD19 ⁺ CD24 ⁺ CD38 ⁺ cell and can suppress the immune response by inhibiting the proliferation of T cells mediated by IL-10 secreted by Breg. It should be appreciated that other Breg subsets exist and are described, for example, in Ding et al. (2015) Human Immunology 76:615-621.

"Breg related disease" refers to a disease or condition related to the regulation of B cells. Breg-associated diseases can be caused by, for example, inhibition of an anti-tumor response by Breg vectors or effector T cell proliferation. The disease of the Breg vector can be cancer. "Breg-related diseases" and "Breg mediated diseases" are used interchangeably herein.

"Patient" includes any human or nonhuman animal. "Non-human animals" include all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. "Patient" and "subject" are used interchangeably herein.

The present invention provides a method of treating a patient having a solid tumor with an antibody that specifically binds to CD38, regardless of whether the tumor cell exhibits CD38. The invention further provides methods for treating a condition having a disease modifying T cell (Treg), bone marrow derived suppressor cell (MDSC), or a disease modulating B cell (Breg) vector. The invention further provides methods for modulating Treg, MDSC, or Breg activity to treat solid tumors that are CD38 positive and/or associated with high levels of these immunosuppressive cells.

The present invention is based, at least in part, on the discovery that the anti-CD38 antibody DARZALEX ^(TM) (daratumumab) has immunomodulatory activity in patients, which reduces the number of immunosuppressive Tregs, MDSCs, and Bregs, increases CD8 the number of ⁺ and CD8 ⁺ T cells to Treg ratio, the facilitate formation of CD8 ⁺ central memory cells, and increased T cell clonal.

Positive clinical evaluation DARZALEX ^TM (Dara mAb) anti-CD38 antibody therapy and other heme and plasma cell tumor efficacy of disorders, including multiple myeloma, by which lines the antibody by antibody effector functions (such as the ADCC, CDC, ACDP, and apoptosis) have the ability to eliminate CD38-positive cells, but their immunomodulatory activity in promoting adaptive immune responses has not been recognized. Other immunomodulatory antibodies (anti-PD1, anti-CTLA4) act by targeting components of the immune system that inhibit anti-tumor responses. For example, anti-PD1 antibodies have been shown to increase T cell proliferation, stimulate antigen-specific memory responses, and partially alleviate the inhibition of effector T cells in vitro Treg vectors (see, e.g., U.S. Patent No. 8,779,105). Two anti-PD-1 antibodies are currently approved for the treatment of melanoma, OPDIVO® (nivolumab) and KEYTRUDA® (pembrolizumab), and these antibodies are in various solid tumors Clinical development, such as non-small cell lung cancer, prostate cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, prostate cancer, fallopian tube cancer, ovarian cancer, pancreatic cancer, breast cancer and brain cancer, kidney cancer, bladder cancer, urethra Cancer, esophageal cancer, and colorectal cancer. The anti-CTLA-4 antibody YERVOY® (ipilimumab) has been approved for the treatment of melanoma. YERVOY® (ipimumab) and another anti-CTLA-4 antibody (tremelimumab) are also being developed for prostate cancer, non-small cell lung cancer, ovarian cancer, gastrointestinal cancer, gastric cancer, colorectal cancer. , kidney cancer, esophageal cancer, and genitourinary cancer.

Without wishing to be bound by any particular theory, based on the immunomodulatory effects observed with the herein DARZALEX ^TM (Dara mAb), DARZALEX ^TM (Dara mAb) and other anti-CD38 antibodies may be tied in the treatment of solid tumors Effective. Patients with CD38-negative solid tumors may also respond to CD38 antibody therapy responses due to the general activation of the immune response observed in patients treated with DARZALEXTM (dalacil ⁾ .

The present invention provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 for a period of time sufficient to treat the solid tumor.

The invention also provides a method of treating a patient having a disease modulating a T cell (Treg) vector, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 for a period of time sufficient to treat The time of the disease in the Treg medium.

The invention also provides a method of treating a condition of a disease having a bone marrow-derived suppressor cell (MDSC) vector, comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 for a sufficient period of time The time to treat the disease of the MDSC vector.

The invention also provides a method of treating a patient having a disease modulating a B cell (Breg) vector comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 for a period of time sufficient to treat The time of the disease in the Breg medium.

The invention also provides a method of inhibiting the activity of a regulatory T cell (Treg) comprising contacting the regulatory T cell with an antibody that specifically binds to CD38.

The invention also provides a method of inhibiting the activity of a bone marrow-derived suppressor cell (MDSC) comprising contacting the MDSC with an antibody that specifically binds to CD38.

The invention also provides a method of inhibiting the activity of a regulatory B cell (Breg) comprising contacting the Breg with an antibody that specifically binds to CD38.

The present invention also provides a method of treating a patient having a solid tumor comprising reducing the number of regulatory T cells (Tregs) in the patient by administering to the patient an antibody that specifically binds to CD38.

The present invention also provides a method of treating a patient having a solid tumor comprising reducing the number of bone marrow-derived suppressor cells (MDSCs) in the patient by administering to the patient an antibody that specifically binds to CD38.

The present invention also provides a method of treating a patient having a solid tumor comprising reducing the number of regulatory B cells (Breg) in the patient by administering to the patient an antibody that specifically binds to CD38.

The invention also provides a method of enhancing an immune response in a patient comprising administering to the patient in need thereof an antibody that specifically binds to CD38 for a time sufficient to enhance the immune response.

In some embodiments, the patient has a viral infection.

The invention also provides a method of treating a patient suffering from a viral infection comprising administering to the patient in need thereof an antibody that specifically binds to CD38 for a time sufficient to treat the viral infection.

In some embodiments, the immune response is an effector T cell (Teff) response.

In some embodiments, the Teff reaction is by CD4 ⁺ T cells or CD8 ⁺ T cells.

In some embodiments, the Teff reaction is mediated by CD4 ⁺ T cells.

In some embodiments, the Teff reaction is mediated by CD8 ⁺ T cells.

In some embodiments, the Teff response is an increase in the number of CD8 ⁺ T cells, an increase in CD8 ⁺ T cell proliferation, an increase in T cell pure line expansion, an increase in CD8 ⁺ memory cell formation, an increase in antigen-dependent antibody production. , or an increase in the production of interleukins, chemokines, or interleukins.

The proliferation of T cells can be measured, for example, by measuring the rate of DNA synthesis using tritiated thymidine, or measuring the production of interferon-γ (IFN-γ) in vitro, or measuring T cells in a cell population of a patient sample using known methods. The absolute number or percentage is evaluated.

Pure lineage amplification can be assessed by, for example, sequencing the TCR of a T cell pool using known methods.

The formation of memory cells can be measured by using, for example, FACS (na Ve) T cells (CD45RO ^- /CD62L ⁺ ) were evaluated for the ratio of memory T cells (CD45RO ⁺ /CD62L ^high ).

Production of interleukins, chemokines, or interleukins (such as interferon-gamma (IFN-γ), tumor necrosis factor-α (TNF-α), IL-1, IL-2, IL-3, IL- 4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-16, IL-18, and IL-23, MIP-1α, MIP-1β, RANTES, CCL4 production) Evaluation is performed using standard methods such as ELISA or ELLISPOT assays.

The production of antigen-specific antibodies can be assessed from samples derived from patients using standard methods such as ELISA or radioimmunoassay (RIA).

The meaning of "increase or increasing" various Teff reactions is easy to understand. In the test sample or in the object when compared to the control, for example For example, in patients treated with anti-CD38 antibodies, when compared to the same patients before treatment, or in groups of patients or patients who respond to anti-CD38 antibody therapy, compared to the same treatment In a group of patients or patients who have not responded, the increase may be increased by at least about 5%, at least about 10%, 25%, 50%, 51%, 52%, 53%, 54%, 55%, 56. %, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 200 %, 250%, 300%, 350%, 400%, or more. Generally, the increase is statistically significant.

Similarly, the meaning of "reduce or reducing" or "decreasing or decrease" the number of Treg, MDSC, and/or Breg is readily understood. In a test sample or in a subject when compared to a control, for example, in a patient treated with an anti-CD38 antibody, when compared to the same patient prior to treatment, or in response to anti-CD38 antibody therapy In a group of patients or patients, the reduction may be at least about 10%, 25%, 50%, 51%, 52% compared to a group of patients or patients who have not responded to the same treatment. , 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69 %, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120% , 130%, 140%, 150%, 200%, 250%, 300%, 350%, 400%, or more. In general, the reduction is statistically significant.

In some embodiments, an antibody that specifically binds to CD38 inhibits the function of an immunosuppressive cell.

In some embodiments, the immunosuppressive cell line modulates T cells (Tregs), bone marrow derived suppressor cells (MDSCs), or modulates B cells (Bregs).

In some embodiments, the Treg is a CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim T cell.

In some embodiments, the CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim cells exhibit Foxp3.

In some embodiments, the CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim T cells exhibit CD38.

The ability of the Treg function, such as its ability to inhibit Teff cells, can be assessed using known methods, such as assessing the ability of Treg to inhibit Teff proliferation in mixed lymphocyte response (MLR).

The Treg function can be inhibited by, for example, reducing the relative number of Tregs when compared to Teff (e.g., increasing the ratio of CD8 ⁺ /Treg cells) by directly killing a subset of Treg or Treg (such as CD38 ⁺ Treg).

In some embodiments, the Treg function is inhibited by killing Treg cells.

In some embodiments, Treg killing is by antibody-induced antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or antibody that specifically binds to CD38 The induced apoptosis is mediated.

In some embodiments, Treg killing is by ADCC medium.

In some embodiments, the CD38 ⁺ Treg is killed.

In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% , 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32 %, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 60% of the Treg were killed.

Since CD38 is only expressed in one part of Treg and MDSC, it is expected that treatment of patients with solid tumors will not result in systemic loss of Treg and MDSC and thus may provide an improved safety profile.

In some embodiments, the MDSC is a CD11b ⁺ HLA-DR ^- CD14 ^- CD33 ⁺ CD15 ⁺ cell.

In some embodiments, CD11b ⁺ HLA-DR ^- CD14 ^- CD33 ⁺ CD15 ⁺ MDSC represents CD38.

The MDSC function can be inhibited, for example, by reducing the number of MDSCs by directly killing the cells.

In some embodiments, the MDSC function is inhibited by killing CD38 ⁺ MDSC.

In some embodiments, MDSC killing is by antibody-induced antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or antibodies that specifically bind to CD38 The induced apoptosis is mediated.

In some embodiments, the MDSC kill is by the ADCC medium.

In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% , 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32 %, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 60% of the MDSC lines were killed.

In some embodiments, the Breg is a CD19 ⁺ CD24 ⁺ CD38 ⁺ cell.

The Breg function can be inhibited, for example, by reducing the number of Bregs by directly killing Breg.

In some embodiments, the Breg function is inhibited by killing CD38 ⁺ Breg.

In some embodiments, Breg killing is by antibody-induced antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or antibodies that specifically bind to CD38 The induced apoptosis is mediated.

In some embodiments, Breg killing is by ADCC medium.

In some embodiments, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% , 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32 %, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, or 60% of the Breg were killed.

Treg plays a key role in maintaining peripheral tolerance. The naturally occurring CD4 ⁺ CD25 ^hi Treg is produced in the thymus and exhibits Foxp3, which is used to establish and maintain transcription factors required for Treg lineage identification and inhibition. Treg can accumulate in disease sites (eg, in tumors) where it inhibits the effector function of tumor antigen-specific T cells, resulting in an insufficient anti-tumor response. The increased density of tumor infiltrating Foxp3 ⁺ Treg is associated with poor prognosis in various solid tumors, including pancreatic cancer, ovarian cancer, and hepatocellular carcinoma. The loss of Treg leads to enhanced anti-tumor immunity and tumor rejection in the murine model, but can also lead to the development of autoimmune diseases.

Bone marrow-derived suppressor cells (MDSCs) are heterogeneous populations of early bone marrow precursor cells, immature granule cells, macrophages, and dendritic cells at different stages of differentiation. They are abundantly accumulated in cancer patients, and they have potent immunosuppressive functions, inhibiting the cytotoxic activity of both natural killer cells (NK) and natural killer T cells (NKT), and by CD8 ⁺ T cell media. Adaptive immune response. Although the mechanism of NK cell inhibition is currently not well understood, the T cell inhibition of MDSC vectors is responsible for multiple pathways, including the production of arginase 1/ARG1 and upregulation of nitric oxide synthase 2 (NOS2). . ARG1 and NOS2 metabolize L-arginine and block the translation of T cell CD3 ζ chain together, or separately, inhibit T cell proliferation, and promote T cell apoptosis. In addition, MDSC secretes immunosuppressive interleukins and induces regulation of T cell development.

MDSC is induced by proinflammatory cytokines and is found to increase in infectious and inflammatory pathological conditions. They accumulate in the blood, bone marrow, and secondary lymphoid organs of tumor-bearing mice, and their presence in the tumor microenvironment indicates a pathogenic role in promoting tumor-associated immunosuppression.

MDSC has been described in patients with colon cancer, melanoma, hepatocellular carcinoma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, renal cell carcinoma, pancreatic cancer, and breast cancer (Mandruzzato et al., ( 2009) J Immunol 182:6562-6568; Liu et al, (2009) J Cancer Res Clin Oncol 136: 35-45; Ko et al, (2009) Clin Cancer Res 15: 2148-2157; Morse et al, (2009) Expert Opin Biol Ther 9: 331-339; Diaz-Montero et al, (2009) Cancer Immunol Immunother 58: 49-59; Corzo et al, (2009) J Immunol 182: 5693-5701). In cancer patients, Diaz et al. (Diaz-Montero et al. (2009) Cancer Immunol Immunother 58: 49-59) suggest that the accumulation of MDSC is associated with more severe disease and poor prognosis.

Tumor infiltrating Breg has been identified in solid tumors, and Breg can promote tumor growth and metastasis by various mechanisms, such as inhibiting the antitumor activity of CD8 ⁺ T cells and NK cells, as for example, Ding et al., (2015) Human Immunology 76 :615-62.

"Antibody-dependent cytotoxicity", "antibody-dependent cellular cytotoxicity", or "ADCC" is a mechanism for inducing cell death depending on the antibody-coated target cells and effector cells having lytic activity (such as nature). Interaction between Fcγ receptors (FcγR) expressed on effector cells between killer cells, monocytes, macrophages, and neutrophils. For example, NK cells express FcγRIIIa, while monocytes exhibit FcγRI, FcγRII, and FcvRIIIa. The death of antibody-coated target cells (such as CD38-expressing cells) occurs due to effector cell activity through the secretion of membrane pore-forming proteins and proteases. To assess the ADCC activity of an antibody that specifically binds to CD38, the antibody can be added to a combination of CD38 expressing cells and immune effector cells that can be activated by the antigen-antibody complex to cause cell lysis of the target cell. Cell lysis is typically detected by a label (eg, a radioactive matrix, a fluorescent dye, or a native intracellular protein) released from the lysed cells. Exemplary effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Exemplary target cells include Tregs or MDSCs that express CD38. In an exemplary assay, target cells were labeled with 20 μCi of ⁵¹ Cr for 2 hours and washed thoroughly. The cell concentration of these target cells can be adjusted to 1 × 10 ⁶ cells/ml, and various concentrations of anti-CD38 antibodies are added. Effect: The target cell ratio was 40:1 added to the target cells to start the assay. After incubation at 37 ° C for 3 hours, the assay was stopped by centrifugation and ⁵¹ Cr released from the lysed cells was measured in a scintillation counter. The percent cytotoxicity can be calculated as the maximum percentage of lysis that can be induced by the addition of 3% perchloric acid to the target cells.

"Antibody-dependent cellular phagocytosis"("ADCP") refers to a mechanism by which internal cells of phagocytic cells, such as macrophages or dendritic cells, are internalized to destroy antibody-coated target cells. ADCP can be assessed by using Treg or MDSCs that express CD38 as target cells (which are engineered to express GFP or other marker molecules). Effect: The target cell ratio can be, for example, 4:1. The effector cells can be cultured together with the target cells for 4 hours in the presence or absence of anti-CD38 antibodies. After the culture, the cells can be separated using an accutase. Macrophages can be identified by conjugated fluorescently labeled anti-CD11b and anti-CD14 antibodies, and the percentage of phagocytosis can be determined using standard methods for GFP fluorescence in such CD11 ⁺ CD14 ⁺ macrophages.

"Complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of an antibody that binds to a target binds to and activates the complement component C1q, which in turn reactivates the complement level. The reaction causes the target cell to die. Activation of complement can also result in the deposition of complement components on the surface of the target cell, which promotes ADCC by binding to complement receptors (e.g., CR3) on white blood cells.

The ability of a single antibody to induce ADCC can be enhanced by engineering its oligosaccharide component. The human IgGl or IgG3 line is N-glycosylated at Asn297 and most of the glycans are in the well-known biantennary G0, G0F, G1, G1F, G2, or G2F form. Antibodies produced from unengineered CHO cells typically have a glycan fucose content of at least about 85%. The dual-antennary complex oligosaccharide attached to the Fc region removes core trehalose to enhance the ADCC of the antibody by improving FcγRIIIa binding without altering antigen binding or CDC activity. Such mAbs can be achieved using different methods that have been reported to result in successful performance of relatively high amounts of defucosylated antibodies (Fc oligosaccharides with a biantennary complex), such as control of osmotic pressure (Konno et al. (2012) Cytotechnology 64: 249-65), using the variant CHO strain Lec13 as a host cell line (Shields et al. (2002) J Biol Chem 277:26733-26740), and using variant CHO strain EB66 as a host cell line (Olivier et al., (2010) MAbs 2(4), Epub ahead of print; PMID: 20562582), using the rat fusion tumor cell line YB2/0 as a host cell line (Shinkawa et al., (2003) J Biol Chem 278) :3466-3473), introducing a short interfering RNA specific for the α1,6-fucosyltrasferase ( FUT8 ) gene (Mori et al. (2004) Biotechnol Bioeng 88:901-908), co-expression or β-1,4- N - acetyl glucosaminyl transferase III (β-1,4-N- acetylglucosaminyltransferase III) and the group's high α- mannosidase II (Golgi α-mannosidase II) or Keefe Kifunensine (a potent alpha-mannosidase I inhibitor) (Ferrara et al., (2006) J Biol Chem 281:503 2-5036; Ferrara et al, (2006) Biotechnol Bioeng 93: 851-861; Xhou et al, (2008) Biotechnol Bioeng 99: 652-65). The ADCC elicited by the anti-CD38 antibodies used in the methods of the invention, as well as in some of the examples of each of the following numbering examples, can also be enhanced by certain substitutions in the antibody Fc. Exemplary substitutions are, for example, substitutions at positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 of the amino acid (residue numbering according to the EU index), such as U.S. Patent No. 6,737,056 Said in the middle.

In some embodiments, an antibody that specifically binds to CD38 comprises a substitution in an antibody Fc.

In some embodiments, the antibody that specifically binds to CD38 comprises a substitution (residue) at amino acid position 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 in antibody Fc. The number is based on the EU index).

In some embodiments, the antibody that specifically binds to CD38 has a biantennary glycan structure with a trehalose content of between about 0% and about 15%, such as 15%, 14%, 13%, 12%, 11 % 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

In some embodiments, the antibody that specifically binds to CD38 has a biantennary glycan structure with a trehalose content of about 50%, 40%, 45%, 40%, 35%, 30%, 25%, 20%, 15 %, 14%, 13%, 12%, 11% 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%.

Substitutions in Fc and reduced trehalose content enhance the ADCC activity of this antibody that specifically binds to CD38.

"Fucose content" means the amount of trehalose monosaccharide in the sugar chain at Asn297. The relative amount of trehalose is the percentage of the structure containing trehalose relative to all saccharide structures. These can be characterized and quantified by a variety of methods, for example: 1) using N-glycosidase F treated samples (eg, complex, heterozygous, and oligo-and high-mannose structures) MALD1-TOF, as described in International Patent Publication No. WO 2008/077546; 2) Enzymatic release of Asn297 glycan, followed by derivatization and detection by fluorescence (HPLC) and/or HPLC-MS (UPLC-PLC) MS) to detect/quantify; 3) complete protein analysis of native or reduced mAbs, cleavage of Asn297 glycans with Endo S or others between the first and second GlcNAc monosaccharides leaving a link to the first GlcNAc The trehalose is treated with or without treatment; 4) the mAb is digested into constituent peptides by enzymatic digestion (eg trypsin or endopeptidase Lys-C) followed by HPLC-MS (UPLC) -MS) isolation, detection and quantification; or 5) specific enzymatic deglycosylation at Asn 297 with PNGase F to separate the mAb oligosaccharide from the mAb protein. The released oligosaccharides can be labeled with fluorophores and separated and identified by a variety of complementary techniques that allow for the comparison of experimental mass and theoretical mass by matrix-assisted laser desorption free (MALDI) mass spectrometry. Characterizing the glycan structure, determining the degree of sialylation by ion exchange HPLC (GlycoSep C), separating and quantifying the oligosaccharide form by normal phase HPLC (GlycoSep N) according to hydrophilicity criteria, and by efficient Capillary electrophoresis-laser induced fluorescence (HPCE-LIF) was used to separate and quantify oligosaccharides.

As used herein, "low fucose" or "low fucose content" refers to an antibody having a trehalose content of from about 0% to about 15%.

As used herein, "normal fucose" or "normal fucose content" means that the antibody has a trehalose content of more than about 50%, usually more than about 60%, 70%, 80%, or More than 85%.

In some embodiments, an antibody that specifically binds to CD38 can induce killing of Treg, MDSC, and/or Breg by apoptosis. Methods for assessing apoptosis are well known and include, for example, Annexin IV staining using standard methods. color. The anti-CD38 antibody used in the method of the present invention may be at about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% Apoptosis was induced in 80%, 85%, 90%, 95%, or 100% of cells.

In some embodiments, Teff or immunosuppressive cells are present in the bone marrow or in peripheral blood.

In some embodiments, Teff or immunosuppressive cells are present in the bone marrow.

In some embodiments, Teff or immunosuppressive cells are present in the peripheral blood.

In some embodiments, an antibody that specifically binds to CD38 increases the ratio of CD8 ⁺ T cells to Treg.

In some embodiments, an antibody that specifically binds to CD38 increases the ratio of CD8 ⁺ central memory cells to CD8 ⁺ naive cells. CD8 ⁺ central memory cells can be identified as CD45RO ⁺ /CD62L ^{+ high} cells. CD8 ⁺ naive cells can be identified as CD45RO-/CD62L ⁺ cells.

In some embodiments, an anti-system non-promoting antibody that specifically binds to CD38.

A non-agonistic anti-system that specifically binds to CD38 means that after binding to CD38, it does not induce significant proliferation of peripheral peripheral blood mononuclear cell samples when compared to proliferation induced by isotype control antibodies or media alone. antibody.

In some embodiments, a non-potentiating antibody that specifically binds to CD38 induces proliferation of peripheral blood mononuclear cells (PBMC) in a statistically insignificant manner. PBMC proliferation can be assessed by culturing the cells from healthy donor-isolated PBMCs in 200 μl RPMI at 1 x 10 ⁵ cells/well in flat-bottom 96-well plates in the presence or absence of test antibodies. After 4 days of culture at 37 ° C, 30 μl of ³ H-thymidine (16.7 μCi/ml) may be added, and incubation may continue for overnight. ³ H-thymidine incorporation can be assessed using a Packard Cobra Plus Horse Counter (Packard Instruments, Meriden, DT, USA) according to the manufacturer's instructions. Data can be calculated as the mean cpm (± SEM) of PBMC obtained from several donors. Statistical significance or insignificance between samples incubated in the presence or absence of a test antibody is calculated using standard methods.

An exemplary anti-CD38 anti-system DARZALEX ^(TM) (dalabiza ⁾ useful in the methods of the invention. DARZALEX ^TM (Dara mAb) contained are shown in SEQ ID NO: heavy chain variable region (VH) 4 and 5 and a light chain variable region (VL) amino acid sequence, respectively, SEQ ID NO:. 6, The heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 of 7, and 8, and the light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 of SEQ ID NOS: 9, 10, and 11, respectively, and The IgG1/κ subtype is described in U.S. Patent No. 7,829,693. The heavy chain amino acid sequence of DARZALEX ^(TM) (dalacil ⁾ is shown in SEQ ID NO: 12 and the light chain amino acid sequence is shown in SEQ ID NO: 13.

In some embodiments, an antibody that specifically binds to CD38 competes for binding to CD38 with an antibody comprising the heavy chain variable region (VH) of SEQ ID NO: 4 and the light chain variable region (VL) of SEQ ID NO: 5.

In some embodiments, an antibody that specifically binds to CD38 binds at least to the region of SKRNIQFSCKNIYR (SEQ ID NO: 2) and EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).

SEQ ID NO: 1

SEQ ID NO: 2 SKRNIQFSCKNIYR

SEQ ID NO: 3 EKVQTLEAWVIHGG

SEQ ID NO: 4

SEQ ID NO: 5

SEQ ID NO:6 SFAMS

SEQ ID NO: 7 AISGSGGGTYYADSVKG

SEQ ID NO:8 DKILWFGEPVFDY

SEQ ID NO: 9 RASQSVSSYLA

SEQ ID NO: 10 DASNRAT

SEQ ID NO: 11 QQRSNWPPTF

SEQ ID NO: 12

SEQ ID NO: 13

A well-known in vitro method can be used to assess competitive binding of an antibody to a reference antibody, such as DARZALEX ^(TM) (Dalimab ^{) having} VL of SEQ ID NO: 4 and VL of SEQ ID NO: 5, for CD38. In an exemplary method, recombinant C38 cells expressing CD38 can be incubated with an unlabeled reference antibody for 15 minutes at 4 °C and then incubated with an excess of fluorescently labeled test antibody for 45 minutes at 4 °C. After washing in PBS/BSA, fluorescence can be measured by standard methods using flow cytometry. In another exemplary method, the extracellular portion of human CD38 can be coated onto the surface of an ELISA plate. An excess of unlabeled reference antibody can be added for about 15 minutes, and then the biotinylated test antibody can be added. After washing in PBS/Tween, conjugated horseradish peroxidase (HRP) streptavidin can be used to detect the binding of the test biotinylated antibody and detect the signal using standard methods. . In such competitive assays, it will be apparent that the reference antibody can be labeled and the test antibody can be unlabeled. When the reference antibody inhibits the binding of the test antibody to CD38, or the test antibody inhibits binding of the reference antibody to CD38 by at least 80%, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 At %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, the test antibody competes with the reference antibody. The epitope of the test antibody can be further defined by known methods such as peptide mapping or hydrogen/hydrazine protection assays, or by crystal structure determination.

Antibodies that bind to the SKRNIQFSCKNIYR (SEQ ID NO: 2) region of human CD38 (SEQ ID NO: 1) and the EKVQTLEAWVIHGG (SEQ ID NO: 3) region can be produced, for example, by using standard methods and methods described herein. Mice were immunized with a peptide having the amino acid sequences shown in SEQ ID NOS: 2 and 3, and using, for example, an ELISA or mutation-inducing study to characterize the resulting antibody for binding to the peptide.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof an anti-CD38 antibody that binds to the human CD38 (SEQ ID NO: 1) SKRNIQFSCKNIYR (SEQ ID NO: 2) region and EKVQTLEAWVIHGG (SEQ ID NO: 3) region. The epitope of the antibody used in the method of the present invention includes some or all of the residues having the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the antibody epitope comprises at least one amino acid in the region of SKRNIQFSCKNIYR (SEQ ID NO: 2) of human CD38 (SEQ ID NO: 1) and at least one amine group in the region of EKVQTLEAWVIHGG (SEQ ID NO: 3) acid. In some embodiments, the antibody epitope comprises at least two amino acids in the region of SKRNIQFSCKNIYR (SEQ ID NO: 2) of human CD38 (SEQ ID NO: 1) and at least two of the regions of EKVQTLEAWVIHGG (SEQ ID NO: 3) Amino acid. In some embodiments, the antibody epitope comprises at least three of at least three amino acids in the region of SKRNIQFSCKNIYR (SEQ ID NO: 2) of human CD38 (SEQ ID NO: 1) and EKVQTLEAWVIHGG (SEQ ID NO: 3) Amino acid.

In some embodiments, the antibody that specifically binds to CD38 comprises the HCDR1, HCDR2, and HCDR3 amino acid sequences of SEQ ID NOs: 6, 7, and 8, respectively.

In some embodiments, the antibody that specifically binds to CD38 comprises the LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NOS: 9, 10, and 11, respectively.

In some embodiments, the antibody that specifically binds to CD38 comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amino acid sequences of SEQ ID NOS: 6, 7, 8, 9, 10, and 11, respectively.

In some embodiments, the antibody that specifically binds to CD38 comprises VH and VL, the VH line having 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4, and The VL line has 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:5.

In some embodiments, the antibody that specifically binds to CD38 comprises VH of SEQ ID NO:4 and VL of SEQ ID NO:5.

In some embodiments, the antibody that specifically binds to CD38 comprises the heavy chain of SEQ ID NO: 12 and the light chain of SEQ ID NO: 13.

Other exemplary anti-CD38 anti-systems that can be used in any of the embodiments of the invention: mAb003, which comprises the VH and VL sequences of SEQ ID NOS: 14 and 15, respectively, and are described in U.S. Patent No. 7,829,693. This VH of mAb003 and the VL can be expressed as IgG1/κ.

SEQ ID NO: 14

SEQ ID NO: 15 mAb024, which comprises the VH and VL sequences of SEQ ID NOS: 16 and 17, respectively, and is described in U.S. Patent No. 7,829,693. This VH of mAb024 and the VL can be expressed as IgG1/κ.

SEQ ID NO: 16

SEQ ID NO: 17

MOR-202 (MOR-03087), which contains the VH and VL sequences of SEQ ID NOS: 18 and 19, respectively, and is described in U.S. Patent No. 8,088,896. This VH of MOR-202 and the VL can be expressed as IgG1/κ.

SEQ ID NO:18

SEQ ID NO: 19 Isaximab (Isatuximab); which comprises the VH and VL sequences of SEQ ID NOS: 20 and 21, respectively, and is described in U.S. Patent No. 8,153,765. VH and VL of exetoximab can be expressed as IgG1/κ.

SEQ ID NO 20:

SEQ ID NO: 21:

Other exemplary anti-CD38 antibodies that can be used in the methods of the present invention include those described in International Patent Publication No. WO05/103083, International Patent Publication No. WO06/125640, International Patent Publication No. WO07/042309, International Patent Publication No. WO 08/047242, or International Patent Publication No. WO 14/178820.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 for a time sufficient to treat the solid tumor, The antibody comprises VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 for a time sufficient to treat the solid tumor, The antibody comprises VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 for a time sufficient to treat the solid tumor, The antibody comprises VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 for a time sufficient to treat the solid tumor, The antibody comprises VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 for a time sufficient to treat the solid tumor, The antibody comprises VH of SEQ ID NO: 20 and VL of SEQ ID NO: 21.

In some embodiments, the solid tumor is melanoma.

In some embodiments, the solid tumor is a lung cancer.

In some embodiments, the solid tumor is squamous non-small cell lung cancer (NSCLC).

In some embodiments, the solid tumor is a non-squamous NSCLC.

In some embodiments, the solid tumor is a lung adenocarcinoma.

In some embodiments, the solid tumor is renal cell carcinoma (RCC) (eg, renal clear cell carcinoma or renal papillary cell carcinoma), or a metastatic disease thereof.

In some embodiments, the solid tumor is a mesothelioma.

In some embodiments, the solid tumor is nasopharyngeal carcinoma (NPC).

In some embodiments, the solid tumor is colorectal cancer.

In some embodiments, the solid tumor is prostate cancer or castration resistant prostate cancer.

In some embodiments, the solid tumor is gastric cancer.

In some embodiments, the solid tumor is ovarian cancer.

In some embodiments, the solid tumor is gastric cancer.

In some embodiments, the solid tumor is liver cancer.

In some embodiments, the solid tumor is pancreatic cancer.

In some embodiments, the solid tumor is thyroid cancer.

In some embodiments, the solid tumor is a head and neck squamous cell carcinoma.

In some embodiments, the solid tumor is an esophageal or gastrointestinal cancer.

In some embodiments, the solid tumor is a breast cancer.

In some embodiments, the solid tumor is a fallopian tube cancer.

In some embodiments, the solid tumor is a brain cancer.

In some embodiments, the solid tumor is urethral cancer.

In some embodiments, the solid tumor is a genitourinary cancer.

In some embodiments, the solid tumor is endometriosis.

In some embodiments, the solid tumor is cervical cancer.

In some embodiments, the solid tumor is a metastatic disease of cancer.

In some embodiments, solid tumors lack detectable CD38 performance.

When compared to controls, such as those detected by anti-CD38 antibodies using well-known methods for performance detected by isotype control antibodies, CD38 expression is statistically present in solid tumor tissue or on cells isolated from solid tumors. Insignificant, solid tumors lack detectable CD38 performance.

The anti-CD38 antibody used in the methods of the invention may also be re-selected from, for example, a phage display library in which the phage system is engineered to express human immunoglobulin or a portion thereof, such as a Fab, single chain antibody (scFv), or unpaired Or paired antibody variable regions (Knappik et al, (2000) J Mol Biol 296: 57-86; Krebs et al, (2001) J Immunol Meth 254: 67-84; Vaughan et al, (1996) Nature Biotechnology 14: 309-314; Sheets et al., (1998) PITAS (USA) 95: 6157-6162; Hoogenboom and Winter, (1991) J Mol Biol 227: 381; Marks et al., (1991) J Mol Biol 222: 581). The CD38 binding variable domain can be isolated from, for example, a phage display library (expressing antibody heavy and light chain variable regions) as a fusion protein having a phage pIX coat protein, as described in Shi et al., (2010) J Mol Biol 397: 385-96 and International Patent Publication No. WO 09/085462. The antibody library can be screened for binding to the human CD38 extracellular domain, and the positive strain obtained is further characterized, the lysate is isolated from the Fab, and subsequently cloned into a full length antibody. Such phage display methods for isolated human antibodies have been established in the art. See, for example, U.S. Patent No. 5,223,409, U.S. Patent No. 5,403,484, U.S. Patent No. 5,571,698, U.S. Patent No. 5,427,908, U.S. Patent No. 5,580,717, U.S. Patent No. 5,969,108, U.S. Patent No. 6,172,197, U.S. Patent No. 5,885,793 U.S. Patent No. 6,521,404, U.S. Patent No. 6,544,731, U.S. Patent No. 6,555,313, U.S. Patent No. 6,582,915, and U.S. Patent No. 6,593,081.

In some embodiments, the anti-CD38 anti-system is isotype of IgGl, IgG2, IgG3, or IgG4.

The Fc portion of the antibody can mediate an effector function of the antibody, such as antibody-dependent cellular mediator cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement dependent cytotoxicity (CDC). This function can be mediated by binding of an Fc effector domain to an Fc receptor on immune cells having phagocytic or lytic activity, or by binding of an Fc effector to a complement system component. In general, effects mediated by cells or complement components that bind to Fc result in inhibition or depletion of target cells, such as CD38 expressing cells. Human IgG isotypes IgG1, IgG2, IgG3, and IgG4 showed differential ability in effector function. ADCC can be mediated by IgGl and IgG3, ADCP can be mediated by IgGl, IgG2, IgG3, and IgG4, and CDC can be mediated by IgGl and IgG3.

An antibody substantially identical to the antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 can be used in the methods of the invention. As used herein, the term "substantially identical" means that the two antibody VH or VL amino acid sequences being compared are identical or have "insubstantial differences". No substantial difference in antibody heavy or light chain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 does not adversely affect antibody performance Affected amino acid substitution. The percent identity can be determined, for example, by pairwise alignment using the preset settings of the AlignX module of Vector NTI v.9.0.0 (Invitrogen, Carlsbad, CA). The protein sequences of the invention can be used as a query sequence to perform searches against published or patent databases to, for example, identify related sequences. To perform the exemplary system XBLAST program BLASTP program or such Retrieval (http _ // www_ncbi_nlm / nih_gov) , or use the default setting of ^{GenomeQuest TM (GenomeQuest, Westborough, MA} ) kit. Exemplary substitutions that can be made to an antibody that specifically binds to CD38 are, for example, conservative substitutions with amino acids having similar charge, hydrophobicity, or stereochemical properties. Conservative substitutions can also be made to improve antibody properties (eg, stability or affinity), or to improve the effector function of the antibody. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions can be made in the heavy or light chain of the anti-CD38 antibody. . In addition, any natural residue in the VH or VL can also be substituted with alanine, as previously described for alanine scanning mutagenesis (MacLennan et al, Acta Physiol Scand Suppl 643: 55- 67, 1998; Sasaki et al., Adv Biophys 35: 1-24, 1998). The desired amino acid substitution can be determined by one of ordinary skill in the art when such substitutions are desired. Amino acid substitution can be carried out, for example, by PCR mutation induction (U.S. Patent No. 4,683,195). Variant libraries can be generated using well-known methods, such as using random (NNK) or non-random codons (eg, DVK codons), which encode 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp), then screen the library of variants to find variants of the desired nature. The resulting variants can be tested in vitro for their ability to bind to CD38, such as to induce ADCC, ADCP, or apoptosis, or to modulate CD38 enzymatic activity, using methods described herein.

In some embodiments, the antibody that specifically binds CD38 may be a range of affinities (K _D) binds human CD38. In accordance with one embodiment of the invention, and in some embodiments each of the following numbered embodiments, the CD38 antibody that specifically binds to CD38 with high affinity, e.g. K _D equal to or less than about 10 ^-7 M, such as But not limited to, 1 to 9.9 (or any range or value such as 1, 2, 3, 4, 5, 6, 7, 8, or 9) x 10 ^-8 M, 10 ^-9 M, 10 ^-10 M , 10 ^-11 M, 10 ^-12 M, 10 ^-13 M, 10 ^-14 M, 10 ^-15 M, or any range or value thereof, as determined by surface plasma resonance or Kinexa method, as in the technical field Those who have the usual knowledge practice. An exemplary affinity is equal to or less than 1 x 10 ^-8 M. Another exemplary affinity is equal to or less than 1 x 10 ^-9 M.

In some embodiments, an anti-system bispecific antibody that specifically binds to CD38. VL and/or VH regions of existing anti-CD38 antibodies or re-identified as described herein The VL and VH regions can be engineered into bispecific full length antibodies. Such bispecific antibodies can be made by modulating the CH3 interaction between the monospecific antibody heavy chains to form bispecific antibodies using techniques such as those described in U.S. Patent No. 7,695,936. International Patent Publication No. WO 04/111233; U.S. Patent Publication No. US2010/0015133; U.S. Patent Publication No. US2007/0287170; International Patent Publication No. WO2008/119353; U.S. Patent Publication No. US2009/0182127; U.S. Patent Publication No. US2010/0286374; Patent Publication No. US 2011/0123532; International Patent Publication No. WO 2011/131746; International Patent Publication No. WO 2011/143545; or US Patent Publication No. US 2012/0149876. Additional bispecific structures that can incorporate the VL and/or VH regions of the antibodies of the invention, such as dual variable domain immunoglobulins (International Patent Publication No. WO 2009/134776), or include various dimerization domains to link differently The structure of the two antibody arms, such as the leucine zipper or the collagen dimerization domain (International Patent Publication No. WO 2012/022811, U.S. Patent No. 5,932,448; U.S. Patent No. 6,833,441).

For example, bispecific antibodies can be produced in vitro in a cell-free environment by introducing asymmetric mutations in the CH3 region of two monospecific homodimeric antibodies, and in reducing conditions (to allow disulfide bonds) Isomerization) The bispecific heterodimeric antibody is formed as two parental monospecific homodimeric antibodies according to the method described in International Patent Publication No. WO 2011/131746. In such methods, the first monospecific bivalent antibody (eg, an anti-CD38 antibody) and the second monospecific bivalent antibody system are engineered to have some substitution in the CH3 domain that promotes heterodimer stability. The anti-systems are incubated together under reducing conditions sufficient for the disulfide isomerization of the cysteine in the hinge region; such that the bispecific antibody is produced by Fab arm exchange. The culture conditions are optimally restored to non-reducing. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, ginseng (2- Carboxyethyl)phosphine (TCEP), L-cysteine and β-mercaptoethanol, the reducing agent is preferably selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and ginseng (2) - Carboxyethyl) phosphine. For example, disulfethitol at a temperature of at least 20 ° C and at least 25 mM of 2-MEA or at least 0.5 mM of dithiothreitol may be used It is present and incubated for at least 90 minutes at a pH of 5 to 8 (for example at a pH of 7.0 or at a pH of 7.4).

An exemplary CH3 mutant line K409R and/or F405L that can be used in the first heavy chain of the bispecific antibody and in the second heavy chain.

The method of the invention can be used to treat animal patients belonging to any classification. Examples of such animals include mammals such as humans, rats, dogs, cats, and farm animals.

Investment/pharmaceutical composition

Antibodies that specifically bind to CD38 can be provided in the form of a suitable pharmaceutical composition comprising an antibody that specifically binds to CD38 and a pharmaceutically acceptable carrier. The carrier can be a diluent, adjuvant, excipient, or vehicle with which the antibody that specifically binds to CD38 is administered. Such vehicles can be liquids such as water and oils, including those from petroleum, animal, vegetable, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They can be sterilized by conventional, well known sterilization techniques such as filtration. The compositions may contain pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, and color formers, as needed to approximate physiological conditions. The concentration of an antibody that specifically binds to CD38 in such pharmaceutical formulations can vary widely, from less than about 0.5% by weight, often from at least about 1% by weight up to 15 or 20%, 25% by weight. 30%, 35%, 40%, 45%, or 50%, and will be selected based primarily on the desired dosage, fluid volume, viscosity, etc., depending on the particular mode of administration selected. Suitable media agent formulation (comprising other human proteins, such as human serum albumin), for example, are described in, for example, based Remington:. The Science and Practice of Pharmacy, 21 st Edition, Troy, DBed, Lipincott Williams And Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, see especially pp. 958-989.

The mode of administration of the antibody that specifically binds to CD38 in the method of the present invention may be in any suitable route, such as parenteral administration, such as intradermal, intramuscular, and peritoneal. Intraperitoneal, intravenous or subcutaneous, pulmonary, transmucosal (oral, nasal, intravaginal, rectal), or other means known to those skilled in the art and well known in the art. Antibodies that specifically bind to CD38 can be administered intratumorally to lymph node drainage sites using known methods for local delivery into tumors.

An antibody that specifically binds to CD38 can be administered to a patient by any suitable route, such as parenterally (by intravenous (iv) infusion or bolus injection), intramuscularly Or subcutaneous or intraperitoneal. The iv infusion can be administered, for example, within 15, 30, 60, 90, 120, 180, 240 minutes, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours. .

The dosage administered to the patient is sufficient to alleviate or at least partially arrest the condition to be treated ("therapeutically effective amount") and can sometimes be from 0.005 mg to about 100 mg/kg, such as from about 0.05 mg to about 30 mg/kg or from about 5 mg to about 25 mg/kg, or about 4 mg/kg, about 8 mg/kg, about 16 mg/kg, or about 24 mg/kg, or for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg/ Kg, but may be even higher, such as about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg/kg .

Fixed unit dose may also be administered, e.g. 50,100,200,500, or lOOOmg, or the dose may be based on the patient's surface area, e.g. 500,400,300,250,200, or 100mg / m ^2. A dose between 1 and 8 times (eg 1, 2, 3, 4, 5, 6, 7, or 8 times) can often be administered, but 9, 10, 11, 12, 13, 14 can also be administered. 15, 16, 17, 18, 19, 20, or more doses.

Administration of antibodies that specifically bind to CD38 in the methods of the invention can be administered on one, two, three, four, five, six, one, two, three, one, five, and six weeks Repeatedly, seven weeks, two months, three months, four months, five months, six months, or more. It is also possible to repeat the treatment process, such as chronic administration. Repeated administration can be at the same dose or at different doses. For example, an antibody that specifically binds to CD38 in the methods of the invention can be administered at 8 mg/kg or at 16 mg/kg at weekly intervals for 8 weeks, followed by biweekly at 8 mg/kg or at 16 mg/kg. The administration was continued for another 16 weeks, followed by intravenous infusion at 8 mg/kg or at 16 mg/kg every four weeks.

An antibody that specifically binds to CD38 can be administered by maintenance therapy in the methods of the invention, such as, for example, once a week for 6 months or longer.

For example, an antibody that specifically binds to CD38 in the method of the present invention may be used as a daily dose in an amount of about 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90, or 100 mg/kg, provided daily, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, Provided at least one of 39, or 40 days, or alternatively, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 after initiation of treatment, Provided at least one of 15, 16, 18, 19, or 20 weeks, or any combination thereof, and using a single or divided dose every 24, 12, 8, 6, 4, or 2 hours, or Any combination.

Antibodies that specifically bind to CD38 in the methods of the invention may also be administered prophylactically to reduce the risk of developing cancer, delay the onset of cancer progression events, and/or reduce the risk of recurrence when the cancer is in remission. This may be particularly useful in patients who are known to have tumors but are difficult to locate tumors due to other biological factors.

Antibodies that specifically bind to CD38 in the methods of the invention can be stored lyophilized and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective for conventional protein formulations and well known freeze-drying and reconstitution techniques can be used.

An antibody that specifically binds to CD38 in the methods of the invention can be administered in combination with a second therapeutic agent.

In the methods of the invention, an antibody that specifically binds to CD38 can be administered with any one or more of a chemotherapeutic drug or other anti-cancer therapeutic known to those of ordinary skill in the art. Chemotherapeutic agents are chemical compounds that are useful in the treatment of cancer, and include growth inhibitors or other cytotoxic agents, and include alkylating agents, antimetabolites, anti-microtubule inhibitors, topoisomerase inhibitors (topoisomerase inhibitors). , receptor tyrosine kinase inhibitors, angiogenesis inhibitors, and the like. Examples of chemotherapeutic agents include: alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan, And piposulfan; aziridine, such as benzodopa, carboquone, meturedopa, and uredopa; Ethylenimine and methylamelamine, including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphonamide Triethylenethiophosphaoramide), and trimethylolomelamine; nitrogen mustard, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine (estramustine), ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, neonitrogen mustard (novembichin) ), cholesterol phenyl b Phenosterine, prednimustine, trofosfamide, uracil mustard; nitrosureas, such as carmustine, Chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysin ), actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, Carabincin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, tertinopyridin ( Detorubicin), 6-diazo-5-oxo-L-normal leucine, doxorubicin, epirubicin, esorubicin, edabubi Star (idarubicin), marcellomycin, mitomycin, mycophenolic acid, nocardia Nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin ), streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; Metabolic drugs, such as methotrexate and 5-FU; folic acid analogs such as denopterin, amine methotrexate, pteropterin, trimetrexate; , such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine (azacitidine), 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enoxa Otacitabine, floxuridine; androgen, such as calpressone, Dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenal agents, such as aminoglutethimide, mitotane , trolostane; folic acid supplements, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; Amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone ); elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoxantrone Mit (mitoguazone); mitoxantrone; mopidanmol; nitracrine; pentostatin; phenamet; pirarubicin ( Pirarubicin); podophyllinic acid; 2-ethyl 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; Triaziquone; 2,2',2"-trichlorotriethylamine;carbamate;vindesine;dacarbazine;mannomustine; dibromomannose Alcohol (mitobronitol); mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");cyclophosphamide; thiophene Thietepa; member of the paclitaxel or taxane family, such as paclitaxel (TAXOL®), docetaxel (TAXOTERE®), and its analogues; chlorambucil; gemcitabine ; 6-thioguanine; guanidinium; amidoxime; platinum analogues, such as cisplatin and carboplatin; vinblastine; platinum; etoposide (etoposide, VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine (vi Norelbine); novelbine; nonovantrone; teniposide; daunomycin; aminopterin; xeloda; Ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecita Bin (capecitabine); receptor tyrosine kinase inhibitors and / or angiogenesis inhibitors, including Nexavar® (sorafenib (sorafenib)), SUTENT® (sunitinib (sunitinib)), VOTRIENT ^TM (Pa pazopanib (pazopanib)), PALLADIA ^TM (Tosi Neeb (toceranib)), ZACTIMA ^TM (vandetanib (vandetanib)), RECENTIN® (cediranib (cediranib)), regorafenib (regorafenib ) (BAY 73-4506), axitinib (axitinib) (AG013736), to he erlotinib (lestaurtinib) (CEP-701) , TARCEVA® ( erlotinib (erlotinib)), IRESSA ^TM (gefitinib Medicine (gefitinib), Gilotrif ^® (afatinib), TYKERB® (lapatinib), neratinib, and the like, and any of the above Acceptable salt Acid, or derivative thereof. Also included in this definition are anti-hormonal agents for modulating or inhibiting the action of hormones on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitory 4 (5) -imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and FARESTON® (toremifene) And anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; A pharmaceutically acceptable salt, acid, or derivative of either. Other conventional cytotoxic compounds as disclosed in Wiemann et al., 1985, in Medical Oncology (Calabresi et al., eds.), Chapter 10, McMillan Publishing, may also be suitable for use in the methods of the present invention.

The CD38 binding antibody may be in combination with the method of the present invention are used in a specific exemplary agents include tyrosine kinase inhibitors and anticancer therapy target, such as IRESSA ^TM (gefitinib) and Tarceva ^® (erlotinib Other antagonists of HER2, HER3, HER4, or VEGF. Exemplary HER2 antagonists include CP-724-714, HERCEPTIN ^TM (trastuzumab (trastuzumab)), OMNITARG ^TM (pertuzumab (pertuzumab)), TAK-165 , TYKERB® ( lapatinib) (EGFR and HER2 inhibitors), and GW-282974. Exemplary HER3 antagonists include anti-Her3 antibodies (see, e.g., U.S. Patent Publication No. 2004/0197332). Exemplary HER4 antagonists include anti-HER4 siRNA (see, eg, Maatta et al, Mol Biol Cell 17: 67-79, 2006). Exemplary VEGF antagonist based Avastin ^TM (bevacizumab (bevacizumab)).

Exemplary agents that can be used in combination with antibodies that specifically bind CD38 in the methods of the invention include standard care drugs for solid tumors, or immunological checkpoint inhibitors.

The second therapeutic agent in the methods of the invention may be an immunological checkpoint inhibitor.

In some embodiments, the immunological checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, or an anti-CTLA-4 antibody.

In some embodiments, the immunological checkpoint inhibitor is an antagonist anti-PD-1 antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2 antibody, an antagonist anti-LAG3 antibody, or an antagonist anti-TIM3 antibody.

In some embodiments, the immunological checkpoint inhibitor is an anti-PD-1 antibody.

In some embodiments, the immunological checkpoint inhibitor is an anti-PD-L1 antibody.

In some embodiments, the immunological checkpoint inhibitor is an anti-PD-L2 antibody.

In some embodiments, the immunological checkpoint inhibitor is an anti-LAG3 antibody.

In some embodiments, the immunological checkpoint inhibitor is an anti-TIM3 antibody.

In some embodiments, the immunological checkpoint inhibitor is an anti-CTLA-4 antibody.

Any antagonistic anti-PD-1 antibody can be used in the methods of the invention. An exemplary anti-PD-1 anti-system, OPVIDO® (Nivoruzumab) and KEYTRUDA® (Pacliizumab) can be used. OPVIDO® (Nivoruzumab) is described, for example, in U.S. Patent No. 8,008,449 (Antibody 5C4) and comprises VH of SEQ ID NO: 24 and VL of SEQ ID NO: 25. KEYTRUDA® (Pacliizumab) is described, for example, in U.S. Patent No. 8,354,509 and includes VL of SEQ ID NO: 22 and VL of SEQ ID NO: 23. The amino acid sequence of nivoluzumab and paclizumab can also be obtained by CAS registration. Additional PD-1 anti-systems that can be used are described in U.S. Patent No. 7,332,582, U.S. Patent Publication No. 2014/0044738, International Patent Publication No. WO 2014/17966, and U.S. Patent Publication No. 2014/035636.

"Antagonist" refers to a molecule that, when bound to a cellular protein, inhibits the reaction or activity induced by at least one natural ligand of the protein. In one molecule, at least one of the reactions or activities is inhibited by at least about 30%, 40%, 45%, 50% more than the at least one reaction or activity is inhibited in the absence of an antagonist (eg, a negative control). , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or when compared to inhibition in the absence of an antagonist, inhibition When the statistics are statistically significant, they are antagonists. Antagonists can be antibodies, soluble ligands, small molecules, DNA, or RNA such as siRNA. For example, a typical response or activity induced by PD-1 binding to its receptor PD-L1 or PD-L2 may be a reduction in antigen-specific CD4 ⁺ or CD8 ⁺ cell proliferation or T cell interferon-gamma (IFN- A decrease in gamma production, which results in inhibition of an immune response against, for example, a tumor. A typical response or activity induced by binding of TIM-3 to its receptor (such as Galectin-9) can result in a decrease in antigen-specific CD4 ⁺ or CD8 ⁺ cell proliferation and a decrease in T cell IFN-γ production. , or a decrease in the surface manifestation of CD137 on CD4 ⁺ or CD8 ⁺ cells, which results in inhibition of an immune response against, for example, a tumor. Therefore, an antagonist PD-1 antibody that specifically binds to PD-1, an antagonist PD-L2, an antagonist antibody that specifically binds to TIM-3 induces an immune response by inhibiting the inhibitor pathway.

SEQ ID NO: 22

SEQ ID NO: 23

SEQ ID NO:24

SEQ ID NO: 25

An anti-PD-L1 antibody that enhances the immune response can be used in the methods of the invention (e.g., antagonist anti-PD-L1 antibodies). Exemplary anti-PD-L1 anti-systems, dvvalumab, etezolizumab, and avelumab, and are described, for example, in U.S. Patent Publication No. 2009/0055944 U.S. Patent No. 8,552,154, U.S. Patent No. 8,217,149, and U.S. Patent No. 8,779,108.

Devaluzumab comprises VH of SEQ ID NO: 26 and VL of SEQ ID NO: 27.

Alitizumab comprises VH of SEQ ID NO: 28 and VL of SEQ ID NO: 29.

Iviral mAb comprises VH of SEQ ID NO: 30 and VL of SEQ ID NO: 31.

SEQ ID NO:26

SEQ ID NO:27

SEQ ID NO:28

SEQ ID NO:29

SEQ ID NO: 30

SEQ ID NO: 31

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH of SEQ ID NO: 4 and The combination of VL of SEQ ID NO: 5 with an anti-PD-1 antibody comprising VH of SEQ ID NO: 24 and VL of SEQ ID NO: 25 for a period of time sufficient to treat the solid tumor.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH of SEQ ID NO: 4 and The combination of VL of SEQ ID NO: 5 with an anti-PD-1 antibody comprising VH of SEQ ID NO: 22 and VL of SEQ ID NO: 23 for a period of time sufficient to treat the solid tumor.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH of SEQ ID NO: 4 and The combination of VL of SEQ ID NO: 5 with an anti-PD-L1 antibody comprising VH of SEQ ID NO: 26 and VL of SEQ ID NO: 27 for a period of time sufficient to treat the solid tumor.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of a specific binding to CD38 The combination of the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 with an anti-PD-L1 antibody comprising VH of SEQ ID NO: 28 and VL of SEQ ID NO: 29 for a sufficient period of time The time to treat the solid tumor.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH of SEQ ID NO: 4 and The combination of VL of SEQ ID NO: 5 with an anti-PD-L1 antibody comprising VH of SEQ ID NO: 30 and VL of SEQ ID NO: 31 for a period of time sufficient to treat the solid tumor.

The invention also provides a method of enhancing an immune response in a patient comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH and SEQ ID of SEQ ID NO:4) The combination of NO: 5 VL) with an anti-PD-1 antibody comprising VH of SEQ ID NO: 24 and VL of SEQ ID NO: 25 for a period of time sufficient to enhance the immune response.

The invention also provides a method of enhancing an immune response in a patient comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH and SEQ ID of SEQ ID NO:4) The combination of NO: 5 VL) with an anti-PD-1 antibody comprising VH of SEQ ID NO: 22 and VL of SEQ ID NO: 23 for a period of time sufficient to enhance the immune response.

The invention also provides a method of enhancing an immune response in a patient comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH and SEQ ID of SEQ ID NO:4) The combination of NO: 5 VL) with an anti-PD-L1 antibody comprising VH of SEQ ID NO: 26 and VL of SEQ ID NO: 27 for a period of time sufficient to enhance the immune response.

The invention also provides a method of enhancing an immune response in a patient comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH and SEQ ID of SEQ ID NO:4) The combination of NO: 5 VL) with an anti-PD-L1 antibody comprising VH of SEQ ID NO: 28 and VL of SEQ ID NO: 29 for a period of time sufficient to enhance the immune response.

The invention also provides a method of enhancing an immune response in a patient comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5) and a combination of an anti-PD-L1 antibody comprising VH of SEQ ID NO: 30 and VL of SEQ ID NO: 31 for a period of time sufficient to enhance The time of the immune response.

The invention also provides a method of treating a patient having colorectal cancer comprising administering to the patient in need thereof a therapeutically effective amount of a combination of an antibody that specifically binds CD38 and an antagonist anti-ID-1 antibody. A period of time sufficient to treat the colorectal cancer.

The invention also provides a method of treating a patient suffering from colorectal cancer comprising administering to a patient in need thereof a therapeutically effective amount of a combination of an antibody that specifically binds CD38 and an antagonist anti-PD-L1 antibody. A period of time sufficient to treat the colorectal cancer.

The invention also provides a method of treating a patient having colorectal cancer comprising administering to the patient in need thereof a therapeutically effective amount of a combination of an antibody that specifically binds CD38 and an antagonist anti-PD-L2 antibody. A period of time sufficient to treat the colorectal cancer.

The invention also provides a method for treating a patient suffering from lung cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a combination of an antibody that specifically binds CD38 and an antagonist anti-PD-1 antibody for a period of time A time sufficient to treat the lung cancer.

The invention also provides a method for treating a patient suffering from lung cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a combination of an antibody that specifically binds CD38 and an antagonist anti-PD-L1 antibody for a period of time A time sufficient to treat the lung cancer.

The invention also provides a method for treating a patient suffering from lung cancer, comprising administering to a patient in need thereof a therapeutically effective amount of a combination of an antibody that specifically binds CD38 and an antagonist anti-PD-L2 antibody for a period of time A time sufficient to treat the lung cancer.

The present invention also provides a method of treating a patient suffering from prostate cancer comprising administering to a patient in need thereof a therapeutically effective amount of a combination of an antibody that specifically binds to CD38 and an antagonistic anti-PD-1 antibody. A period of time sufficient to treat the prostate cancer.

The present invention also provides a method of treating a patient suffering from prostate cancer comprising administering to a patient in need thereof a therapeutically effective amount of a combination of an antibody that specifically binds CD38 and an antagonist anti-PD-L1 antibody. A period of time sufficient to treat the prostate cancer.

The present invention also provides a method of treating a patient suffering from prostate cancer comprising administering to a patient in need thereof a therapeutically effective amount of a combination of an antibody that specifically binds CD38 and an antagonist anti-PD-L2 antibody. A period of time sufficient to treat the prostate cancer.

An anti-LAG-3 antibody that enhances the immune response can be used in the methods of the invention. Exemplary anti-LAG-3 anti-systems that can be used are described, for example, in International Patent Publication No. WO 2010/019570.

An anti-CTLA-4 antibody that enhances the immune response can be used in the methods of the invention. An exemplary anti-CTLA-4 anti-system, ipilimumab, can be used.

Anti-PD-1, anti-PD-L1, anti-PD-L2, anti-LAG3, anti-TIM3, and anti-CTLA-4 antibodies useful in the methods of the invention can also be regenerated using the methods described herein.

In some embodiments, an anti-PD1 antibody comprising VH of SEQ ID NO: 32 and VL of SEQ ID NO: 33 can be used.

In some embodiments, an anti-PD1 antibody comprising VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35 can be used.

In some embodiments, an anti-TIM-3 antibody comprising VH of SEQ ID NO: 36 and VL of SEQ ID NO: 37 can be used.

In some embodiments, an anti-TIM-3 antibody comprising VH of SEQ ID NO: 38 and VL of SEQ ID NO: 39 can be used.

SEQ ID NO:32

SEQ ID NO:33

SEQ ID NO: 34

SEQ ID NO: 35

SEQ ID NO:36

SEQ ID NO:37

SEQ ID NO:38

SEQ ID NO:39

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH of SEQ ID NO: 4 and The combination of VL of SEQ ID NO: 5 with an anti-PD-1 antibody comprising VH of SEQ ID NO: 32 and VL of SEQ ID NO: 33 for a period of time sufficient to treat the solid tumor.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH of SEQ ID NO: 4 and The combination of VL of SEQ ID NO: 5 with an anti-PD-1 antibody comprising VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35 for a period of time sufficient to treat the solid tumor.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5) is combined with an anti-TIM-3 antibody comprising VH of SEQ ID NO: 36 and VL of SEQ ID NO: 37 for a time sufficient to treat the solid tumor.

The invention also provides a method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 (which comprises VH of SEQ ID NO: 4 and The combination of VL of SEQ ID NO: 5 with an anti-TIM-3 antibody comprising VH of SEQ ID NO: 38 and VL of SEQ ID NO: 39 for a period of time sufficient to treat the solid tumor.

In the methods of the invention, a combination of an antibody that specifically binds to CD38 and a second therapeutic agent can be administered for any given period of time. For example, an antibody that specifically binds to CD38 and a second therapeutic agent can be administered to a patient on the same day, and even in the same intravenous infusion. However, the antibody that specifically binds to CD38 and the second therapeutic agent can also be administered on alternate days, or alternate weeks or months, and the like. In some methods, an antibody that specifically binds to CD38 and a second therapeutic agent can be administered at a time close enough to allow simultaneous (eg, in serum) treatment of the patient at a detectable level (eg, in serum). in. In some methods, the entire process of treatment with an antibody that specifically binds to CD38, which consists of several doses over a period of time, is preceded by or during the course of treatment with a second therapeutic agent (which consists of several doses) Rear. A recovery period of 1, 2, or several days or weeks may be used between administration of an antibody that specifically binds to CD38 and a second therapeutic agent.

An antibody that specifically binds to CD38 or a combination of an antibody that specifically binds to CD38 and a second therapeutic agent can be combined with any form of radiation therapy (including external beam radiation, intensity modulated radiation therapy (IMRT), focused radiation, and any Forms of radiosurgery (including Gamma Knife, Cyberknife, Linear Accelerator (Linac), and interstitial radiation (eg implanted radioactive seeds, GliaSite balloons)), and/or with surgery Apply together.

Focused radiation methods that can be used include stereotactic radiosurgery, fractional stereotactic radiosurgery, and intensity modulated radiation therapy (IMRT). It is apparent that stereotactic radiosurgery involves accurately delivering radiation to tumor tissue (eg, brain tumors) while avoiding surrounding non-tumor, normal tissue. The dose of radiation applied using stereotactic radiosurgery may vary, typically from 1 Gy to about 30 Gy, and may include intermediate ranges including, for example, 1 to 5, 10, 15, 20, 25, up to 30 Gy. Because of the non-invasive fixation device, stereotactic radiation does not need to be delivered in a single treatment. The treatment plan can be repeated day after day, whereby a plurality of fractionated radiation doses are delivered. When used to treat a tumor over a period of time, the radiosurgery is referred to as "fractionated stereotactic radiosurgery" or FSR. In contrast, stereotactic radiosurgery refers to a one-session treatment. Fractional stereotactic radiosurgery may result in a high therapeutic rate, ie, a high rate of tumor cell killing and a low impact on normal tissues. Tumors and normal tissues respond differently to high single dose radiation to multiple small doses of radiation. A single high dose of radiation may kill more normal tissue than several small doses of radiation. Therefore, multiple small doses of radiation can kill more tumor cells without harming normal tissue. The dose of radiation applied using fractional stereotactic radiation may vary from 1 Gy to about 50 Gy and may include intermediate ranges including, for example, 1 to 5, 10, 15, 20, 25, 30, 40, up to 50 Gy. Low fractionated dose. Intensity modulated radiation therapy (IMRT) can also be used. IMRT is an advanced mode of high precision three dimensional conformal radiation therapy (3DCRT) that uses a computer controlled linear accelerator to deliver precise radiation doses to a specific range within a malignant tumor or tumor. In the 3DCRT, the contour of each radiation beam is shaped into a contour suitable for the target using a multi-leaf collimator (MLC) (from the beam's eye view). BEV) viewpoint, whereby several beams are generated. The IMRT adjusts the intensity of the radiation beam in multiple small quantities so that the radiation dose more precisely conforms to the three-dimensional (3-D) shape of the tumor. Therefore, IMRT enables higher radiation doses to be concentrated into areas within the tumor while minimizing doses to surrounding normal critical structures. IMRT increases the ability to conform a therapeutic amount to the shape of a concave tumor, for example, when the tumor is entangled in a fragile structure such as the spinal cord or major organs or blood vessels.

Subcutaneous administration of a pharmaceutical composition comprising an antibody that specifically binds to CD38 and a hyaluronanase

The antibody that specifically binds to CD38 can be administered subcutaneously in the form of a pharmaceutical composition comprising an antibody that specifically binds to CD38 and a hyaluronidase.

The concentration of the antibody that specifically binds to CD38 in the pharmaceutical composition administered subcutaneously may be about 20 mg/ml.

The pharmaceutical composition for subcutaneous administration may comprise between about 1,200 mg and 1,800 mg of an antibody that specifically binds to CD38.

The pharmaceutical composition for subcutaneous administration may comprise about 1,200 mg of an antibody that specifically binds to CD38.

The pharmaceutical composition for subcutaneous administration may comprise about 1,600 mg of an antibody that specifically binds to CD38.

The pharmaceutical composition for subcutaneous administration may comprise about 1,800 mg of an antibody that specifically binds to CD38.

The pharmaceutical composition administered subcutaneously can comprise a hyaluronidase between about 30,000 U and 45,000 U.

The pharmaceutical composition for subcutaneous administration may comprise about 1,200 mg of an antibody that specifically binds to CD38 and about 30,000 U of hyaluronidase.

The pharmaceutical composition for subcutaneous administration may comprise about 1,800 mg of an antibody that specifically binds to CD38 and about 45,000 U of hyaluronidase.

The pharmaceutical composition for subcutaneous administration may comprise about 1,600 mg of an antibody that specifically binds to CD38 and about 30,000 U of hyaluronidase.

The pharmaceutical composition for subcutaneous administration may comprise about 1,600 mg of an antibody that specifically binds to CD38 and about 45,000 U of hyaluronidase.

The pharmaceutical composition for subcutaneous administration may comprise hyaluronan rHuPH20 having the amino acid sequence of SEQ ID NO:40.

rHuPH20 is a recombinant hyaluronic acid enzyme (HYLENEX® recombinant) and is described in International Patent Publication No. WO 2004/078140.

Hyaluronanase degrades the enzyme of hyaluronic acid (EC 3.2.1.35) and reduces the viscosity of hyaluronic acid in the extracellular matrix, thereby increasing tissue permeability.

SEQ ID NO:40

The pharmaceutical composition comprising the antibody that specifically binds to CD38 and the hyaluronidase can be administered on one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six Repeatedly after weeks, seven weeks, two months, three months, four months, five months, six months, or longer. It is also possible to repeat the treatment process, such as chronic administration. Repeated administration can be at the same dose or at different doses. For example, a pharmaceutical composition comprising an antibody that specifically binds to CD38 and a hyaluronan can be administered once a week for eight weeks, followed by two weeks for a period of 16 weeks, followed by four weeks. The pharmaceutical composition to be administered may comprise about 1,200 mg of an antibody that specifically binds to CD38 and about 30,000 U of hyaluronidase, wherein the concentration of the antibody that specifically binds to CD38 is about 20 mg/ml in the pharmaceutical composition. The pharmaceutical composition to be administered may comprise about 1,800 mg of specific binding to CD38 The antibody and about 45,000 U of hyaluronidase. The pharmaceutical composition to be administered may comprise about 1,600 mg of an antibody that specifically binds to CD38 and about 30,000 U of hyaluronidase. The pharmaceutical composition to be administered may comprise about 1,600 mg of an antibody that specifically binds to CD38 and about 45,000 U of hyaluronidase.

A pharmaceutical composition comprising an antibody that specifically binds to CD38 and a hyaluronan can be administered subcutaneously to the abdominal region.

The pharmaceutical composition comprising the antibody that specifically binds to CD38 and the hyaluronidase can be administered in a total volume of about 80 ml, 90 ml, 100 ml, 110 ml, or 120 ml.

At the time of administration, 20 mg/ml of an antibody that specifically binds to CD38 (in 25 mM sodium acetate, 60 mM sodium chloride, 140 mM D-mannitol, 0.04% polysorbate 20, pH 5.5) and rHuPH20 (1.0 mg/) can be administered. mL (75-150 kU/mL) was mixed with 10 mM L-histamine, 130 mM NaCl, 10 mM L-methionine, 0.02% polysorbate 80, pH 6.5), after which the mixture was administered to the subject.

Although the present invention has been described in terms of general terms, the embodiments of the present invention are further disclosed in the following examples, which should not be construed as limiting the scope of the claims.

Example 1. General materials and methods Sample collection and processing

Peripheral blood and bone marrow aspirate were collected in a heparinized tube immediately following the base period prior to the first infusion and the time point indicated during the treatment period. Most of the samples were evaluated using real-time flow cytometry 24 to 48 hours after collection when they arrived at the central laboratory. Peripheral blood mononuclear cells (PBMC) were obtained from whole blood, isolated by density gradient centrifugation, and stored frozen until analysis. For T cell activation, homozygous, and CD38 ⁺ Treg inhibition assays, frozen PBMC samples were used and samples were analyzed before and after treatment.

Flow cytometry analysis of these samples was performed at the BARC global central laboratory using pre-validated immunophenotyping assays to assess NK cells, T cells, B cells, myeloma cells (CD138 ⁺ ), And CD38 performance. Briefly, blood samples and bone marrow samples were stained with the following multi-fluorescent dye antibody groups: cell lineage group: PerCPCy5.5α-CD19 (pure line HIB19; Becton Dickinson [BD]), APCα-CD24 (SN3; eBioscience), PC7α -CD3 (UCHT-1; Beckman Coulter), V500α-CD16 (3G8; BD), and PEα-CD56 (MY; BD); regulatory T cells (T _reg ) group: APCα-CD25 (2A3; BD), PEα- CD127 (HIL-7R-M21; BD), APC-H7α-HLA-DR (G46-6; BD), and PerCPα-CD4 (L200; BD); initial (naive)/memory T cell group: APC-H7α- CD4 (RPA-T4; BD), PerCP-Cy5.5α-CD8 (RPA-T4 BD), PEα-CD62L (SK11; BD), and APCα-CD45RA (HI100; BD). The CD38 expression was assessed using Alexa 647-labeled antibody mAb 003, which is described in U.S. Patent No. 7,829,693, having the VH and VL sequences of SEQ ID NO: 14 and SEQ ID NO: 15. Blood samples were prepared using different lysis-cleaning methods. Bone marrow extract samples were subjected to membrane or intracellular staining using various antibodies. Becton Dickinson FACS lysis solution was used to lyse red blood cells in peripheral blood samples, and the Fix and Perm cell permeation reagent from Invitrogen was used for intracellular staining of bone marrow extract samples. Stained samples were obtained on a FACS Canto II flow cytometer and the data was analyzed using FacsDiva software. The absolute count of the immune cell population in the blood sample and the percentage of lymphocytes in the bone marrow sample were determined at all time points tested.

T cell receptor (TCR) sequencing

T cell diversity was analyzed by using deep sequencing of TCR rearrangement using the genetic DNA of PBMC samples to assess the pureness of CD8 ⁺ T cells. Adaptive Biotechnologies Immunoseq ^TM measured using a commercially available system for sequencing the TCR, and an analysis system of a multiplex polymerase chain reaction using pre (PCR) assay (TR2015CRO-V-019) for which the like-based primer and reverse by the forward primer The forward and reverse primers directly target the family of variable (V) genes (forward primers) and junction (J) genes (reverse primers). Each of the V and J gene primers serves as a priming pair to amplify the somatic cell recombinant TCR, and each primer contains a specific universal DNA sequence. After the initial PCR amplification, each amplification product is amplified a second time with a forward primer and a reverse primer containing a universal sequence and a transfer sequence required for Illumina DNA sequencing (adaptor) Sequence).

T cell response to viral antigens and alloantigens

The patient's PBMC were plated on a 96-well plate (2 x 10 ⁵ cells/well) and stimulated for 5 days with a mixture of 23 major histocompatibility gene complexes (MHC) class I restricted viral peptides. (cocktail), derived from human cytomegalovirus (CMV), Esplanade-Barr virus (EBV), and influenza virus (2 μg/ml; CEF peptide pool; PANATecs ^® ); or an equal number of healthy donors 25-Gy irradiated allogeneic PBMC. Unstimulated PBMC and PBMC stimulated with anti-CD3/CD28 coated beads served as negative controls and positive controls, respectively. On day 5, cell-free supernatant interferon gamma (IFN-γ) was measured by sandwich enzyme-linked immunosorbent assay (ELISA; human IFN gamma ELISA Ready-SET-Go; eBioscience) and it served as T cell activation Alternative sign.

Regulation of T cell (Treg) inhibition of effector cell function: carboxyluciferin amber imidate (CFSE) dilution assay

Healthy donor PBMC with PerCP-Cy5.5α-CD3 (SK7; BD), KOα-CD45, (J33; Beckman Coulter), V450α-CD4 (SK3; BD), PEα-CD25 (M-A251, BD) PE Cy7α-CD127 (HIL-7R-M21; BD), and APCα-CD38 (HB-7; BD) were labeled and sorted by FACS Aria (BD). The sorted effector cells were labeled with carboxyfluorescein amber ylide (CFSE; eBioscience) and coated with anti-CD3/CD28 beads in CD38 ⁺ Treg or CD38 ^- Treg (Treg to effector cell ratio 1 :1) Stimulation in RPMI plus 10% fetal calf serum in the presence or absence. After 72 hours, flow cytometry was performed and the dilution percentage of CFSE was used as a surrogate for T cell proliferation.

Bone marrow-derived suppressor cell (MDSC) phenotype and DARZALEX ^TM (Dalabizumab) Medium ADCC

Three normal healthy donor PBMCs were co-cultured with myeloma cell lines (RPMI8226, U266, H929) for six days and evaluated for the production of granule globular MDSC (G-MDSC) (CD11b ⁺ CD14 ^- HLA ^- DR ^- CD15 ⁺ CD33 ⁺ ), as described in Gorgun et al., Blood 121: 2975-87, 2013. G-MDSC was not present in normal healthy PBMC, however after co-cultivation with all three myeloma cell lines, G-MDSC was present at 5 to 25% of the total PBMC population (data not shown). For assessment of circling flow measurement of G-MDSC cells (gating is) include CD11b ⁺ policy as a first circle, followed by CD14 ^- and HLA ^- DR ^- circle, and then followed by CD15 ⁺ and CD33 ⁺ circle. G-MDSCs were sorted by cells and assessed for CD38 expression levels and sensitivity to ADCC of DARZALEX ^(TM) (duracil) media. To assess the effect of DARZALEX ^(TM) (dalabizumab) on ADCC/CDC of MDSC, serum containing complement or isotype control was added to the ADCC assay.

Initial and memory T cell analysis

Patients from heparinized peripheral blood samples obtained, after each infusion DARZALEX ^TM (Dara mAb). Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll-Hypaque density gradient centrifugation and stored in liquid nitrogen in cryopreservation medium (addition of 10% human serum and 10% dimethylidene RPMI) . For FACS analysis, PBMC were thawed, and the 2 × 10 ⁶ cells / group resuspended with 0.05% azide and 0.1% HAS of phosphate buffered saline (PBS).

data analysis

All data analysis and correlation diagrams are generated using only R software (R: A Language and Environment for Statistical Computing, R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2011, ISBN 3-900051-07-0 http_ //_www_R-project_org/). All treated subjects with evaluable responses were included in the data analysis. Consistently, the responders were defined as subjects that had the best response to sCR, VGPR, and PR according to IRC, and non-responders were defined as subjects that had the best response to MR, SD, and PD according to IRC.

Different statistical comparisons include: (i) the base level between the responder and the non-responder, (ii) the base period of the responder and the non-responder, and (iii) between the responder and the non-responder. Percent change, (iv) change in the ratio of the base period to treatment. The comparisons first included the use of the Shapiro-Wilk test for normality verification (Royston). (1995) Remark AS R94: A remark on Algorithm AS 181: The W test for normality. Applied Statistics, 44, 547-551). It was found that almost all data did not have a normal distribution. The difference level test includes a non-parametric Wilcox rank sum test (Hollander and Wolfe (1973), Nonparametric Statistical Methods. New York: John Wiley & Sons. Pages 27-33 (single sample), 68 -75 (two-sample)) and t-test after Box Cox conversion (Weisberg, S. (2014) Applied Linear Regression, Fourth Edition, Wiley Wiley, Chapter 7). For the Box-Kax conversion, the decimal (1e-07) is added to a value equal to zero. In all cases, the two checks are consistent. Unless otherwise indicated, the Wilburson rating and the assay p-value are shown in the table throughout this specification. When testing for differences in the base period of the responders and non-responders, each subject was tested for a two-group pairing test, and in all other cases, a two-group unpaired test was performed.

Because the samples of the various lymphocyte clusters were not taken at the same time point for different dosing schedules, population modeling was performed. The model fit is performed on the level of the visit. Modeling of the population and the activation of NK cells involves the inclusion of a threshold stick model (Lutz et al., "Statistical model to estimate a threshold dose and its confidence limits for the analysis of sublinear dose-response relationships, exemplified for Mutagenicity data." Mutation Research/Genetic Toxicology and Environmental Mutagenesis 678.2 (2009): 118-122.). A linear mixed-effects model with random intercepts and slopes was fitted to B cells, T cell subsets, and white blood cells, mononuclear spheres, neutrophils, and lymphocytic disease populations (Bates et al., ( 2014). "lme4: Linear mixed-effects models using Eigen and S4." ArXiv e-print; submitted to the Journal of Statistical Software , http:_//_arxiv_org/abs/_1406.5823). This linear mixed modeling system was performed on the relative days after the start of treatment (ADY). This linear mixed model fit is performed on log-transformed reaction variables. In the case where the reaction variable value is equal to 0, 0.1 is added to all reaction variable values to allow modeling on a logarithmic scale.

Example 2. Study 54776414MMY2002 Design (SIRIUS)

The target group of study 54747414MMY2002 (SIRIUS) is a patient with advanced multiple myeloma who has previously received at least 3 therapies, including proteasome inhibitors (PI) and immunomodulatory drugs (IMiD), or Both PI and IMiD are double refractory. The primary endpoint/final analysis response assessment was based on an independent review committee (IRC) and computerized algorithm evaluation using the 2011 IMWG guidelines (clinical research report: an open label, multicenter, phase 2 trial) Exploring the efficacy and safety of DARZALEXTM (Dalabizumab) in subjects with multiple myeloma who have previously received at least 3 therapies (including proteasome inhibitors and IMiD) or Proteasome inhibitors and IMiD are dually refractory (EDMS-ERI-92399922; de Weers et al. (2011) J Immunol 186(3): 1840-1848).

These assessments included: overall response rate (ORR), duration of response, response time and optimal response, clinical benefit rate, time to progression (TTP), progression-free survival (PFS), and overall survival (OS).

This study, a total of 124 objects by DARZALEX ^TM (Dara mAb) therapy (de Weers et al., (2011) J Immunol 186 ( 3): 1840-1848). Eighteen subjects were treated at 8 mg/kg and 106 subjects were treated at 16 mg/kg. Dosing schedule based follows: A Group: DARZALEX ^TM (Dara mAb) 16mg / kg: Cycle 1 and Cycle 2: a first 1,8,15, and 22 days (weekly), the cycle period of 3 to 6: The first 1 and 15 days (every other week), period 7+: day 1 (every 4 weeks). Each cycle is 4 weeks.

Group B: DARZALEX ^TM (Dara mAb) 8mg / kg: 1+ period: 1 day (every 4 weeks).

The primary goal of the study was to determine the efficacy of two treatment regimens for DARZALEXTM (Dalabizumab ⁾ , as measured by ORR (CR+PR), in subjects with multiple myeloma, who had previously Accepting at least 3 therapies, including PI and IMiD, or the disease of these subjects is dually refractory for PI and IMiD (Clinical Research Report: An Open-label, Multi-Center, Phase 2 trial, which explores multiple cases The efficacy and safety of DARZALEXTM (Dalabiza ⁾ in subjects with myeloma who have previously received at least 3 therapies (including proteasome inhibitors and IMiD) or such subjects for proteasome inhibitors and IMiD In terms of double refractory. EDMS-ERI-92399922).

Secondary objectives of this study included assessment of the safety and tolerability of DARZALEXTM (dalalim ⁾ , additional measures of efficacy (eg, clinical benefit, TTP, PFS, and OS) along with assessment of pharmacokinetics, immunogens Sexual, pharmacodynamic, and biomarkers that predict the response to DARZALEXTM (dalabizumab ⁾ . Additional research-related information is available from clinical research protocols (clinical research report: an open-label, multicenter, phase 2 trial exploring DARZALEXTM (dalabizumab ⁾ in subjects with multiple myeloma Efficacy and safety, these subjects have previously received at least 3 therapies (including proteasome inhibitors and IMiD) or these subjects are dually refractory to proteasome inhibitors and IMiD. EDMS-ERI-92399922).

In the first stage of Part 1, one subject (6%) in the 8 mg/kg group responded, and 5 subjects (31%) responded in the 16 mg/kg group. Therefore, in the second and second portions of the first part, only the 16 mg/kg group was amplified.

In the 16 mg/kg group, 31 subjects achieved PR or better response based on IRC assessment; ORR line 29% (95% CI: 21%, 39%). Three subjects (3%) achieved sCR and 13 subjects (12%) achieved VGPR or better.

Example 3. DARZALEX ^TM Effect of (dalalim) on T cell expansion and activity in patients enrolled in the 54747414MMY2002 study (SIRIUS)

CD38 is expressed on a variety of immune cells and hematopoietic cells. Extensive immunodissection was performed by flow cytometry to examine the effect of DARZALEX ^(TM) (dalacil) on immune cell subsets and the association of baseline levels of these cells with clinical response. Evaluation of various cell populations in peripheral blood and bone marrow extracts, including T cells (CD3 ⁺ , CD4 ⁺ , CD8 ⁺ , and regulatory T cells ) by flow cytometry after baseline and DARZALEX ^TM treatment (Treg)), B cells (CD19 ⁺ ), NK cells, mononuclear spheres (CD14 ⁺ ), white blood cells, and neutrophils to monitor changes in these cell populations in responders and non-responders.

Lymphocytes, white blood cells, mononuclear balls, and neutrophils

White blood cells, lymphocytes, mononuclear spheres, and neutrophil counts in peripheral blood of responders and non-responders were studied. In order to 8mg / kg and 16mg / kg dose of total lymphocytes were found in patients with DARZALEX ^TM (Dara mAb) therapy (Fig. 1). Linear mixed-effects modeling revealed an increase of 0.8 × 10 ⁶ cells/μL (CI = 0.06, 0.11) on a logarithmic scale every 100 days. A slight increase in mononuclear and white blood cells was found, which was significantly increased by 0.03×10 ⁶ cells/μL (CI=0.01, 0.04) and 0.03×10 ⁶ cells/μL on a logarithmic scale for each 100 days (CI= 0.01, 0.05). Although the neutrophil reduction was noted in some patients, the median neutrophil count was consistent with the baseline and the change was not significant.

The baseline levels of each of these cell populations were compared between the reaction groups. The use of the Wilcoxon signed-rank test did not reveal evidence of differences in the base level of any of these cell types across the response group ( Table 1 ).

NK cell

Total NK cells (CD16 ⁺ CD56 ⁺ ) and activated NK cells (CD16 ⁺ CD56 ^dim ) decreased over time in the case of DARZALEXTM (dalacil) treatment (data not shown).

B cell

Absolute counts of B cells (CD45 ⁺ CD3 ^- CD19 ⁺ ) in peripheral blood or bone marrow extracts over time during treatment with DARZALEXTM (dalacil ⁾ were measured in responders and non-responders. B cells increased slightly in whole blood and remained unchanged in bone marrow aspirate. Linear mixed modeling of B cells in peripheral blood revealed a minimal increase of 0.1 × 10 ⁶ cells/μL on a logarithmic scale at 100 days in the course of DARZALEX ^TM treatment [CI=0.04, 0.16 ]. The percentage of B cells (CD45 ⁺ CD3 ^- CD19 ⁺ / lymphocytes) in bone marrow aspirate during daramumab treatment did not change in responders or non-responders (p = 0.1 and 0.4, respectively). In addition, no evidence of a different B cell count at the base stage was found between the responder and the non-responder (p=0.5).

T cells increased in the case of lymphocytes noted DARZALEX ^TM (Dara mAb) Healing (FIG. 1), even if the B cells show only a minimal increase (see above). For further investigation, various T cell populations (CD3 ⁺ , CD4 ⁺ , CD8 ⁺ T cells, regulatory T cells) in peripheral blood and bone marrow were studied.

After treatment with DARZALEXTM (dalabizumab ⁾ , CD3 ⁺ , CD4 ⁺ , and CD8 ⁺ T cells in peripheral blood increased (both absolute counts of lymphocytes/μl and percentage). Figure 2 shows the percentage change in absolute counts of CD3 ⁺ T cells (CD45 ⁺ CD3 ⁺ ) from the peripheral phase in peripheral blood of each patient over time. FIG black line shows the absolute count of all patients of median × 10 ⁶ cells / μL. Only more than 2 observations of the visit were included in the figure. Figure 3 shows the % change in absolute count of CD4 ⁺ T cells (CD45 ⁺ CD3 ⁺ CD4 ⁺ ) from the peripheral phase in peripheral blood of each patient over time. The black line in the figure shows the median of all patients. Only more than 2 observations of the visit were included in the figure. Figure 4 shows the % change in absolute count of CD8 ⁺ T cells (CD45 ⁺ CD3 ⁺ CD8 ⁺ ) from the peripheral phase in peripheral blood of each patient over time. The black line in the figure shows the median of all patients. Only more than 2 observations of the visit were included in the figure. Linear mixed model of absolute counts of the peripheral blood revealed, on average, total T cells (CD45 ⁺ CD3 ⁺⁾ after DARZALEX ^TM (Dara mAb) therapy based on each of 0.13 × 100 days increased in the logarithmic scale 10 ⁶ cells/μl (CI=0.1, 0.15). CD8 ⁺ T cells were found to increase significantly by 0.16 × 10 ⁶ cells/μl (CI = 0.13, 0.19) on a logarithmic scale every 100 days. CD4 ⁺ cells were found to increase moderately by 0.11 × 10 ⁶ cells/μl (CI = 0.09, 0.13) on a logarithmic scale every 100 days.

For each of the T cell subsets, the responders showed that the absolute percentage change from the base period was higher than that of the non-responders (CD3 ⁺ p = 3.2993e-05; CD4 ⁺ p = 3.486e-05; CD8 ⁺ p = 2.7172e) -05; regulatory T cells p=0.002). Table 2 shows the results of the Wilburson symbol level check to compare the percentage change from the base period for the absolute count of each T cell subpopulation in the peripheral blood between the responder and the non-responder.

Similarly in the bone marrow, total T cells (CD45 ⁺ CD3 ⁺ as a percentage of lymphocytes) and CD8 ⁺ T cells (CD45 ⁺ CD3 ⁺ CD8 ⁺ as a percentage of lymphocytes) were found in responders and non-responders in DARZALEX ^TM (dalacil) was significantly increased during treatment (CD3 ⁺ responder p = 3.8147e-06, no responder p = 9.8225e-05; CD8 ⁺ responder p = 3.8147e-06, no responder p =0.0003). The median CD4 ⁺ T cells in the bone marrow did not change in any of the clinical response groups. Table 3 shows the results of Wilburson symbol level determination of various T cells which are % of lymphocytes in the bone marrow. Figure 5 shows the percentage (%) of CD45 ⁺ CD3 ⁺ cells over time during DARZALEX ^(TM) (dalabizumab) treatment (reactors and non-responders are included in the graph). Figure 6 shows % of CD45 ⁺ CD3 ⁺ CD8 ⁺ cells over time during DARZALEX ^TM (dalabizumab) treatment (reactors and non-responders are included in the chart).

Although both responders and non-responders showed increased T cells in peripheral blood and bone marrow, responders had the largest percentage change from baseline. In order to distinguish between responders and non-responders with different levels of CD3 ⁺ , CD4 ⁺ , and CD8 ⁺ T cells prior to treatment with DARZALEX ^(TM ), the base period measurements of each subgroup in peripheral blood were compared.

According to the Wilkason symbolic level test, the absolute T cell count in the base phase in peripheral blood ( Table 4 ) or the percentage of T cells in the bone marrow from the total lymphocytes ( Table 5 ), between responders and non-responders There are no statistically significant differences.

T regulatory cell

The Treg cell line was identified as a CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim cell population in the sample. The ratio of CD8 ⁺ T cells to Treg over time in peripheral blood and bone marrow in patients treated with DARZALEX ^(TM) (dalacil ^{) was} assessed. The ratio of peripheral blood and bone marrow increased. Figure 7A shows the median value of CD8 ⁺ /Treg and CD8 ^{+ /} CD4 ⁺ cell ratios in peripheral blood of all patients at each time point. Figure 7B shows the median value of CD8 ⁺ /Treg and CD8 ⁺ /CD4 ⁺ T cell ratios in the bone marrow of all patients at each time point. The change in the absolute count ratio of CD8 ⁺ Treg and CD8 ⁺ /CD4 ⁺ in peripheral blood as a function of treatment time ( Table 6 ) and bone marrow ( Table 7 ) was significant according to the Wilburson symbol level test.

In the combined data analysis of the SIRIUS and GEN501 studies (Example 6), the median ratio of CD8 ⁺ /CD4 ⁺ and CD8 ⁺ /Treg cells in peripheral blood was at week 8 (CD8 ⁺ /CD4 ⁺ p = 5.1 ×) ^10-5, and the CD8 ⁺ / Treg of p = 1.8 × 10 ^-7) and at 16 weeks (CD8 ⁺ / CD4 ⁺ sum p = 0.00017, and CD8 ⁺ / Treg of p = 4.1 × 10 ^-7) due to the increase of. Similarly, in the bone marrow, the median ratio of CD8 ⁺ /CD4 ⁺ and CD8 ⁺ /Treg cells was increased during treatment (12 + 1 week cycle) compared to the base phase (CD8 ⁺ /CD4 ⁺ p = 0.00016, and CD8 ⁺ /Treg p = 2.8 × 10 ^-7 ). No significant differences were observed between responders and non-responders.

Example 4. Research Design (GEN501)

Research GEN501 (NCT00572488) assessment of double refractory MM patients as monotherapy DARZALEX ^TM (Dara monoclonal antibody). Sample isolation, processing, and statistical analysis are as described in Example 1 and Example 2. This study has been described in Lokhorst et al., N Eng J Med 373: 1207-19, 2005.

Briefly, the GEN501 line DARZALEXTM (Dalabizumab) was first studied in human-first (first-in-human) clinical studies in subjects with MM. It is a safety study in which the 1/2-stage dose is gradually increased. The study is divided into two parts. Part 1 is a study of the progressive increase in open-label doses; Part 2 is an open-label, multi-group, one-arm study based on the dose levels established in Part 1.

In Part 1, 10 dose levels of DARZALEX ^(TM) (dalabizumab ^{) were} evaluated: 0.005, 0.05, 0.10, 0.50, 1, 2, 4, 8, 16, and 24 mg/kg. The two lowest dose populations were each assigned 1 (+3) subjects, and the standard 3 (+3) subjects were dosed to the remaining 8 dose groups. Part 2 is an open label study that includes two dose levels, 8 mg/kg and 16 mg/kg. Part 1 includes 32 objects, and Part 2 includes 72 objects.

Example 5. DARZALEX ^TM (Dalabizumab) treatment induces T cell purity in patients

In view of the note in the peripheral blood and bone marrow in MY2002 amplification study CD8 ⁺ T cells, the assay used Immunoseq ^TM T cell receptor (TCR) of the next-generation high-throughput sequencing, to determine amplify CD8 ⁺ T Whether the cell is purely intrinsic is an indication of an adaptive immune response. A total of 17 patient samples enrolled in the GEN501 study were evaluated (reaction n=6, ie, PR; no responder n=11, ie, MR, SD, PD).

TCR sequencing revealed, DARZALEX ^TM (Dara mAb) treatment significantly increases the homogenous nature of the disease. Figure 8A shows the correlation between T cell homogeneity after DARZALEX ^(TM) (dalabizumab) treatment (p=0.0056). Figure 8B shows the fold change in pureness in individual patients. Responders are marked with stars. This data indicates that the T cell expansion noted in the case of DARZALEX ^(TM) (dalabizumab) treatment can be purely intrinsic.

Reactors have greater total amplification in the TCR depot compared to non-responders (as measured by abundance changes; CIA). Figure 8C shows the CIA% of individual patients. Group A: Responders, Group B: No responders. A statistically significant difference was observed between responders and non-responders (p=0.037). Figure 8D shows the sum of absolute abundance changes (CIA) of each expanded T cell colony in the responder and non-responder. Figure 8E shows the maximum CIA% for each individual patient. Group A: Responders, Group B: No responders. A statistically significant difference was observed between responders and non-responders (p=0.048). Figure 8F shows the maximum CIA for individual cell lines in the responders (Group A) and non-responders (Group B).

CIA identifies significant differences in pure abundance between two samples and absolute abundance changes for each expanded strain by using Fisher's exact test (DeWit et al. J. Virol. 2015). Summing to get.

Example 6. DARZALEX ^TM (Dalabizumab) immunomodulatory effects in patients enrolled in the GEN501 study

Various T and B cell populations in the responders and non-responders registered with GEN501 were evaluated.

Lymphocyte

Similar to SIRIUS (MMY2002) study, during DARZALEX ^TM (Dara mAb) therapy, peripheral blood and bone marrow of lymphocytes were increased. This increase is due to an increase in the number of both CD4 ⁺ and CD8 ⁺ cells.

CD8 ⁺ Central memory cell

The CD8 ⁺ T cell phenotype over time in patients treated with DARZALEX ^(TM) (dalacil ^{) was} studied in a subset of 17 patients enrolled in the GEN 501 study. CD8 ⁺ cells from patients were identified as either initial (CD45RO-/CD62L ⁺ ) (T _N ) cells or central memory (T _CM ) (CD45RO ⁺ /CD62L ^{+ high} ) cells using standard protocols.

Figure 9A shows % of CD8 ⁺ naive cells (% of CD8 ⁺ cells), and Figure 9B shows % of CD8 ⁺ central memory cells. DARZALEX ^TM (Dara mAb) therapy significantly reduced the amount of the initial CD8 ⁺ T cells of (8 weeks p = 1.82 × 10 ^-4), and the amount of increase of CD8 ⁺ memory T cells (week 8 of p = 4.88 ×10 ^-2 ). This suggests that the initial cytotoxic T cells are converted to memory T cells, which can be activated against a particular antigen. A white square indicates at least a minimum response ( Patients with MR), and black squares indicate patients with stable disease or disease progression. Significantly greater reductions in CD8 ⁺ naive T cells were evident in patients responding to treatment (data not shown). Figure 9C shows that DARZALEX ^(TM) (dalacil) treatment increases the percentage of HLA class I-restricted T cells that drive viral-specific and alloreactive T cell responses. Figure 9D shows that amplifying effector memory T cells exhibit low levels of CD38. It is important to note that these T cells exhibit normal and even increased functional activity against viral peptides and alloantigens (see Example 8 ). Based on these functional results, we conclude that there is an amplification of the antigen-producing T cells against the virus and alloantigen, or an improvement in activity during the treatment of DARZALEX ^(TM) (dalabizumab ⁾ . These data indicate that, unlike regulatory cell subsets, effector T cells do not require CD38 expression to function properly and expand.

CD38 positive regulatory T cells

Robust cytotoxic T cells of the cell expansion and increased activity was observed, along with several subsets of immunosuppressive CD38 cell indicates performance with recent literature, facilitates verifying DARZALEX ^TM (Dara mAb) regulatory cell population of regulatory T (Treg) cells , bone marrow-derived suppressor cells (MDSC), and the effects of regulating B cells (Breg).

T cells (Treg) (CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim ) were isolated using standard procedures. The frequency of Treg was analyzed using flow cytometry.

Prior to Treg activation, a subset of peripheral Tregs (10% + 10%) exhibited high levels of CD38. Figure 10A, the top panel shows the frequency of Treg in the CD3 ⁺ CD4 ⁺ cell population (P4 cell population) at the base phase. Figure 10A , the lower panel shows a subset of Tregs (P5 cell population) that exhibit high CD38. These CD38 ⁺ Tregs are highly sensitive to DARZALEX ^(TM) (Dalabizumab) treatment and exhibit a significant and almost immediate decline after the first dose of DARZALEX ^(TM) (dazumab ⁾ (patient n=17; first Week at the base period P = 8.88 × 10 ^-16 ). The frequency of Treg after DARZALEX ^(TM) (dalabizumab) treatment is shown in Figure 10B , top panel (P4 cell population). 10B, the following figure shows, after the first (1 ^st) DARZALEX ^TM (Dara mAb) infusion, CD38 ^high Treg (P5 Cells) Treg population of most significant line losses. These CD38 ⁺ Tregs were depleted throughout the treatment of DARZALEX ^TM (dalabizumab ⁾ ( p = 8.88 × 10 ^-16 , 1.11 × 10 ^-15 at the bases of days 1, 4, and 8 respectively), and 1.50×10 ^-11 ). Figure 1OC shows % of CD38 ^high Treg in total CD3 ⁺ cells after 6 months of base phase, week 1, week 4, week 8, relapse, and end of treatment (EOT). The CD38 ^high Treg returned to the base phase at this point in time. Changes in CD38 ⁺ Treg were similar between patients who responded to treatment and those who did not respond, however, CD8 ⁺ T cells at week 8 were shown to respond to patients treated with DARZALEX ^TM (dalacil ⁾ : The Treg ratio was significantly higher ( P = 0.00955; Figure 10D ).

In order to assess the loss of CD38 ⁺ Treg DARZALEX ^TM (Dara mAb) therapy of possible biological relevance, autologous CD3 ⁺ CD38 ⁺ Treg of CD38 on T cells in ^- assessment of Treg suppression. In a series of experiments using samples from multiple healthy donors, CD38 ⁺ Treg inhibited T cell proliferation (9.9% cell proliferation was observed) than CD38 ^- Treg (53.2% cell proliferation observed) or negative control (observation) To 74.9% cell proliferation) is more robust ( Fig. 10E ).

Because MDSCs are not easily detectable in frozen PBMC samples, CD38 ⁺ granule globular MDSCs (CD11b ⁺ CD14 ^- HLA-DR ^- CD15 ⁺ CD33 ⁺ ) are produced in vitro from PBMCs from patients at the base stage and have patients receiving a DARZALEX ^TM (Dara mAb) infusion and isolated. Figure 11 shows the flow cytometry profile of the identified MDSC ( Figure 11 , upper profile, boxed cell population). About half of the MDSCs showed CD38 ( Figure 11 , middle panel ; circled P7 cell population). In patients treated with DARZALEXTM (dalacil ⁾ , the CD38 ^high MDSC line was almost depleted ( Figure 11 , lower panel ; circled P7 cell population).

Patients and in non-responders to treatment with at least a minimum rest (Minimal Repose) in, CD38 ^high lineage nonspecific MDSC in the case DARZALEX ^TM (Dara mAb) treatment of the loss over time. Figure 12 shows that at 1 week, 4 weeks, or 8 weeks of treatment, the percentage of CD38 ^high MDSC was reduced to nearly 0% in patients. The CD38 ^high line non-specific MDSC returns to the base phase after the end of treatment.

Patients with the largest CD38 ⁺ population within lineage non-specific MDSCs demonstrated the best and most durable response to DARZALEX ^(TM) (dalacil) treatment. Figure 13 shows that patients with the highest percentage of CD38 ^high MDSC (as shown in Figure 11) and classified as patients with PR or MR have no progression of at least 8 months for patients 2, 4, 15, 16, or 17 Progression-Free Survival (PFS).

The CD38 ^high lineage non-specific MDSC is also sensitive to in vitro DARZALEX ^(TM) (dalabizum)-induced ADCC. The ADCC assay was performed using CD38 ^high MDSC and Doddy cells from two donors as control target cells with an effector:target cell ratio of 50:1. Figure 14 shows the results of an experiment from a donor. DARZALEX ^TM (Dara mAb) induced dissociation of the MDSC cell.

CD38 ⁺ Breg was measured in DARZALEX ^TM (dalacil) treated patients (n=16) and similar to CD38 ⁺ Treg, CD38 ⁺ Breg was depleted after the first dose of DARZALEX ^TM (dalabiza ⁾ (Phase 1 vs. base period, p = 0.0018; paired Wilcardson grades) and continued low when the patient was under treatment ( Figure 15A ). When stimulated, FACS sorted Breg produced IL-10 ( Fig. 15B ).

Taken together, these observations indicate that the loss of immunosuppressive CD38 ⁺ MDSC, Breg, and Treg is a significant contributor to changes in T cell populations and pure lineage induced by DARZALEXTM (dalabizumab ⁾ .

Example 7. CD38 ⁺  MDSC cells are present in cancer patients

Flow cytometry was used to study the percentage of MDSC (Lin ^- CD14 ^- HLADR ^{low / -} ) and its equivalent CD38 performance in peripheral blood of patients with NSCLC or prostate cancer.

In the analysis samples from NSCLC and prostate cancer patients, the percentage of MDSC was between about 10% and 37% and between about 10% and 27%, respectively, of PBMC. CD38 is expressed in 80 to 100% of Lin ^- CD14 ⁺ HLADR ^{- / low} MDSC of PBMC of NSCLC patients, and is identified in 70 to 100% of MDSCs of PBMC of prostate cancer patients.

Example 8. DARZALEX ^TM (Dalabizumab) enhances antiviral T cell responses

To further assess the effect DARZALEX ^TM (Dara mAbs) on T cell activation and functionality, in patients with a range of clinical outcomes DARZALEX ^TM (Dara mAb) therapy (n = 7), the reaction is measured in IFN-γ production by peripheral T cells in viruses and alloantigens. Patients with PR or better response show a significant increase in IFN-γ secretion in response to viral and alloantigens after treatment with DARZALEXTM (dalacil ⁾ , at least one point during the treatment period As such, it indicates that T cell function is impaired by low CD38 expression (see Example 6, Figure 9C). Similar to TCR pure lineage data, this increase was more pronounced in patients who responded to DARZALEXTM (dalabizumab ⁾ than in those who did not respond. Figure 16A shows the antiviral response of a representative patient with VGPR. Figure 16B shows the antiviral response of a representative patient with CR. Figure 16C shows the antiviral response of a representative patient with PD. Figure 16D shows the antiviral response of a representative patient with MR. In the figure, the error bars represent the standard error of the mean of the duplicate cultures. The asterisk indicates a statistically significant change between the indicated comparisons. Shows the best response according to the criteria of the Independent Review Board. Consistent with these results, viral-reactive T cells in patients with VGPR (Fig. 16E) or CR (Fig. 16F) exhibited an increase in proliferative capacity during treatment with DARZALEX ^(TM) (dalabizumab ⁾ .

Example 9. Immune Cell Subtypes Expressing CD38 to DARZALEX ^TM Mechanism of sensitivity of (dalabizumab)

Data indicating GEN501 SIRIUS and research, in DARZALEX ^TM (Dara mAb) therapy, immune cell lines showed some loss of CD38 (NK cells, regulatory T cells (of Treg), modulation of B cell (Breg), and bone marrow Derived inhibitory cells (MDSC), however, the number of other immune cells that express CD38 is increased (cytotoxic T cells and helper T cells).

To address the mechanism of sensitivity, assess the level of CD38 expression in various immune cell subsets in healthy donors and in multiple myeloma patients enrolled in the GEN501 or SIRIUS study. Figure 17A shows a distribution map of CD38 expression in immune cells of healthy donors, and Figure 17B shows a distribution map of CD38 expression in immune cells of multiple myeloma patients. In healthy donors, CD38 is most expressed on NK cells, followed by mononuclear spheres, B cells, and T cells. In patients with multiple myeloma, CD38 is highest on plasma cells, followed by a subset of B cells, NK cells, monocytes, B cells, and T cells. Figure 17C shows a comparison of the mean fluorescence intensity (MFI) of CD38 of NK cells, Treg, Breg, B cells, and T cells in patients with relapsed and refractory myeloma, showing that NK cells are highest after plasma cells. Horizontal CD38, followed by regulation of T cells (Treg) and regulation of B cells (Breg).

In addition to CD38 expression, other cell surface proteins such as complement inhibitory proteins (CIP; CD46, CD55, CD59) may also result in sensitivity or resistance to DARZALEX ^(TM) (dalabizumab ⁾ . In vitro assessment of CIP in immune cell subsets revealed that NK cells exhibited very low levels of CD59 and CD55, while other T cell populations and B cell populations showed much higher levels. This may also result in variability in the sensitivity of the DARZALEXTM (Dalabizumab ^{) of} immune cell subtypes (data not shown).

discuss

This study by reducing CD38 ⁺ immunosuppressive cell population and accompanying the induced secondary and cytotoxic T cell amplification reaction to produce IFN-γ of virus peptides of and increases TCR homogenous nature of the described previously known DARZALEX ^TM ( The immunomodulatory effect of daramumab, which indicates an improvement in the adaptive immune response.

This study demonstrated, MDSC and Breg CD38 expression and vulnerable DARZALEX ^TM (Dara mAb) therapy on. These cells are known to be present in the tumor microenvironment and result in tumor growth, immune evasion, angiogenesis, metastasis, and the production of inhibitory interleukins. In addition to these CD38 ⁺ suppressor cell subsets, a novel regulatory T cell subset (CD4 ⁺ CD25 ⁺ CD127 ^dim ) was also identified, which also exhibited high levels of CD38 and demonstrated superior autologous T cell inhibitory ability. These cells are also sensitive to DARZALEX ^(TM) (dalabizumab ⁾ and these cells are significantly reduced in the treated patients. Elimination of these CD38 ⁺ immunoregulatory cells by DARZALEX ^(TM) (Dalabizumab ⁾ reduces local immunosuppression in the myeloma microenvironment and allows positive immune effector cells to expand and result in an anti-tumor response.

In fact, a significant increase in the broad T cell population including CD4 ⁺ and CD8 ^{+ was} observed in peripheral blood and in the bone marrow (i.e., tumor). Specific CD8 ⁺ subsets by DARZALEX ^TM (Dara mAb) therapy changes, including significantly reduced naive T cells and concomitant significant increase of effector memory CD8 ⁺ T cells, indicating that effector T cells is shifted to undergo antigen phenotype This phenotype retains immunological memory and is responsive to tumor antigens. The ratio of CD8 ⁺ :CD4 ⁺ and CD8 ⁺ :Treg also increased significantly under treatment, which exhibited a shift in positive immunomodulators to negative immunomodulators.

To assess that the amplified CD4 ⁺ and CD8 ⁺ T cells are essentially pure, the T cell depot of the patient subset is examined. Even in patients with SD of the optimum reaction or progress of patients, T cell clonal exemplary case also DARZALEX ^(TM) (Dara mAb) treatment of the significant increase. Therefore, the pure increase in T cells cannot be simply attributed to a reduction in tumor burden. However, the deviation of clonal T cells in patients with a good clinical response of the larger, and the associated increase in CD8 ⁺ T cells, which indicated that under DARZALEX ^TM (Dara mAb) therapy was observed T cell expansion Increased antigen driven. This is significant in the patient population, which is pre-treated in large numbers (median number of 5 previous treatments) and is not expected to establish a strong anti-tumor immune response. In addition to the pure increase in TCR, patients responsive to DARZALEX ^(TM) (Dalabizumab ⁾ showed an increased T cell response to pre-existing viruses and alloantigens, indicating rescue of immune system recovery from immunosuppressive status.

Treatment with DARZALEX ^(TM) (Dalabizumab) resulted in immunosuppressive MDSC and modulation of T cell and B cell reduction. These reductions are accompanied by amplification of CD4 ⁺ T helper cells and CD8 ⁺ cytotoxic T cells. T cell homologous and functional antiviral responses as measured by IFN-[gamma] production are also increased under treatment with DARZALEX ^(TM) (dalabizumab ⁾ . These observations indicate that T cells continue to function properly despite the low performance of CD38, and suggest that an increase in T cell response may be due to the loss of regulatory cells. Furthermore, these T cell expansion, activity, and homologous changes may be more pronounced in patients who respond to DARZALEX ^(TM) (dalabizumab ⁾ than those who do not respond. Many of these changes with the reversal of the associated recurrence DARZALEX ^TM (Dara mAb) therapy. This indicates that through additional DARZALEX ^TM (Dara mAb) of immunomodulation of previously uncharacterized mechanisms of action, which may lead to clinical response and DARZALEX ^TM (Dara mAb) effect.

Recently, antibodies that promote anti-tumor immune responses, rather than directly targeting cancer, have demonstrated efficacy in a range of environments. Antibodies that inhibit CTLA-4 and PD-1 promote T cell expansion and enhance T cell activation, leading to prolonged survival in patients with advanced solid tumors and hematological malignancies such as Hodgkin's lymphoma And delay the recurrence of the disease. By enhancing anti-cancer immunity, these immunomodulatory antibodies may not only induce clinical response, but also prevent disease recurrence.

Example 10. In the 54674714MMY2002 (SIRIUS) part 2 clinical study with a single dose of DARZALEX ^TM Serum proteosome analysis of multiple myeloma patients with (dalabizum) exchange Biomarker sample collection and processing

Peripheral blood samples were collected in standard serum separation tubes (2.5 mL to 5 mL) and serum samples were aliquoted and shipped to SomaLogic, Inc (Boulder, CO) for multi-analyte serum protein profiling.

Serum protein profiling was performed at SomaLogic using a pre-validated SOMAscan assay that measures 1129 protein analytes using molecules based on SOMAmer affinity. The SOMAmer reagent is a single-stranded DNA-based protein affinity reagent. The assay used a small amount of input sample (150 [mu]L plasma) and converted the protein signal to a SOMAmer signal, which was quantified by a custom DNA microarray.

Each SOMAmer contains 4 functional parts:

1. A unique protein recognition sequence

2. Biotin for capture

3. Photocleavable linkers

4. Fluorescent molecules for detection

This unique protein recognition sequence uses DNA and incorporates chemically modified nucleotides that mimic the side chain of the amino acid, which amplifies the diversity of standard aptamers and enhances the specificity and affinity of protein-nucleic acid interactions (Gold et al., PLoS) One 5: e15004, 2010). The appropriate system is selected by SELEX. The SOMAmer reagent is selected using proteins that exhibit the original configuration. Because such SOMAmer reagents require a complete tertiary protein structure to bind. The unfolded or denatured putatively inactive protein is not detected by the SOMAmer reagent.

The primary mixture of SOMAmer reagents was grouped for sample type and dilution. The reagent is pre-bound to the streptavidin beads prior to sample culture. The protein binds to the homologous SOMAmer in the sample during equilibration, washes, is incubated with NHS-biotin, washed, and then the beads are exposed to UV light to cleave the photocleavable linker. The eluate contains a SOMAmer reagent that binds to the protein labeled by biotin. Streptavidin captures and subsequent washing removes unbound SOMAmer reagent. In the final elution, the SOMAmer molecules are released from their homologous proteins by denaturing conditions. The final eluate was mixed into a custom Agilent DNA microarray, while the SOMAmer molecule fluorophore was quantified by relative fluorescence units (RFU). The RFU is proportional to the amount of protein in the sample.

Samples of the MMY2002 study were tested in two major batches. The first batch of 180 samples contained serum samples from 90 subjects on the first day of the first cycle (C1D1, base period) and C3D1 (day 1 of the third cycle). 180 samples were analyzed together on 3 separate SomaScan disks. The second batch of samples included 50 C1D1 samples, including 35 replicate samples from the first batch.

data analysis Input data set and definition

Treated subjects with evaluable responses are included in the data analysis. In the full report, responders were defined as subjects with sCR, VGPR, and PR overall optimal response (according to IRC, for MMY2002), and disease stable (SD) subjects were defined as having minimal response (MR) or SD. Subjects, and non-responders were defined as subjects with overall overall response to disease progression (PD) (according to IRC, for MMY2002).

Somalogic data preprocessing Batch calibration

Samples of the first and second batches of MMY2002 were tested on two different versions of the SOMAscan platform. The difference between the two versions is small and includes three SOMAmer sequences that vary from version to version (CTSE: 3594-6_1->3594-6_5, FCN1:3613-62_1->3613-62_5, BMPER: 3654-27_1->3654-27_4). These are all removed from the analysis.

According to SomaLogic's standard inter-disk calibration workflow, the plate-wide of each SOMAmer is defined by calculating the ratio of the measured values of the seven intra-panel calibrators to the specific overall reference value of the Master-mix. The scaling factor is calibrated so the measurements of the three first batches are calibrated. The plate-specific scale factor of each SOMAmer reagent was equally applied to each sample on the disc.

Given the different SOMAscan platform versions of the first and second batches, using a modified implementation of SomaLogic's standard inter-disk calibration workflow by considering the ratio of repeated measurements across 35 samples across batches Systematic inter-batch variability correction. For each SOMAmer, calculate the ratio of the first batch of post-calibration measurements for each of the 35 replicate samples divided by the pre-calibration measurements for the second batch ( ri,j ). The median of these 35 ratios is used to define the modified SOMAmer specific calibration scale factor for the second batch of samples. . These calibration scaling factors are then similarly implemented in the standard SOMAscan program.

Once the corrected calibration scale factor is calculated, the distribution of all scale factors for each batch of the analysis is plotted to assess the presence of the outliers. Due to poor reproducibility, nine SOMAmers with very large or very small calibration values (> 0.25 and < 3) were removed from the analysis.

After batch calibration and SOMAmer filtration of MMY2002, log2 conversion was applied to all protein concentration values of MMY2002 to make the data more normal and improve the performance of the parametric statistical assay.

Mixed variable correction

Estimation of the portion of the data set variability explained by meta-variables (eg demographics, response levels, and sample time points) by principal component analysis of a centered and proportional data set And identification of possible confounding factors. A simple linear model was fitted to identify the highest level of PC that was significantly associated with each variable of interest. The significance of these associations was determined using the Wald test, and the portion of the PC variability explained by the model was estimated by fitting R2. For the MMY2002 data, the site ID was found to be related to PC1 and explained the largest part of the data set variability ( 7.37%, p-value = 3.71 x 10-9). In order to reduce the influence of the sample-derived part-related effects in the data, ComBat28 was used to correct the site ID effect.

Repeat sample merging

The data of 35 samples repeated between the first batch and the second batch of MMY2002 were combined by calculating the average of each protein.

Differential Protein Concentration Analysis responders to non-responders

A statistical comparison of the distribution of protein concentrations in the base phase and in the treatment of DARZALEXTM responders in non-responders was performed using two complementary methods: (i) Wilkason grades and assays ( Hollander and Wolfe, Ninparametirc Statistical Methods. New York: John Wiley & Sons. 1973.27-33 (one sample), 68-75 (two samples), for each SOMAmer, and (ii) Limma analysis (Ritchie, ME, etc.) Human, Nucleic Acids Res. 2015; 20:43(7):e47), for all SOMAmers simultaneously. All p-values use Benjamini-Hochberg (BH) for multi-hypothesis corrections. Method adjustments (Benjamini and Hochberg, (1995) JRStatist.Soc. B.57: 289-300; R: A Language and Environment for Statistical Computing, R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria.2011 ;ISBN 3-900051-07-0). When the adjusted p-value <0.05, reject the zeroing hypothesis of indifference performance.

The base period of treatment

Three alternative statistical methods were used to compare base protein levels to therapeutic protein levels: (i) bidirectional repeated measures ANOVA6, (ii) Wilcardson symbol level assay, and (iii) Friedman test ( Johnson et al. (2007) Biostatistics 8(1): 118-127). All p-values were adjusted to control FDR using the BH method for multiple hypothesis corrections (Benjamini and Hochberg, JRStatist. Soc. B. 57: 289-300, 1995). In addition to treatment significance, two-way repeated measures ANOVA (Chambers et al, Analysis of variance; designed experiments: Chapter 5. Statistical Models in S, Editors JM Chambers and TJ Hastie. Wadsworth & Brookes/Cole. 1992) are also used to determine each SOMAmer Whether a significant point in time occurs: reaction level interaction. The modified Wilburson ratings and assays are used as post-test applications to clearly determine whether responders and non-responders exhibit different therapeutic effects by calculating the protein concentration value and the base protein concentration value for each subject. The difference and the Will Carson level and verification. The significance value was adjusted using the BH method, and the zero return hypothesis was rejected when the adjusted p-value was <0.05.

Grading system training

Baseline protein level MMY2002 data was used to establish a reaction prediction grading system. The 10-fold cross-validation approach is repeated 30 times using four different machine learners: Support Vector Machine (SVM), Random Forest (Random Forest) , RF), Naïve Baye (NB), and j48 decision tree. For each learner, the training program begins with a 10 fold of the balance of the data set (outer loop). One of these folds is provided as a test subgroup, while the remaining nine are passed to the inner loop as a training subgroup. Within the inner loop, the training subgroup is again divided into balanced 10 folds, which produce a training inner set and a test inner set. For this Each of the training episodes performs learner training and repeats the procedure 30 times for each subgroup within the outer loop. The accuracy of each inner loop learner in predicting the test set is used to select features and optimize model parameters. Once the 30 (30x) internal cycles of each training group are completed, an evaluation of the performance of the outer loop (using optimized parameters and features) is performed for each corresponding test group. The entire outer loop procedure was then repeated 30 times to generate 30 reaction predictions for each sample in the data set. Approximate values for the performance of new test cases from the final model of the AUC, Sensitivity, and Specificity Statistics obtained from this cycle method (trained in the complete raw data set).

Results of the MMY2002 study

Various comparisons were made, including treatment-induced response-dependent changes in protein expression. One of the proteins showing a decrease in performance over time in the responder was PD-L1; however, in the non-responder, the PD-L1 protein expression increased over time. The engagement of PD-L1 on T cells causes a decrease in T cell function and an increase in Treg development. Figure 18 shows the protein expression profiles of PD-L1 in responders, non-responders, and patients with stable disease in the first and third cycles.

The binding of PD-L1 to its receptor PD-1 inhibits the anti-tumor response and drives T cells to be non-reactive and depleted. Although not wishing to be bound by any particular theory, a down-regulation of PD-L1 may also result in an improvement in the enhanced anti-tumor immune response of solid tumors following CD38 treatment.

<110> Janssen Biotech, Inc. He Ahmadi, Tahamtan, Casneuf, Tineke, Lokhorst, Henk Silk (Mutis, Tuna) Amisa (Sasser, Amy)

<120> Immunomodulation and treatment of solid tumors using antibodies that specifically bind to CD38

<130> JBI5067ARNP

<141> along with this article

<140> To be assigned

<150> 15/191808

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<151> 2016-06-24

<150> 62/331489

<151> 2016-05-04

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<151> 2015-12-04

<150> 62/250566

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Claims (83)

A method of treating a patient having a solid tumor comprising administering to the patient in need thereof a therapeutically effective amount of an antibody that specifically binds to CD38 for a period of time sufficient to treat the solid tumor. The method of claim 1, wherein the antibody that specifically binds to CD38 elicits an immune response in the patient. The method of claim 2, wherein the immune response is an effector T cell (Teff) reaction. The method of claim 3, wherein the Teff reaction is by CD4 ⁺ T cells or CD8 ⁺ T cells. The method of claim 4, wherein the Teff reaction is mediated by the CD8 ⁺ T cells. The method of claim 3, wherein the Teff reaction is an increase in the number of CD8 ⁺ T cells, an increase in CD8 ⁺ T cell proliferation, an increase in T cell pure line expansion, an increase in CD8 ⁺ memory cell formation, and an antigen dependence. Increased production of sex antibodies, increased production of interleukins, increased production of chemokines, or increased production of interleukins. (by these cells) The method of claim 1, wherein the antibody that specifically binds to CD38 inhibits the function of the immunosuppressive cell. The method of claim 7, wherein the immunosuppressive cell line modulates T cells (Tregs). The method of claim 8, wherein the Tregs are CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim T cells. The method of claim 9, wherein the Treg exhibits CD38. The method of claim 10, wherein the function of the Tregs is inhibited by killing the Tregs. The method of claim 11, wherein the killing of the Treg is mediated by antibody-dependent cellular cytotoxicity (ADCC). The method of claim 7, wherein the immunosuppressive cell line is a bone marrow-derived suppressor cell (MDSC). The method of claim 13, wherein the MDSC is a CD11b ⁺ HLADR ^- CD14 ^- CD33 ⁺ CD15 ⁺ cell. The method of claim 14, wherein the CD11b ⁺ HLADR ^- CD14 ^- CD33 ⁺ CD15 ⁺ cells exhibit CD38. The method of claim 15, wherein the function of the MDSC is suppressed by killing the MDSC. The method of claim 16, wherein the killing of the MDSC is by the ADCC. The method of claim 7, wherein the immunosuppressive cell line modulates B cells (Breg). The method of claim 18, wherein the Breg is a CD19 ⁺ CD24 ⁺ CD38 ⁺ cell. The method of claim 19, wherein the Breg function is inhibited by killing the Breg. The method of claim 20, wherein killing the Breg is by means of ADCC. The method of claim 7, wherein the immunosuppressive cells are present in the bone marrow or in peripheral blood. The method according to claim 1, wherein the solid tumor is melanoma, lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, colorectal cancer, prostate cancer, castration-resistant prostate cancer, gastric cancer (stomach) Cancer), ovarian cancer, gastric cancer, liver cancer, pancreatic cancer, thyroid cancer, squamous cell carcinoma of the head and neck, esophageal or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urinary tract cancer, urogenital cancer , endometriosis, cervical cancer, or metastatic disease of the cancer. The method of claim 23, wherein the solid tumor lacks detectable CD38 expression. The method of claim 1, wherein the specific binding to CD38 is an anti-systemic non-promoting antibody. The method of claim 25, wherein the non-potentiating antibody induces proliferation of a sample of peripheral blood mononuclear cells in vitro in a statistically insignificant manner. The method of claim 1, wherein the antibody that specifically binds to CD38 and the antibody comprising the heavy chain variable region (VH) of SEQ ID NO: 4 and the light chain variable region (VL) of SEQ ID NO: Competition is combined with CD38. The method of claim 27, wherein the antibody that specifically binds to CD38 binds at least to the region of SKRNIQFSCKNIYR (SEQ ID NO: 2) and EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1). The method of claim 28, wherein the antibody that specifically binds to CD38 comprises a heavy chain complementarity determining region (HCDR) 1, HCDR2, HCDR3 of SEQ ID NOs: 6, 7, 8, 9, 10, and 11, respectively. Light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3 amino acid sequence. The method of claim 29, wherein the antibody that specifically binds to CD38 comprises VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5. The method of claim 23, wherein the antibody that specifically binds to CD38 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: a) VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15. b) VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17; c) VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; or d) VH and SEQ ID of SEQ ID NO: 20. NO: VL of 21 . The method of claim 31, wherein the antibody that specifically binds to CD38 comprises: a) the VH of the SEQ ID NO: 14 and the VL of the SEQ ID NO: 15; b) the VH of the SEQ ID NO: 16 and VL of SEQ ID NO: 17; c) VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; or d) VH of SEQ ID NO: 20 and VL of SEQ ID NO: 21. The method of claim 1, wherein the anti-system that specifically binds to CD38 is administered in combination with a second therapeutic agent. The method of claim 33, wherein the second therapeutic agent is a chemotherapeutic agent, a target anticancer therapy, a standard care drug for treating solid tumors, or an immunological checkpoint inhibitor. The method of claim 34, wherein the immunological checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-LAG3 antibody, an anti-TIM3 antibody, or an anti-CTLA-4 antibody. The method of claim 35, wherein the immunological checkpoint inhibitor is an anti-PD-1 antibody. The method of claim 36, wherein the anti-PD-1 antibody comprises a) VH of SEQ ID NO: 22 and VL of SEQ ID NO: 23; b) VH of SEQ ID NO: 24 and SEQ ID NO: VL; c) VL of SEQ ID NO: 32 and VL of SEQ ID NO: 33; or d) VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35. The method of claim 35, wherein the immunological checkpoint inhibitor is an anti-PD-L1 antibody. The method of claim 38, wherein the anti-PD-L1 antibody comprises a) VH of SEQ ID NO: 26 and VL of SEQ ID NO: 27; b) VH of SEQ ID NO: 28 and SEQ ID NO: 29 VL; or c) VH of SEQ ID NO: 30 and VL of SEQ ID NO: 31. The method of claim 35, wherein the immunological checkpoint inhibitor is an anti-PD-L2 antibody. The method of claim 35, wherein the immunological checkpoint inhibitor is an anti-LAG3 antibody. The method of claim 35, wherein the immunological checkpoint inhibitor is an anti-TIM-3 antibody. The method of claim 42, wherein the anti-TIM-3 antibody comprises a) VH of SEQ ID NO: 36 and VL of SEQ ID NO: 37; or b) VH of SEQ ID NO: 38 and SEQ ID NO: 39 VL. The method of claim 33, wherein the second therapeutic agent is administered simultaneously, sequentially, or separately. The method of claim 1, wherein the anti-system that specifically binds to CD38 is administered intravenously. The method of claim 1, wherein the anti-CD38-specific anti-system is administered subcutaneously in a pharmaceutical composition comprising the antibody that specifically binds to CD38 and hyaluronan. The method of claim 46, wherein the hyaluronanase is rHuPH20 of SEQ ID NO:40. The method of claim 1, wherein the patient is treated with radiation therapy or has been treated with radiation therapy. The method of claim 1, wherein the patient has undergone surgery or will undergo surgery. A method of inhibiting the activity of an immunosuppressive cell comprising contacting the immunosuppressive cell with an antibody that specifically binds to CD38. The method of claim 50, wherein the immunosuppressive cell line is Treg. The method of claim 51, wherein the Tregs are CD3 ⁺ CD4 ⁺ CD25 ⁺ CD127 ^dim T cells. The method of claim 50, wherein the immunosuppressive cell line is MDSC. The method of claim 53, wherein the MDSC is a CD11b ⁺ HLADR ^- CD14 ^- CD33 ⁺ CD15 ⁺ cell. The method of claim 50, wherein the immunosuppressive cell line is Breg. The method of claim 55, wherein the Breg is a CD19 ⁺ CD24 ⁺ CD38 ⁺ cell. The method of claim 50, wherein the specific binding to CD38 is an anti-systemic non-promoting antibody. The method of claim 57, wherein the non-potentiating antibody induces proliferation of a sample of peripheral blood mononuclear cells in vitro in a statistically insignificant manner. The method of claim 58, wherein the antibody that specifically binds to CD38 competes for binding to CD38 with an antibody comprising VL of SEQ ID NO: 4 and VL of SEQ ID NO: 5. The method of claim 59, wherein the antibody that specifically binds to CD38 binds at least to the region of SKRNIQFSCKNIYR (SEQ ID NO: 2) and EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1). The method of claim 60, wherein the antibody that specifically binds to CD38 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amines of SEQ ID NOS: 6, 7, 8, 9, 10, and 11, respectively. Base acid sequence. The method of claim 61, wherein the antibody that specifically binds to CD38 comprises VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5. The method of claim 50, wherein the antibody that specifically binds to CD38 comprises the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: a) VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15. b) VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17; c) VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; or d) VH and SEQ ID of SEQ ID NO: 20. NO: VL of 21 . The method of claim 63, wherein the antibody that specifically binds to CD38 comprises: a) the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15; b) the VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17; c) VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; or d) VH of SEQ ID NO: 20 and VL of SEQ ID NO: 21. A method of enhancing an immune response in a patient comprising administering to the patient an antibody that specifically binds to CD38. The method of claim 65, wherein the patient has a cancer or a viral infection. The method of claim 65, wherein the specific binding to CD38 is an anti-systemic non-promoting antibody. The method of claim 67, wherein the non-potentiating antibody induces proliferation of a sample of peripheral blood mononuclear cells in vitro in a statistically insignificant manner. The method of claim 68, wherein the antibody that specifically binds to CD38 competes for binding to CD38 with an antibody comprising VL of SEQ ID NO: 4 and VL of SEQ ID NO: 5. The method of claim 69, wherein the antibody that specifically binds to CD38 binds at least to the region of SKRNIQFSCIYR (SEQ ID NO: 2) and EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1). The method of claim 70, wherein the antibody that specifically binds to CD38 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amines of SEQ ID NOS: 6, 7, 8, 9, 10, and 11, respectively. Base acid sequence. The method of claim 71, wherein the antibody that specifically binds to CD38 comprises VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5. The method of claim 65, wherein the antibody that specifically binds to CD38 comprises the following HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: a) VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15. b) VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17; c) VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; or d) VH and SEQ ID of SEQ ID NO: 20. NO: VL of 21 . The method of claim 73, wherein the antibody that specifically binds to CD38 comprises: a) the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15; b) the VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17; c) VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; or d) VH of SEQ ID NO: 20 and VL of SEQ ID NO: 21. A method of treating a patient having a viral infection comprising administering to the patient an antibody that specifically binds to CD38 for a time sufficient to treat the viral infection. The method of claim 75, wherein the specific binding to CD38 is an anti-systemic non-promoting antibody. The method of claim 76, wherein the non-potentiating antibody induces proliferation of a sample of peripheral blood mononuclear cells in vitro in a statistically insignificant manner. The method of claim 77, wherein the antibody that specifically binds to CD38 competes for binding to CD38 with an antibody comprising VL of SEQ ID NO: 4 and VL of SEQ ID NO: 5. The method of claim 78, wherein the antibody that specifically binds to CD38 binds at least to the region of SKRNIQFSCIYR (SEQ ID NO: 2) and EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1). The method of claim 79, wherein the antibody that specifically binds to CD38 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 amines of SEQ ID NOS: 6, 7, 8, 9, 10, and 11, respectively. Base acid sequence. The method of claim 80, wherein the antibody that specifically binds to CD38 comprises VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5. The method of claim 75, wherein the antibody that specifically binds to CD38 comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: a) VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15. b) VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17; c) VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; or d) VH and SEQ ID of SEQ ID NO: 20. NO: VL of 21 . The method of claim 82, wherein the antibody that specifically binds to CD38 comprises: a) the VH of SEQ ID NO: 14 and the VL of SEQ ID NO: 15; b) the VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17; c) VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19; or d) VH of SEQ ID NO: 20 and VL of SEQ ID NO: 21.