Immunomodulation and treatment of solid tumors with antibodies that specifically bind cd38

Abstract

This invention relates to a method for immunomodulation and treatment of patients with solid tumors using antibodies that specifically bind to CD38.

Description

[0001] This application is a divisional application of the invention application filed on June 24, 2016, with Chinese national application number 201680048975.9, entitled "Immunomodulation and Treatment of Solid Tumors with Antibodies that Specifically Bind CD38". Technical Field

[0002] This invention relates to a method for immunomodulation and treatment of solid tumors using antibodies that specifically bind to CD38. Background Technology

[0003] The immune system is tightly controlled by a network of co-stimulatory and co-inhibitory ligands and receptors. These molecules provide secondary signals for T cell activation and a balanced network of positive and negative signals to maximize immune responses against infection and tumors while limiting self-immunity (Wang et al., (March 7, 2011, e-paper)). J Exp Med 208(3):577-92; Lepenies et al., (2008) Endocr MetabImmune Disord Drug Targets 8:279-288).

[0004] With the anti-CTLA-4 and anti-PD-1 antibody YERVOY ® (ipilimumab), KEYTRUDA ® (pembrolizumab) and OPDIVO ® The approval of nivolumab, along with immune checkpoint therapies targeting synergistic inhibitory pathways in T cells to enhance anti-tumor immune responses in the treatment of solid tumors, has advanced clinical care for cancer patients. While anti-PD-1 / PD-L1 antibodies have demonstrated encouraging clinical responses in patients with multiple solid tumors, response rates remain considerably low, at approximately 15% to 20% in pre-treated patients (Swaika et al., (2015)). Mol Immunol doi:10.1016 / j.molimm.2015.02.009).

[0005] While natural killer (NK) cells, dendritic cells (DCs), and effector T cells can drive effective antitumor responses, tumor cells often induce an immunosuppressive microenvironment that favors the development of immunosuppressive populations of immune cells, such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), or regulatory B cells (Bregs). These immunosuppressive populations are associated with tumor immune tolerance and immunotherapy regimen failure in cancer patients and experimental tumor models.

[0006] Therefore, there is still a need to develop new cancer immunotherapies that induce adaptive immune responses against tumors or target immunosuppressive immune cells. Summary of the Invention

[0007] The present invention provides a method for treating a patient with a solid tumor, the method comprising administering a therapeutically effective amount of a CD38-specific antibody to the patient in need.

[0008] The present invention also provides a method for treating a patient with a regulatory T cell (Treg)-mediated disease, the method comprising administering a therapeutically effective amount of a CD38-specifically binding antibody to the patient in need.

[0009] The present invention also provides a method for treating a patient with a bone marrow-derived suppressor cell (MDSC) mediated disease, the method comprising administering a therapeutically effective amount of a CD38-specifically binding antibody to a patient in need.

[0010] The present invention also provides a method for treating a patient with a regulatory B-cell (Breg)-mediated disease, the method comprising administering a therapeutically effective amount of a CD38-specific antibody to the patient in need.

[0011] The present invention also provides a method for inhibiting the activity of regulatory T cells (Tregs), the method comprising contacting Tregs with an antibody that specifically binds to CD38.

[0012] The present invention also provides a method for inhibiting the activity of bone marrow-derived suppressor cells (MDSCs), the method comprising contacting MDSCs with an antibody that specifically binds to CD38.

[0013] The present invention also provides a method for inhibiting the activity of regulatory B cells (Breg), the method comprising contacting Breg with an antibody that specifically binds to CD38.

[0014] The present invention also provides a method for enhancing the immune response in a patient, the method comprising administering to the patient an antibody that specifically binds to CD38.

[0015] The present invention also provides a method for treating a patient with a solid tumor, the method comprising reducing the number of Treg cells in the patient by administering an antibody that specifically binds to CD38.

[0016] The present invention also provides a method for treating a patient with a solid tumor, the method comprising reducing the number of bone marrow-derived suppressor cells (MDSCs) in the patient by administering an antibody that specifically binds to CD38.

[0017] The present invention also provides a method for inhibiting the activity of immunosuppressive cells, the method comprising contacting immunosuppressive cells with an antibody that specifically binds to CD38.

[0018] The present invention also provides a method for treating a patient suffering from a viral infection, the method comprising administering an antibody that specifically binds to CD38 to a patient in need of such treatment. Attached Figure Description

[0019] Figure 1 DARZALEX is shown to respond to doses of 8 mg / kg (upper limit) or 16 mg / kg (lower limit). ™ (Daralimumab) treatment resulted in a median increase in lymphocytes over time, which returned to baseline at the end of treatment. Study: SIRIUS. X-axis indicates time, representing treatment cycles and the number of days of administration within each cycle (C1D1: Day 1 of Cycle 1; C1D4: Day 4 of Cycle 1, etc.). SCR: Baseline; EOT: End of Treatment; WK: Week; POST-WK: Post-treatment at the specified week; post-PD FU: Follow-up after progression. Areas highlighted in gray indicate the 25th to 27th percentile (IQR) interval of data points at each visit for responders.

[0020] Figure 2 The results for each individual patient, using DARZALEX, are shown. ™ Patients treated with daratumumab had CD3 levels in their peripheral blood. + The percentage change (%) of absolute T cell count relative to baseline (light gray line). Study: SIRIUS (MMY2002). The X-axis indicates time, expressed as treatment cycles and the number of days of administration within each treatment cycle (C1D1: Day 1 of Cycle 1; C1D4: Day 4 of Cycle 1, etc.). WK: Week; POST-WK: Post-treatment at the specified week; POST-PD FU: Follow-up after progression. The black line shows the median % change for all patients.

[0021] Figure 3 The results for each individual patient, using DARZALEX, are shown. ™ Patients treated with daratumumab had CD4 levels in their peripheral blood. + The percentage change (%) of the absolute T cell count relative to baseline (light gray line). Study: SIRIUS. The X-axis indicates time, expressed as treatment cycles and the number of days of administration within each treatment cycle (C1D1: Day 1 of Cycle 1; C1D4: Day 4 of Cycle 1, etc.). WK: Week; POST-TMT: Post-treatment. The black line shows the median % change for all patients.

[0022] Figure 4 The results for each individual patient were shown using DARZALEX. ™ Patients treated with daratumumab had CD8 levels in their peripheral blood. +The percentage change (%) of absolute T cell count relative to baseline (light gray line). Study: SIRIUS. The X-axis indicates time, expressed as treatment cycles and the number of days of administration within each treatment cycle (C1D1: Day 1 of Cycle 1; C1D4: Day 4 of Cycle 1, etc.). WK: Weeks; Pre-PD FU: Follow-up before progression; Post-PD FU: Follow-up after progression. The black line shows the median % change for all patients.

[0023] Figure 5 DARZALEX at doses of 8 mg / kg or 16 mg / kg is shown. ™ During daratumumab treatment, CD45 in bone marrow aspirate + CD3 + Cell count (measured as a percentage of lymphocytes) increases over time. As shown in the figure, this graph includes both responders and non-responders. Study: SIRIUS. The X-axis indicates time, representing the treatment cycle and the number of days of administration within each treatment cycle (C2D22: day 22 of cycle 2, etc.). SCR: Baseline; Post-PD FU1: Follow-up after progression. Areas highlighted in gray indicate the 25th to 27th percentile (IQR) intervals of data points at each visit for non-responders at 8 mg / kg, responders at 16 mg / kg, or non-responders at 16 mg / kg. NR: Non-responders; R: Responders.

[0024] Figure 6 DARZALEX at doses of 8 mg / kg or 16 mg / kg is shown. ™ During daratumumab treatment, CD45 in bone marrow aspirate + CD3 + CD8 + Cell count (measured as a percentage of lymphocytes) increases over time. As shown in the figure, this graph includes both responders and non-responders. Study: SIRIUS. The X-axis indicates time, representing the treatment cycle and the number of days of administration within each treatment cycle (C2D22: day 22 of cycle 2, etc.). SCR: Baseline; Post-PD FU1: Follow-up after progression. Areas highlighted in gray indicate the 25th to 27th percentile (IQR) intervals of data points at each visit for non-responders at 8 mg / kg, responders at 16 mg / kg, or non-responders at 16 mg / kg. NR: Non-responders; R: Responders.

[0025] Figure 7A Shown in DARZALEX ™Peripheral blood CD8 levels during daratumumab treatment, expressed as median across all treated patients. + / Treg and CD8 + / CD4 + The cell ratio increased over time. Time points: C1D1: Day 1 of cycle 1; C3D1: Day 1 of cycle 3; C4D1: Day 1 of cycle 4. Study: SIRIUS. SRC: Baseline.

[0026] Figure 7B Shown in DARZALEX ™ CD8+ levels in bone marrow aspirates during daratumumab treatment, expressed as median across all treated patients. + The ratio of / Treg cells increased over time. Time points: C1D1: Day 1 of cycle 1; C3D1: Day 1 of cycle 3; C4D1: Day 1 of cycle 4. Study: SIRIUS.

[0027] Figure 8A The figure shows the CD8 abundance change % (CIA) in responders compared to non-responders, as measured using specific clonal cell abundance change % (CIA). + Increased T-cell clonality. Study: GEN501, a subgroup of 17 patients.

[0028] Figure 8B Shown in DARZALEX ™ CD8 counts in individual patients before and after daratumumab treatment + Fold change in T-cell clonality. Respondents are indicated by an asterisk. Clonality is measured as the fold change (CIA) of the abundance of a specific clone. Study: GEN501, 17 patient subgroup.

[0029] Figure 8C The study showed that responders (group A) had greater total amplification in the TCR library, measured using CIA (abundance variation), compared to non-responders (group B). P = 0.037. Study: GEN501, 17 patient subgroups.

[0030] Figure 8D The sum of absolute changes (CIA) in the abundance of each expanded T cell clone is shown in responders and non-responders. P = 0.035 between responders (Group A) and non-responders (Group B). Study: GEN501, 17 patient subgroups.

[0031] Figure 8E The maximum CIA of a single T-cell clone is shown in responders (Group A) and non-responders (Group B). Study: GEN501, 17 patient subgroups.

[0032] Figure 8FThe study showed that responders (group A) had larger single-clonal amplifications compared to non-responders (group B), measured using maximum %CIA. P = 0.0477. Study: GEN501, 17 patient subgroups.

[0033] Figure 9A The results show the effects of DARZALEX at baseline, or at week 2, week 4, or week 8 of treatment, or after relapse. ™ CD8+ levels in peripheral blood of non-responders (NR, black square) and patients with at least minimal response (MR, white square) of daratumumab + Percentage of initial cells (%). Study: GEN501, 17 patient subgroup. **p=1.82×10 -4 .

[0034] Figure 9B The results show the effects of DARZALEX at baseline, or at week 2, week 4, or week 8 of treatment, or after relapse. ™ CD8+ levels in peripheral blood of non-responders (NR, black square) and patients with at least minimal response (MR, white square) of daratumumab + Percentage of central memory cells (Tem). Study: GEN501, 17 patient subgroup. *p=4.88×10 -2 .

[0035] Figure 9C The results show HLA class I-restricted CD8 levels in peripheral blood at baseline, or at week 1, week 4, or week 8 of treatment, or after relapse. + The percentage of T cells increased. Study: GEN501, 17 patient subgroups.

[0036] Figure 9D This shows CD38 levels at baseline or during treatment. + CD8 in peripheral blood + Naïve T cells and CD8 + It is expressed at low levels in central memory cells (Tem). Study: GEN501, 17 patient subgroups. MFI: mean fluorescence intensity.

[0037] Figure 10A Histograms of FACS analysis are shown, illustrating the Treg (CD3) levels in multiple myeloma patients at baseline. + CD3 + CD4 + CD25 + CD127 dim The frequency of ) (top histogram, P4 cell population) and CD38 in the Treg population +Frequency of Treg cells (bottom histogram, P5 cell population). Study: GEN501, subgroup of 17 patients.

[0038] Figure 10B The histogram of FACS analysis is shown, which is displayed in DARZALEX. ™ Treg (CD3) levels in multiple myeloma patients after daratumumab treatment + CD3 + CD4 + CD25 + CD127 dim The frequency of ) (top histogram, P4 cell population) and CD38 in the Treg population + Treg frequency (bottom histogram, P5 cell population)). DARZALEX ™ (Daratumumab) treatment depletes CD38 + Treg. Study: GEN501, 17 patient subgroup.

[0039] Figure 10C The results show the use of DARZALEX at baseline, or at week 1, week 4, week 8 after relapse, or at the end of treatment (EOT) at 6 months. ™ Patients treated with daratumumab had CD38 levels in their bodies. high CD3 + CD4 + CD25 + CD127 dim Treg frequency. CD38 high Treg frequency varies with DARZALEX ™ (Daralimumab) treatment reduced the size of the neck and restored it to baseline at EOT. Y-axis: derived from CD3 + T cells CD38 high CD3 + CD4 + CD25 + CD127 dim % of Tregs. Study: GEN501, 17 patient subgroups.

[0040] Figure 10D The results show CD8 levels in responders and non-responders at baseline, week 1, week 4, and week 8 of treatment. + / Treg cell ratio. At week 8 of treatment, CD8+ levels were observed in responders. + The / Treg cell ratio was significantly higher in patients than in non-responders (p=0.00955). Study: GEN501, 17 patient subgroups.

[0041] Figure 10E It shows the connection with CD38− Compared to Treg or negative controls, in the presence of CD38 + In the case of Treg cells, effector cell proliferation was more effectively suppressed. Error bars indicate standard errors. An asterisk represents a significant change. Samples were obtained from multiple healthy donors. Cell proliferation was assessed by diluting carboxyfluorescein succinimide ester (CFSE).

[0042] Figure 11 The image shows bone marrow-derived suppressor cells (MDSCs) present in patients with multiple myeloma (top image, cells in boxes), and approximately half of these cells express CD38 (middle image, cells in boxes). This was observed using DARZALEX. ™ In patients treated with a single infusion of daratumumab, the CD38high MDSC cell population was depleted (bottom image, cells in boxes). Study: GEN501, subgroup of 17 patients.

[0043] Figure 12 This shows the use of DARZALEX ™ After 1, 4, or 8 weeks of treatment with daratumumab, CD38high MDSCs (CD11b) + HLADR - CD14 - CD33 + CD15 + The number of CD38-high MDSCs decreased compared to baseline and returned to near baseline after the end of treatment (EOT). Relapsed patients still showed reduced CD38-high MDSCs. Black squares: non-responders; white squares: against DARZALEX. ™ (Daralimumab) treatment resulted in patients with at least a minimal response. Vertical lines indicate the median for each group. Patients 2, 4, 15, 16, and 17 exhibited high initial CD38high MDSC cell populations. Study: GEN501, subgroup of 17 patients.

[0044] Figure 13 Patients with the highest CD38-high MDSC count (patients 2, 4, 15, 16, and 17) were shown to have the highest progression-free survival (PFS). These patients responded to DARZALEX. ™ (Daralimumab) treatment resulted in either a partial response (PR) or a minimal response (MR). SD: stable disease; PD: progressive disease. The X-axis shows the PFS for each individual patient number.

[0045] Figure 14 The MDSC is shown to be a DARZALEX ™ (Daralimumab)-induced ADCC sensitivity. Daudi cells were used as a positive control for target cells in the assay. Cell lysis percentage was measured.

[0046] Figure 15A The results showed that during weeks 1, 4, and 8 of treatment with DARZALEX... ™ CD38 in patients treated with daratumumab + Breg is depleted.

[0047] Figure 15B CD38 is shown + Breg secretes IL-10 upon stimulation.

[0048] Figure 16A The results show the levels of CMV, EBV, and influenza virus-specific (CEF) IFN-γ at specified time points during baseline and treatment, as indicated by data from DARZALEX. ™ Antiviral response was measured in PBMCs of patients with VGPR treated with daratumumab. OD: Optical density. White bar: Negative control; Black bar: Increased CEF; Dashed line: Allogeneic PBMCs only. Asterix indicates statistically significant changes. Pre 4, 8, 10 = Week 4, Week 8, or Week 10 of treatment.

[0049] Figure 16B The results show the levels of CMV, EBV, and influenza virus-specific (CEF) IFN-γ at specified time points during baseline and treatment, as indicated by data from DARZALEX. ™ Antiviral response was measured by the production of PBMCs in patients with complete remission (CR) treated with daratumumab. OD: Optical density. White bars: negative control; black bars: increased CEF; dashed lines: allogeneic PBMCs only. Asterix indicates statistically significant changes. Pre 4, 8, 10 = week 4, week 8, or week 10 of treatment.

[0050] Figure 16C The results show the levels of CMV, EBV, and influenza virus-specific (CEF) IFN-γ at specified time points during baseline and treatment, as indicated by data from DARZALEX. ™ Antiviral response was measured in PBMCs of patients with PD treated with daratumumab. OD: Optical density. White bar: Negative control; Black bar: Increased CEF; Dashed line: Allogeneic PBMCs only. Ns: Not significant. Pre 4, 8 = Week 4 or Week 8 of treatment.

[0051] Figure 16D The results show the levels of CMV, EBV, and influenza virus-specific (CEF) IFN-γ at specified time points during baseline and treatment, as indicated by data from DARZALEX. ™Antiviral response was measured in PBMCs of patients with MR treated with daratumumab. OD: Optical density. White bar: Negative control; Black bar: Increased CEF; Dashed line: Allogeneic PBMCs only. Ns: Not significant. Pre 4, 8 = Week 4 or Week 8 of treatment.

[0052] Figure 16E The results from DARZALEX are shown at specified times at baseline and during treatment. ™ Percentage (%) of proliferating viral reactive T cells in PBMCs of patients with VGPR treated with daratumumab. White bars: negative control; black bars: increased CEF. Asterix indicates statistically significant changes. Pre 4, 8, 10 = week 4, week 8, or week 10 of treatment.

[0053] Figure 16F The results from DARZALEX are shown at specified times at baseline and during treatment. ™ Percentage (%) of proliferating viral reactive T cells in PBMCs of patients with complete remission (CR) treated with daratumumab. White bars: negative control; black bars: increased CEF. Asterix indicates statistically significant changes. Pre 4, 8, 10 = week 4, week 8, or week 10 of treatment.

[0054] Figure 17A Histograms of FACS analysis are shown, illustrating CD38 expression levels on natural killer (NK) cells, monocytes, B cells, and T cells from healthy donors.

[0055] Figure 17B Histograms of FACS analysis are shown, illustrating CD38 expression levels on plasma cells, natural killer (NK) cells, monocytes, B cells, and T cells from multiple myeloma patients.

[0056] Figure 17C The mean fluorescence intensity (MFI) of CD38 in CD38+ Treg, Breg, NK, B cells, and T cells from patients with relapsed and refractory multiple myeloma is compared. CD38 expression levels were lower in B cells and T cells compared to CD38+ Treg, Breg, and NK cells.

[0057] Figure 18 The data shows that PD-L1 protein was downregulated over time in PBMC samples from responders (R) and upregulated over time in PBMC samples from non-responders (NR). SD: stable disease. C1D1: Day 1 of cycle 1; C3D1: Day 1 of cycle 3. The Y-axis shows the log2 protein concentration values. Detailed Implementation

[0058] As used in this specification and the appended claims, unless otherwise expressly stated, the singular forms “a,” “an,” and “described” include plural references. Thus, for example, a reference to “a cell” includes a combination of two or more cells, and so on.

[0059] "CD38" refers to the human CD38 protein (synonyms: ADP-ribosylcyclase 1, cADPr hydrolase 1, cyclic ADP-ribosyl hydrolase 1). Human CD38 has the amino acid sequence shown in GenBank accession number NP_001766 and SEQ ID NO: 1. As is well known, CD38 is a single-pass type II membrane protein, containing amino acid residues 1-21 representing the cytoplasmic domain, amino acid residues 22-42 representing the transmembrane domain, and residues 43-300 representing the extracellular domain of CD38.

[0060] SEQ ID NO: 1 mancefspvsgdkpccrlsrraqlclgvsilvlilvvvlavvvprwrqqwsgpgttkrfpetvlarcvkyteihpemrhvdcqsvwdafkgafiskhpcniteedyqplmklgtqtvpcnkillwsrikdlahqftqvqrdmftledtll gyladdltwcgefntskinyqscpdwrkdcsnnpvsvfwktvsrrfaeaacdvvhvmlngsrsrskifdknstfgsvevhnlqpekvqtleawvihggredsrdlcqdptikelesiiskrniqfsckniyrpdkflqcvknpedssctsei As used herein, “antibody” broadly refers to and includes immunoglobulin molecules, specifically including monoclonal antibodies (including murine monoclonal antibodies, human monoclonal antibodies, humanized monoclonal antibodies, and chimeric monoclonal antibodies), antibody fragments, bispecific or multispecific antibodies, dimer, tetramer, or polymeric antibodies, single-chain antibodies, domain antibodies, and any other modified configuration of immunoglobulin molecules containing antigen-binding sites with desired specificity.

[0061] Immunoglobulins can be designated into five major types based on the amino acid sequence of their heavy chain constant domain: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Based on the amino acid sequence of their constant domain, the antibody light chain of any vertebrate species can be designated into one of two completely different types: κ and λ.

[0062] "Antibody fragments" refer to a portion of an immunoglobulin molecule that retains heavy chain antigen-binding sites and / or light chain antigen-binding sites, such as heavy chain complementarity-determining regions (HCDRs) 1, 2, and 3, light chain complementarity-determining regions (LCDRs) 1, 2, and 3, heavy chain variable regions (VH), or light chain variable regions (VL). Antibody fragments include: Fab fragments, which are monovalent fragments composed of VL, VH, CL, and CH1 domains; F(ab)2 fragments, which are bivalent fragments containing two Fab fragments linked by disulfide bonds at the hinge region; Fd fragments, which consist of VH and CH1 domains; Fv fragments, which consist of the VL and VH domains of a single arm of the antibody; and domain antibody (dAb) fragments (Ward et al., Nature 341:544-546, 1989), which consist of a 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, wherein, where the VH and VL domains are expressed by separate single-chain antibody constructs, the VH / VL domains pair intramolecularly or intermolecularly to form monovalent antigen-binding sites, such as single-chain Fv (scFv) or bivalent antibodies; as described, for example, in International Patent Publications WO1998 / 44001, WO1988 / 01649, WO1994 / 13804 and WO1992 / 01047. These antibody fragments are obtained using techniques well known to those skilled in the art, and fragments with the same mode of efficacy as full-length antibodies are screened.

[0063] "Isolated antibodies" refer to antibodies or antibody fragments that are substantially free of other antibodies with different antigen specificities (e.g., isolated antibodies that specifically bind to CD38 are substantially free of antibodies that specifically bind to antigens other than human CD38). However, isolated antibodies that specifically bind to CD38 may exhibit cross-reactivity with other antigens, such as orthologs of human CD38, such as those found in cynomolgus monkeys. Macaca fascicularis(Crab-eating macaque) CD38. In the case of bispecific antibodies, a bispecific antibody specifically binds to two antigens of interest and is substantially free of antibodies that specifically bind to antigens other than the two antigens of interest. Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals. "Isolated antibody" encompasses antibodies isolated to higher purities, such as antibodies with 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% purity.

[0064] "Specific binding," "specifically binding," or simply "binding" refers to an antibody binding to an antigen or an epitope within that antigen with a higher affinity than it has for other antigens. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -8 M or smaller (e.g., about 1×10⁻⁶) -9 M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller, or approximately 1×10 -12 The equilibrium dissociation constant (K) of M or less D ) binds to the antigen or an epitope within the antigen, usually this K D This antibody binds to the K+ of non-specific antigens (such as BSA, casein). D At least one percent. The dissociation constant can be measured using standard procedures. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to antigens from other species (homologous) (such as humans or monkeys, e.g., cynomolgus monkeys). Macaca fascicularis (cynomolgus, cyno), chimpanzee ( Pan troglodytes (chimpanzee, chimp) or marmoset ( Callithrix jacchus (Common marmoset, marmoset) The same antigens exhibit cross-reactivity. While monospecific antibodies specifically bind to one antigen or one epitope, bispecific antibodies specifically bind to two different antigens or two different epitopes.

[0065] The antibody variable region consists of a "framework" region interrupted by three "antigen-binding sites". Different terms are used to define antigen-binding sites: three complementarity-determining regions (CDRs) in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat, (1970)). J Exp Med132:211-50; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991); The "hypervariant region," "HVR," or "HV" refers to a structurally hypervariable region within the variable domain of an antibody, as defined by Chothia and Lesk (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 "Specific Determining Residue Use" (SDRU) (Almagro, (2004) Mol Recognit 17:132-43). The International Immunogenetics (IMGT) database (http: / / www_imgt_org) provides standardized numbers and definitions for antigen-binding sites. The correspondence between CDR, HV, and IMGT descriptions is described in Lefranc et al., (2003). Dev Comparat Immunol It is described in 27:55-77.

[0066] As used in this article, “Chothia residue” refers to the antibody VL and VH residues numbered according to Al-Lazikani (Al-Lazikani et al., (1997)). J Mol Biol 273:927-48).

[0067] A "frame" or "frame sequence" is the remaining sequence of the variable region other than those sequences defined as antigen-binding sites. Because antigen-binding sites can be defined by various terms as described above, the precise amino acid sequence of a frame depends on how the antigen-binding site is defined.

[0068] "Humanized antibodies" are antibodies whose antigen-binding sites are derived from non-human species and whose variable region framework is derived from human immunoglobulin sequences. Humanized antibodies may contain substitutions in the framework region, such that the framework may not be an exact copy of the expressed human immunoglobulin sequence or germline gene sequence.

[0069] "Human antibody" refers to an antibody having both a heavy chain variable region and a light chain variable region, wherein both the framework and the antigen-binding site are derived from human-origin sequences. If the antibody contains a constant region, that constant region is also derived from a human-origin sequence.

[0070] Human antibodies contain heavy-chain or light-chain variable regions of sequences “derived” from human origin, wherein the variable regions of the antibody are derived from systems using human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include libraries of human immunoglobulin genes displayed on bacteriophages, and transgenic nonhuman animals, such as mice carrying human immunoglobulin gene loci. When compared to human germline immunoglobulin genes or rearranged immunoglobulin genes, human antibodies may contain amino acid differences due to, for example, naturally occurring somatic mutations or intentionally introduced substitutions in the framework or antigen-binding site, or both. Typically, the amino acid sequence of a “human antibody” has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence encoded by human germline immunoglobulin genes or rearranged immunoglobulin genes. In some cases, "human antibodies" may contain a common frame sequence obtained from human frame sequence analysis (e.g., Knappik et al., (2000)). J Mol Biol (as described in 296:57-86); or incorporated into synthetic HCDR3 in human immunoglobulin gene libraries displayed on bacteriophages (e.g., Shi et al., (2010)). J Mol Biol (As described in 397:385-96 and International Patent Publication WO2009 / 085462).

[0071] Human antibodies derived from human immunoglobulin sequences can be generated using systems such as phage display to bind synthetic CDRs and / or synthetic frameworks, or they can be mutagenized in vitro to improve antibody properties, thereby obtaining antibodies that are not naturally present in the whole human antibody lineage in vivo.

[0072] The definition of "human antibody" does not include antibodies whose antigen-binding sites are derived from non-human species.

[0073] "Recombinant antibodies" include all antibodies prepared, expressed, created, or isolated by recombinant methods, such as antibodies isolated from animals (e.g., mice or rats), i.e., transgenic or transchromosomal human immunoglobulin gene sequences, or antibodies isolated from hybridomas prepared therefrom (described further below); antibodies isolated from host cells transformed to express antibodies; antibodies isolated from recombinant combined antibody libraries; and antibodies prepared, expressed, created, or isolated by any other method involving splicing human immunoglobulin gene sequences with other DNA sequences, or antibodies generated in vitro using Fab arm exchange, such as bispecific antibodies.

[0074] "Monoclonal antibody" refers to a formulation of an antibody molecule consisting of a single molecule. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope, or, in the case of bispecific monoclonal antibodies, dual binding specificity for two different epitopes. Therefore, "monoclonal antibody" refers to a group of antibodies having a single amino acid composition in each heavy chain and each light chain, except for possible well-known alterations, such as the removal of the C-terminal lysine from the antibody heavy chain. Monoclonal antibodies may have heterogeneous glycosylation within the antibody group. Monoclonal antibodies can be monospecific or multispecific, or monovalent, bivalent, or multivalent. The term "monoclonal antibody" includes bispecific antibodies.

[0075] An epitope refers to the portion of an antigen that specifically binds to an antibody. Epitopes typically consist of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groups, such as amino acids or polysaccharide side chains, and may possess specific three-dimensional structural features and specific charge characteristics. Epitopes can be composed of continuous and / or discontinuous amino acids forming conformational spatial units. For discontinuous epitopes, amino acids from different portions of the linear sequence of the antigen are close together in three-dimensional space due to the folding of the protein molecule.

[0076] A “variant” is a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide due to one or more modifications (e.g., substitution, insertion, or deletion).

[0077] "In combination with" means to administer two or more therapeutic agents together, as a single agent simultaneously, or as a single agent sequentially in any order to the patient. Generally, each agent will be administered according to a dosage and / or time schedule determined for that agent.

[0078] "Treatment" or "treatment" refers to therapeutic agents used to slow (relieve) undesirable physiological changes or diseases, such as the development or spread of tumors or tumor cells, or to provide beneficial or desired clinical outcomes during treatment. Beneficial or desired clinical outcomes include symptom relief, disease severity reduction, disease stability (i.e., no worsening), delayed or slowed disease progression, no metastasis, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean extended survival compared to the expected survival of the treated individual without treatment. Those who require treatment include those who already have undesirable physiological changes or diseases, and those who are predisposed to developing such changes or diseases.

[0079] "Therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and time period. Therapeutic effective dose can vary depending on factors such as an individual's disease state, age, sex, and weight, as well as the ability of the therapeutic agent or combination of agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of agents include, for example, improvements in the patient's overall health, reduction in tumor burden, inhibition or slowing of tumor growth, and / or the absence of metastasis of cancer cells to other parts of the body.

[0080] "Growth inhibition" (e.g., involving tumor cells) refers to a measurable decrease or delay in the growth of tumor cells or tumor tissue in vitro or in vivo upon exposure to a therapeutic agent or combination of therapeutic agents, compared to a decrease or delay in the growth of the same tumor cells or tumor tissue in the absence of the therapeutic agent or combination of therapeutic agents. Inhibition of tumor cell or tumor tissue growth in vitro or in vivo may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.

[0081] "Regulatory T cells," or "Tregs," or simply "Tregs" refer to T lymphocytes that regulate the activity of other T cells and / or other immune cells (usually by inhibiting their activity). Tregs can be CD3+ cells. + CD4 + CD25 + CD127 dim T cells. It should be understood that Tregs are not entirely limited to this phenotype and can also express Foxp3.

[0082] "Effective T cells," or "Teffs," or "Teff" refer to T lymphocytes that perform immune responses, such as killing tumor cells and / or activating anti-tumor immune responses, to eliminate tumor cells from the body. Teffs can be CD3+. + and CD4 + or CD8 + Teff can secrete, contain, or express markers such as IFN-γ, granzyme B, and ICOS. It should be understood that Teff is not entirely limited to these phenotypes.

[0083] "Treg function" or "Treg role" refers to the inhibitory function of Tregs involved in regulating the host immune response and / or preventing autoimmunity. The function of Tregs can include inhibiting the activity of CD8+ receptors. + Antitumor responses induced by T cells, natural killer (NK) cells, MØ cells, B cells, or dendritic cells (DCs), or suppression of effector T cell proliferation.

[0084] "Inhibition of Treg function" or "inhibition of Treg function" refers to a reduction in the level of Treg function in an animal or human subject, which can be determined by conventional techniques known in the art, either in vitro or in vivo. The level of Treg function may be reduced, for example, by at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%. "Inhibition of Treg function" includes reducing the number of Tregs, for example, by killing Tregs via antibody effector functions such as antibody-dependent cell cytotoxicity (ADCC).

[0085] "Myeloid-derived suppressor cells" or "MDSCs" refer to a specialized population of cells derived from hematopoietic lineages that express the macrophage / monocyte marker CD11b and the granulocyte marker Gr-1 / Ly-6G. The phenotype of MDSCs can be, for example, CD11b... + HLA-DR - CD14 - CD33 + CD15 + MDSCs express low levels or are undetectable expression of mature MHC class II antigen-presenting cell markers and F480. MDSCs are immature cells of the bone marrow lineage and can further differentiate into several cell types, including macrophages, neutrophils, dendritic cells, monocytes, or granulocytes. MDSCs are naturally present in normal adult bone marrow of humans and animals or in conventional hematopoietic sites such as the spleen.

[0086] "Inhibition of MDSC function" or "inhibition of MDSC function" refers to a reduction in the level of MDSC function in vitro or in vivo in an animal or human subject, which can be determined by conventional techniques known in the art. The level of MDSC function may be reduced, for example, by at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%. "Inhibition of MDSC function" includes reducing the number of MDSCs, for example, by killing MDSCs through antibody effector functions such as ADCC. MDSCs can inhibit T cell responses such as proliferation, clonal expansion, or cytokine production through various mechanisms, such as the production of reactive oxygen species, nitrite peroxide, increased arginase metabolism due to high levels of arginase, and increased nitrous oxide synthase. MDSCs can respond to IFN-γ and several cytokines, such as IL-4 and IL-13. IFN-γ can activate MDSCs that induce the activity of nitric oxide synthase 2 (NOS2). Alternatively, Th2 cytokines such as interleukin-4 (IL-4) and IL-13 can activate MDSCs that induce arginase-1 (ARG1) activity. L-arginine metabolism by NOS2 or ARG1 can lead to inhibition of T cell proliferation, and the activity of these two enzymes together leads to T cell apoptosis by producing reactive nitric oxide substances.

[0087] "Treg-related diseases" refer to diseases or disorders associated with T regulatory cells (Tregs). Treg-related diseases can be caused by Treg function, such as suppression of antitumor responses or inhibition of effector T cell proliferation. Treg-mediated diseases can be cancer. "Treg-related diseases" and "Treg-mediated diseases" are used interchangeably in this article.

[0088] "Enhanced effector T cell response" or "enhanced T cell response" refers to the enhancement or stimulation of effector T cells in vivo or in vitro in animal or human subjects to achieve sustained or amplified biological function or to renew or reactivate depleted or inactivated T cells. An exemplary T cell response is CD8. + T cell proliferation, secretion of interferon-gamma, antigen responsiveness, or clonal expansion. The methods for measuring this enhancement are known to those skilled in the art.

[0089] “MDSC-related disease” refers to a disease or disorder associated with bone marrow-derived suppressor cells (MDSCs). MDSC-related disease can be caused by the suppression of MDSC function, such as antitumor responses or effector T cell proliferation. MDSC-mediated disease can be cancer. The terms “MDSC-related disease” and “MDSC-mediated disease” are used interchangeably in this document.

[0090] "Regulatory B cells," or "Bregs," refer to B lymphocytes that suppress the immune response. Bregs can include CD19 cells. + CD24 + CD38 + Breg cells, and can suppress the immune response by inhibiting T cell proliferation mediated by IL-10 secreted by Breg cells. It should be understood that other Breg subgroups exist and, for example, Ding et al. (2015) Human Immunology It is described in 76: 615-621.

[0091] "Breg-related disease" refers to diseases or disorders associated with regulatory B cells. Breg-related diseases can be caused by, for example, Breg-mediated antitumor responses or suppression of effector T cell proliferation. Breg-mediated diseases can be cancer. "Breg-related disease" and "Breg-mediated disease" are used interchangeably in this article.

[0092] "Patient" includes any human or non-human animal. "Non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. "Patient" and "recipient of treatment" are used interchangeably in this article.

[0093] This invention provides a method for treating patients with solid tumors using antibodies that specifically bind to CD38, regardless of whether the tumor cells express CD38. This invention also provides a method for treating patients with diseases mediated by regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), or regulatory B cells (Bregs). This invention also provides a method for modulating Treg, MDSC, or Breg activity to treat CD38-positive solid tumors and / or those associated with high levels of these immunosuppressive cells.

[0094] This invention is at least in part based on the discovery of the anti-CD38 antibody DARZALEX. ™ (Daratumumab) reduces the number of immunosuppressive Tregs, MDSCs, and Bregs in patients and increases CD8+. + The number of T cells and CD8 + The ratio with Treg, promoting CD8 + It promotes the formation of central memory cells and enhances the immunomodulatory activity of T cell clonal expansion.

[0095] DARZALEX ™Daremumab and other anti-CD38 antibodies are being clinically evaluated for their ability to eliminate CD38-positive cells (such as ADCC, CDC, ACDP, and apoptosis) through antibody effector functions in the treatment of heme malignancies and plasma cell disorders (including multiple myeloma), 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 suppress anti-tumor responses. For example, anti-PD1 antibodies have been shown to increase T cell proliferation, stimulate antigen-specific memory responses, and partially alleviate Treg-mediated suppression of effector T cells in vitro (e.g., see U.S. Patent 8,779,105). Currently, two anti-PD-1 antibodies (OPDIVO) are being evaluated. ® (nivolumab) and KEYTRUDA ® Pembrolizumab is approved for the treatment of melanoma, and these antibodies are in clinical trials for various solid tumors such as non-small cell lung cancer, prostate cancer, head and neck cancer, stomach cancer, fallopian tube cancer, ovarian cancer, pancreatic cancer, breast cancer, brain cancer, kidney cancer, bladder cancer, urethral cancer, esophageal cancer, and colorectal cancer. Anti-CTLA-4 antibody YERVOY ® (ipilimumab) has been approved for the treatment of melanoma. YERVOY ® (ipilimumab) and another anti-CTLA-4 antibody, tremelimumab, are also being developed for prostate cancer, non-small cell lung cancer, ovarian cancer, gastrointestinal cancer, stomach cancer, colorectal cancer, kidney cancer, esophageal cancer, and genitourinary cancer.

[0096] Not wishing to be bound by any particular theory, based on the DARZALEX described in this article ™ The immunomodulatory effects observed with daratumumab, DARZALEX ™ (Daratumumab) and other anti-CD38 antibodies are effective in treating solid tumors. Due to the use of DARZALEX... ™ Widespread activation of the immune response has been observed in patients treated with daratumumab, and patients with CD38-negative solid tumors may also respond to anti-CD38 antibody therapy.

[0097] The present invention provides a method for treating a patient with a solid tumor, the method comprising administering to the patient in need a therapeutically effective amount of a CD38-specific antibody sufficient to treat the solid tumor for a period of time.

[0098] The present invention also provides a method for treating a patient with a regulatory T cell (Treg)-mediated disease, the method comprising administering to the patient in need a therapeutically effective amount of a CD38-specific antibody sufficient to treat the Treg-mediated disease for a period of time.

[0099] The present invention also provides a method for treating a patient with a bone marrow-derived suppressor cell (MDSC) mediated disease, the method comprising administering to the patient in need a therapeutically effective amount of a CD38-specific antibody sufficient to treat the MDSC-mediated disease for a duration.

[0100] The present invention also provides a method for treating a patient with a regulatory B cell (Breg)-mediated disease, the method comprising administering to the patient in need a therapeutically effective amount of a CD38-specific antibody sufficient to treat the Breg-mediated disease for a duration.

[0101] The present invention also provides a method for inhibiting the activity of regulatory T cells (Tregs), the method comprising contacting the regulatory T cells with an antibody that specifically binds to CD38.

[0102] The present invention also provides a method for inhibiting the activity of bone marrow-derived suppressor cells (MDSCs), the method comprising contacting MDSCs with an antibody that specifically binds to CD38.

[0103] The present invention also provides a method for inhibiting the activity of regulatory B cells (Breg), the method comprising contacting Breg with an antibody that specifically binds to CD38.

[0104] The present invention also provides a method for treating a patient with a solid tumor, the method comprising reducing the number of regulatory T cells (Tregs) in the patient by administering an antibody that specifically binds to CD38.

[0105] The present invention also provides a method for treating a patient with a solid tumor, the method comprising reducing the number of bone marrow-derived suppressor cells (MDSCs) in the patient by administering an antibody that specifically binds to CD38.

[0106] The present invention also provides a method for treating a patient with a solid tumor, the method comprising reducing the number of regulatory B cells (Breg) in the patient by administering an antibody that specifically binds to CD38.

[0107] The present invention also provides a method for enhancing an immune response in a patient, the method comprising administering to a patient in need an antibody that specifically binds to CD38 for a duration sufficient to enhance the immune response.

[0108] In some implementation schemes, the patient has a viral infection.

[0109] The present invention also provides a method for treating viral infections in patients, the method comprising administering to a patient in need an antibody that specifically binds to CD38 for a duration sufficient to treat the viral infection.

[0110] In some implementations, the immune response is an effector T cell (Teff) response.

[0111] In some implementations, the Teff response is generated by CD4. + T cells or CD8 + T cell mediated.

[0112] In some implementations, the Teff response is generated by CD4. + T cell mediated.

[0113] In some implementations, the Teff response is provided by CD8. + T cell mediated.

[0114] In some implementations, the Teff response is CD8. + Increased number of T cells, CD8 + Increased T cell proliferation, increased T cell clonal expansion, CD8 + Increased formation of memory cells, increased production of antigen-dependent antibodies, or increased production of cytokines, chemokines, or interleukins.

[0115] T cell proliferation can be assessed, for example, by measuring the rate of DNA synthesis using tritium-labeled thymidine, or by measuring the production of in vitro interferon-γ (IFN-γ), or by measuring the absolute number or percentage of T cells in a cell population from a patient sample using known methods.

[0116] Clonal expansion can be evaluated, for example, by sequencing TCRs from a T cell library using known methods.

[0117] Memory cell formation can be measured using, for example, FACS to measure naïve T cells (CD45RO). - / CD62L + ) and memory T cells (CD45RO) + / CD62L high The ratio is used for evaluation.

[0118] The production of cytokines, chemokines, or interleukins (such as interferon-γ (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, and CCL4) can be assessed using standard methods such as ELISA or ELLISPOT assays.

[0119] The production of antigen-specific antibodies can be assessed from samples from patients using standard methods such as ELISA or radioimmunoassay (RIA).

[0120] The meaning of "increased" or "increased" Teff responses is easy to understand. An increase compared to a control in the test sample or treated individuals (e.g., in patients treated with anti-CD38 antibodies compared to the same patients before treatment, or in patients or patient groups that responded to anti-CD38 antibody treatment compared to patients or patient groups that did not respond to the same treatment) can be 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%, 6... Increases of 8%, 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 are considered statistically significant.

[0121] Similarly, it is easy to understand the meaning of "reduced," "decreased," "reduced," or "reduced" numbers of Tregs, MDSCs, and / or Bregs. The reduction compared to a control in the test sample or treated individuals (e.g., in patients treated with anti-CD38 antibodies compared to the same pre-treatment patients, or in patients or patient groups that responded to anti-CD38 antibody treatment compared to patients or patient groups that did not respond to the same treatment) can be 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 above. Generally, reduction is considered statistically significant.

[0122] In some implementations, antibodies that specifically bind to CD38 inhibit the function of immunosuppressive cells.

[0123] In some implementations, the immunosuppressive cells are regulatory T cells (Tregs), bone marrow-derived suppressor cells (MDSCs), or regulatory B cells (Bregs).

[0124] In some implementations, Treg is CD3 + CD4 + CD25 + CD127 dim T cells.

[0125] In some implementations, CD3 + CD4 + CD25 + CD127 dim Cells express Foxp3.

[0126] In some implementations, CD3 + CD4 + CD25 + CD127 dim T cells express CD38.

[0127] Treg functions, such as their ability to suppress Teff cells, can be assessed using known methods, such as assessing the ability of Tregs to suppress Teff proliferation in mixed lymphocyte responses (MLRs).

[0128] For example, it can be achieved by directly killing Tregs or subgroups of Tregs (such as CD38). + To reduce the relative number of Tregs compared to Teffs (e.g., increase CD8), + The ratio of Treg cells to Treg cells was used to suppress Treg function.

[0129] In some implementations, Treg function is suppressed by killing Treg cells.

[0130] In some implementations, Treg killing is mediated by antibody-induced antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or apoptosis induced by antibodies that specifically bind to CD38.

[0131] In some implementations, Treg killing is mediated by ADCC.

[0132] In some implementations, CD38 is killed. + Treg.

[0133] In some implementations, 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 Tregs are killed.

[0134] Since CD38 is expressed only in a subset of Tregs and MDSCs, treatment of patients with solid tumors is expected not to lead to systemic depletion of Tregs and MDSCs, thus providing improved safety.

[0135] In some implementations, MDSC is CD11b + HLA-DR - CD14 - CD33 + CD15 + cell.

[0136] In some implementations, CD11b + HLA-DR - CD14 - CD33 + CD15 + MDSC expresses CD38.

[0137] MDSC function can be inhibited, for example, by directly killing cells to reduce the number of MDSCs.

[0138] In some implementations, by killing CD38 + MDSC is used to suppress the MDSC function.

[0139] In some implementations, MDSC killing is mediated by antibody-induced antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or apoptosis induced by antibodies that specifically bind to CD38.

[0140] In some implementations, MDSC killing is mediated by ADCC.

[0141] In some implementations, 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 MDSCs are killed.

[0142] In some implementations, Breg is CD19 + CD24 + CD38 + cell.

[0143] Breg function can be suppressed, for example, by directly killing Bregs to reduce their number.

[0144] In some implementations, by killing CD38 + Breg is used to suppress Breg function.

[0145] In some implementations, Breg killing is mediated by antibody-induced antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or apoptosis induced by antibodies that specifically bind to CD38.

[0146] In some implementations, Breg killing is mediated by ADCC.

[0147] In some implementations, 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 Bregs are killed.

[0148] Tregs play a crucial role in maintaining peripheral self-tolerance. Naturally occurring CD4... + CD25 hi Tregs produce and express Foxp3 in the thymus, a transcription factor required to establish and maintain the Treg lineage's recognition and repressive functions. Tregs can accumulate at disease sites (e.g., tumors), where they suppress the effector function of tumor antigen-specific T cells, leading to insufficient antitumor responses. Tumor-infiltrating Foxp3 + Increased Treg density is associated with poor prognosis in various solid tumors, including pancreatic cancer, ovarian cancer, and hepatocellular carcinoma. Treg depletion leads to enhanced antitumor immunity and tumor rejection in mouse models, but can also contribute to the development of autoimmune diseases.

[0149] Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of early bone marrow progenitors, immature granulocytes, macrophages, and dendritic cells at different stages of differentiation. They accumulate in large numbers in cancer patients and possess potent immunosuppressive effects, inhibiting the cytotoxic activity of natural killer (NK) cells and natural killer T (NKT) cells and suppressing CD8+ cell proliferation. +T cell-mediated adaptive immune responses. Although the mechanisms of NK cell suppression are not fully understood, multiple pathways can respond to MDSC-mediated T cell suppression, including the production of arginase 1 / ARG1 and the upregulation of nitric oxide synthase 2 (NOS2). ARG1 and NOS2 metabolize L-arginine and, together or separately, block the translation of the CD3ζ chain on T cells, inhibit T cell proliferation, and promote T cell apoptosis. Alternatively, MDSCs secrete immunosuppressive cytokines and induce regulatory T cell development.

[0150] MDSCs are induced by pro-inflammatory cytokines and their numbers are found to increase under 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 has been recognized as contributing to the pathogenicity of tumor-associated immunosuppression.

[0151] MDSC was observed in patients with colon cancer, melanoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, 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 ResClin 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 This is described in 182:5693-5701. In cancer patients, Diaz et al. (Diaz-Montero et al., (2009)) Cancer ImmunolImmunother 58: 49-59) suggests that the accumulation of MDSCs is associated with later-stage disease and poor prognosis.

[0152] Tumor-infiltrating Bregs have been identified in solid tumors, and these Bregs can promote tumor growth and metastasis through various mechanisms, such as inhibiting CD8. + The antitumor activity of T cells and NK cells, as shown, for example, in Ding et al. (2015). Human Immunology As described in 76:615-62.

[0153] Antibody-dependent cell cytotoxicity (ADCC) is a mechanism that induces cell death by interacting with antibody-coated target cells and lytic effector cells (such as natural killer cells, monocytes, macrophages, and neutrophils) via Fcγ receptors (FcγR) expressed on the effector cells. For example, NK cells express FcγRIIIa, while monocytes express FcγRI, FcγRII, and FcvRIIIa. The activity of effector cells, through the secretion of pore-forming proteins and proteases, leads to the death of antibody-coated target cells, such as those expressing CD38. To assess the ADCC activity of antibodies that specifically bind to CD38, the antibody can be co-introduced with immune effector cells that express CD38. These effector cells can be activated by the antigen-antibody complex, thereby causing cell lysis of the target cells. Cell lysis is typically detected based on the release of markers (such as radioactive substrates, fluorescent dyes, or native intracellular proteins) from the lysed cells. Exemplary effector cells used in this type of assay include peripheral blood mononuclear cells (PBMCs) and NK cells. Exemplary target cells include Tregs or MDSCs expressing CD38. In the exemplary assay, 20 µCi of... 51 Cr-labeled target cells for 2 hours, followed by thorough rinsing. The target cell concentration can be adjusted to 1×10⁻⁶. 6 Cells / ml were added, and various concentrations of anti-CD38 antibody were added. Target cells were added at an effector cell:target cell ratio of 40:1 to initiate the assay. After incubation at 37°C for 3 hours, the assay was terminated by centrifugation, and the amount of cells released from the lysed cells was measured using a scintillation counter. 51 Cr. The percentage of cytotoxicity can be calculated as the maximum lysis induced by adding 3% perchloric acid to the target cells.

[0154] Antibody-dependent phagocytosis (ADCP) refers to the mechanism by which antibody-coated target cells are eliminated through internalization by phagocytes (such as macrophages or dendritic cells). ADCP can be assessed using Treg cells or MDSCs expressing CD38 as target cells engineered to express GFP or other marker molecules. The effector cell:target cell ratio can be, for example, 4:1. Effector cells and target cells can be incubated together for 4 hours with or without anti-CD38 antibody. After incubation, cells can be separated using accutase. Macrophages can be identified using fluorescently labeled anti-CD11b and anti-CD14 antibodies, and standard methods based on CD11 can be used. + CD14 + The percentage of GFP fluorescence in macrophages determines the phagocytic percentage.

[0155] Complement-dependent cytotoxicity (ADCC) or "CDC" refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody binds to and activates the complement component C1q. C1q then activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells, which promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0156] The ability of monoclonal antibodies to induce ADCC can be enhanced by engineering their oligosaccharide components. Most glycans of human IgG1 or IgG3 are N-glycosylated in the form of bibranched G0, G0F, G1, G1F, G2, or G2F at Asn297. Antibodies produced from unengineered CHO cells typically have a glycan content of at least 85% fucose. Removing the core fucose from bibranched complex-type oligosaccharides linked to the Fc region can enhance antibody ADCC via improved FcγRIIIa binding without altering antigen binding or CDC activity. Such mAbs can be achieved using various methods reported to induce relatively high defucosylation of antibodies with bibranched complex-type Fc oligosaccharides, such as control of culture osmolarity (Konno et al., (2012)). Cytotechnology (64:249-65), using the variant CHO line Lec13 as the host cell line (Shields et al., (2002)). J Biol Chem 277:26733-26740), using the variant CHO line EB66 as the host cell line (Olivier et al., (2010)). MAbs 2(4), Electronic version before printing; PMID: 20562582), using the rat hybridoma cell line YB2 / 0 as the host cell line (Shinkawa et al., (2003)). J Biol Chem 278:3466-3473), introducing specific targets for α1,6-fucosyltransferase ( FUT8 Small interfering RNAs of genes (Mori et al., (2004)) Biotechnol Bioeng 88:901-908) or co-expression of β-1,4- N - Acetaminoglycopene transferase III and Golgi α-mannosidase II or a potent α-mannosidase I inhibitor, kifunensine (Ferrara et al., (2006)). J Biol Chem 281:5032-5036; Ferrara et al., (2006) Biotechnol Bioeng 93:851-861; Xhou et al., (2008) Biotechnol Bioeng(99:652-65). ADCC induced by the anti-CD38 antibody in some embodiments of each of the methods used in the present invention and in all numbered embodiments listed below can also be enhanced by certain substitutions in the antibody Fc. Exemplary substitutions are, for example, substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 (residues numbered according to the EU index), as described in U.S. Patent 6,737,056.

[0157] In some implementations, the antibody that specifically binds to CD38 contains a substitution in the antibody Fc.

[0158] In some implementations, the antibody that specifically binds to CD38 contains substitutions at amino acid positions 256, 290, 298, 312, 356, 330, 333, 334, 360, 378, or 430 (residues numbered according to the EU index) in the antibody Fc.

[0159] In some embodiments, the antibody that specifically binds to CD38 has a bibranched glycan structure with a fucose content of about 0% to about 15% (e.g., 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0%).

[0160] In some implementations, the antibody that specifically binds to CD38 has a bibranched polysaccharide structure with a fucose content of approximately 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%.

[0161] The substitution and reduced fucose content in Fc can enhance the ADCC activity of antibodies that specifically bind to CD38.

[0162] "Fucose content" refers to the amount of fucose monosaccharide within the sugar chain at Asn297. The relative amount of fucose is the percentage of fucose-containing structures relative to all sugar structures. These can be characterized and quantified by various methods, such as: 1) using MALDI-TOF with samples treated with N-glycosidase F (e.g., complex structures, mixed structures, oligomannose structures, and high-mannose structures), as described in International Patent Publication WO2008 / 077546; 2) enzymatic release of Asn297 glycans, followed by derivatization and detection / quantification by HPLC (UPLC) and / or HPLC-MS (UPLC-MS) with fluorescence detection; 3) complete protein analysis of native or reduced mAb with or without treatment of Asn297 glycans with Endo S or other enzymes, wherein the other enzymes cleave between the first GlcNAc monosaccharide and the second GlcNAc monosaccharide, leaving fucose linked to the first GlcNAc; 4) digestion of mAb into component peptides by enzymatic digestion (e.g., trypsin or endopeptidase Lys-C), followed by separation, detection, and quantification by HPLC-MS (UPLC-MS); or 5) using PNGase F in Asn297 glycans. Specific enzymatic deglycosylation was performed at 297 sites to separate mAb oligosaccharides from mAb proteins. The released oligosaccharides can be fluorophoretically labeled, separated, and identified using various complementary techniques. These techniques allow for: precise characterization of the glycan structure by comparing experimental and theoretical mass using matrix-assisted laser desorption / ionization (MALDI) mass spectrometry; determination of sialylation levels using ion-exchange HPLC (GlycoSep C); separation and quantification of oligosaccharide forms based on hydrophilicity standards using normal-phase HPLC (GlycoSep N); and separation and quantification of oligosaccharides using high-performance capillary electrophoresis-laser-induced fluorescence (HPCE-LIF).

[0163] As used in this article, "low fucose" or "low fucose content" means that the fucose content of the antibody is approximately 0% to 15%.

[0164] As used in this article, “normal fucose” or “normal fucose content” means that the fucose content of the antibody is approximately higher than 50%, typically higher than 60%, 70%, 80%, or 85%.

[0165] In some embodiments, antibodies that specifically bind to CD38 can induce the killing of Tregs, MDSCs, and / or Bregs via apoptosis. Methods for assessing apoptosis are well known and include, for example, annexin IV staining using standard methods. Anti-CD38 antibodies used in the methods of this invention can induce apoptosis in approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of cells.

[0166] In some implementations, Teff or immunosuppressive cells are present in the bone marrow or peripheral blood.

[0167] In some implementations, Teff, or immunosuppressive cells, are present in the bone marrow.

[0168] In some implementations, Teff or immunosuppressive cells are present in peripheral blood.

[0169] In some implementations, antibodies that specifically bind to CD38 increase CD8. + The ratio of T cells to Tregs.

[0170] In some implementations, antibodies that specifically bind to CD38 increase CD8. + Central memory cells and CD8 + The initial cell ratio. CD8 + Central memory cells can be identified as CD45RO + / CD62L +high Cells. CD8 + Initial cells can be identified as CD45RO- / CD62L + cell.

[0171] In some implementations, the antibody that specifically binds to CD38 is a non-stimulatory antibody.

[0172] Non-agonist antibodies that specifically bind to CD38 are those that do not induce significant proliferation of peripheral blood mononuclear cell samples after binding to CD38 in vitro, compared to proliferation induced by isotype control antibodies or cultures alone.

[0173] In some implementations, non-agonist antibodies that specifically bind to CD38 induce peripheral blood mononuclear cell (PBMC) proliferation in a statistically insignificant manner. PBMC proliferation can be assessed by the following steps: isolating PBMCs from a healthy donor, and then, with or without the test antibody, in 200 µl RPMI at a concentration of 1 × 10⁻⁶. 5 Cells were cultured in flat-bottomed 96-well plates. After incubation at 37°C for four days, 30 µl of the solution could be added.3 H-thymidine (16.7 µCi / ml), continue incubation overnight. Evaluation can be performed using a Packard Cobraγ counter (Packard Instruments, Meriden, DT, USA) according to the manufacturer's instructions. 3 Binding of H-thymidine. Data can be calculated as the mean cpm (±SEM) of PBMCs obtained from several donors. Statistical significance or insignificance between samples cultured in the presence or absence of the test antibody was calculated using standard methods.

[0174] An exemplary anti-CD38 antibody that can be used in the method of the present invention is DARZALEX. ™ (Darezumab). DARZALEX ™ (Daratumumab) comprises the heavy chain variable region (VH) and light chain variable region (VL) amino acid sequences shown in SEQ ID NO: 4 and 5, respectively, heavy chain complementarity-determining regions 1 (HCDR1), HCDR2, and HCDR3 in SEQ ID NO: 6, 7, and 8, and light chain complementarity-determining regions 1 (LCDR1), LCDR2, and LCDR3 in SEQ ID NO: 9, 10, and 11, respectively, and is an IgG1 / κ subtype, as described in U.S. Patent 7,829,693. DARZALEX ™ The heavy chain amino acid sequence of (daratumumab) is shown in SEQ ID NO:12, and the light chain amino acid sequence is shown in SEQ ID NO:13.

[0175] In some embodiments, antibodies that specifically bind to CD38 compete with antibodies containing a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5 for binding to CD38.

[0176] In some embodiments, the antibody that specifically binds to CD38 binds at least to the SKRNIQFSCKNIYR region (SEQ ID NO: 2) and the EKVQTLEAWVIHGG region (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).

[0177] SEQ ID NO: 1 mancefspvsgdkpccrlsrraqlclgvsilvlilvvvlavvvprwrqqwsgpgttkrfpetvlarcvkyteihpemrhvdcqsvwdafkgafiskhpcniteedyqplmklgtqtvpcnkillwsrikdlahqftqvqrdmftledtllgyladdltwcgefntskinyqscpdwrkdcsnnpvsvfwktvsrrfaeaacdvvhvmlngsrskifdknstfgsvevhnlqpekvqtleawvihggredsrdlcqdptikelesiiskrniqfsckniyrpdkflqcvknpedssctsei SEQ ID NO: 2 SKRNIQFSCKNIYR SEQ ID NO: 3 EKVQTLEAWVIHGG SEQ ID NO: 4 EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSS SEQ ID NO: 5 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK 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 EVQLLESGGGLVQPGGSLRLSCAVSGFTFNSFAMSWVRQAPGKGLEWVSAISGSGGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCAKDKILWFGEPVFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 13 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Well-known in vitro methods can be used to assess the binding of an antibody to a reference antibody, such as DARZALEX which has the VH of SEQ ID NO: 4 and the VL of SEQ ID NO: 5 ™(Daralimumab) competitively binds to CD38. In one exemplary method, CHO cells expressing recombinant CD38 are incubated with an unlabeled reference antibody at 4°C for 15 minutes, followed by incubation with an excess of fluorescently labeled test antibody at 4°C for 45 minutes. After washing in PBS / BSA, fluorescence can be measured by flow cytometry using standard methods. 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 over approximately 15 minutes, followed by the addition of a biotinylated test antibody. After washing in PBS / Tween, binding to the biotinylated test antibody can be detected using horseradish peroxidase (HRP)-conjugated streptavidin, and the signal can be detected using standard methods. It is evident that in competitive assays, the reference antibody may be labeled, while the test antibody may be unlabeled. When the reference antibody inhibits the binding of the test antibody, the test antibody competes with the reference antibody, or the test antibody inhibits the binding of the reference antibody to CD38 by at least 80%, such as 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The epitopes of the test antibody can be further defined using known methods, such as peptide mapping or hydrogen / deuterium protection assays, or by crystal structure determination.

[0178] Antibodies binding to the regions SKRNIQFSCKNIYR (SEQ ID NO: 2) and EKVQTLEAWVIHGG (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1) can be generated, for example, by immunizing mice with peptides having the amino acid sequences shown in SEQ ID NO: 2 and 3 using standard methods and those described herein, and characterizing the binding of the obtained antibodies to the peptides using, for example, ELISA or mutagenesis studies.

[0179] The present invention also provides a method for treating a patient with a solid tumor, comprising administering to a patient in need an anti-CD38 antibody that binds to the SKRNIQFSCKNIYR region (SEQ ID NO: 2) and the EKVQTLEAWVIHGG region (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1). The epitope of the antibody used in the method of the present invention comprises some or all 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 SKRNIQFSCKNIYR region (SEQ ID NO: 2) and at least one amino acid in the EKVQTLEAWVIHGG region (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1). In some embodiments, the antibody epitope comprises at least two amino acids in the SKRNIQFSCKNIYR region (SEQ ID NO: 2) and at least two amino acids in the EKVQTLEAWVIHGG region (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1). In some embodiments, the antibody epitope comprises at least three amino acids in the region SKRNIQFSCKNIYR (SEQ ID NO: 2) of human CD38 (SEQ ID NO: 1) and at least three amino acids in the region EKVQTLEAWVIHGG (SEQ ID NO: 3).

[0180] In some embodiments, the antibody that specifically binds to CD38 comprises the amino acid sequences HCDR1, HCDR2, and HCDR3, respectively, SEQ ID NO: 6, 7, and 8.

[0181] In some embodiments, the antibody that specifically binds to CD38 comprises the amino acid sequences LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 9, 10, and 11, respectively.

[0182] In some embodiments, the antibody that specifically binds to CD38 comprises the amino acid sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, SEQ ID NO: 6, 7, 8, 9, 10, and 11.

[0183] In some embodiments, the antibody that specifically binds to CD38 comprises VH having 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4, and VL having 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 5.

[0184] In some embodiments, the antibody that specifically binds to CD38 comprises VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5.

[0185] In some embodiments, the antibody that specifically binds to CD38 comprises a heavy chain of SEQ ID NO: 12 and a light chain of SEQ ID NO: 13.

[0186] Other exemplary anti-CD38 antibodies that can be used in any embodiment of the present invention are: mAb003 contains VH and VL sequences of SEQ ID NO: 14 and 15, respectively, and is described in U.S. Patent 7,829,693. The VH and VL sequences of mAb003 can be expressed as IgG1 / κ.

[0187] SEQ ID NO: 14 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAFSWVRQAPGQGLEWMGRVIPFLGIANSAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCARDDIAALGPFDYWGQGTLVTVSSAS SEQ ID NO: 15 DIQMTQSPSSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPRTFGQGTKVEIK; mAb024 contains VH and VL sequences of SEQ ID NO: 16 and 17, respectively, and is described in U.S. Patent 7,829,693. The VH and VL sequences of mAb024 can be expressed as IgG1 / κ.

[0188] SEQ ID NO: 16 EVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVRQMPGKGLEWMGIIYPHDSDARYSPSFQGQVTFSADKSISTAYLQWSSLKASDTAMYYCARHVGWGSRYWYFDLWGRGTLVTVSS SEQ ID NO: 17 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEP EDFAVYYCQQRSNWPPTFGQGTKVEIK; MOR-202 (MOR-03087), which contains VH and VL sequences of SEQ ID NO: 18 and 19 respectively, and is described in U.S. Patent 8,088,896. The VH and VL of MOR-202 can be expressed as IgG1 / κ.

[0189] SEQ ID NO: 18 QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYYMNWVRQAPGKGLEWVSGISGDPSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLPLVYTGFAYWGQGTLVTVSS SEQ ID NO: 19 DIELTQPPSVSVAPGQTARISCSGDNLRHYYVYWYQQKPGQAPVLVIYGDSKRPSGIPERFSGSNSGNTATLTISGTQAEDEADYYCQTYTGGASLVFGGGTKLTVLGQ; Isatuximab contains VH and VL sequences, respectively, SEQ ID NO: 20 and 21, as described in U.S. Patent 8,153,765. The VH and VL sequences of Isatuximab can be expressed as IgG1 / κ.

[0190] SEQ ID NO 20: QVQLVQSGAEVAKPGTSVKLSCKASGYTFTDYWMQWVKQRPGQGLEWIGTIYPGDGDTGYAQKFQGKATLTADKSSKTVYMHLSSLASEDSAVYYCARGDYYGSNSLDYWGQGTSVTVSS SEQ ID NO:21: DIVMTQSHLSMSTSLGDPVSITCKASQDVSTVVAWYQQKPGQSPRRLIYSASYRYIGVPDRFTGSGAGTDFTFTISSVQAEDLAVYYCQQHYSPPYTFGGGTKLEIK Other exemplary anti-CD38 antibodies that can be used in the methods of the present invention include those described in International Patent Publications WO05 / 103083, WO06 / 125640, WO07 / 042309, WO08 / 047242 or WO14 / 178820.

[0191] The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody sufficient to treat the solid tumor for a duration, the antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5.

[0192] The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody sufficient to treat the solid tumor for a duration, the antibody comprising VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15.

[0193] The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody sufficient to treat the solid tumor for a duration, the antibody comprising VH of SEQ ID NO: 16 and VL of SEQ ID NO: 17.

[0194] The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody sufficient to treat the solid tumor for a duration, the antibody comprising VH of SEQ ID NO: 18 and VL of SEQ ID NO: 19.

[0195] The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody sufficient to treat the solid tumor for a duration, the antibody comprising VH of SEQ ID NO: 20 and VL of SEQ ID NO: 21.

[0196] In some implementations, the solid tumor is melanoma.

[0197] In some implementation schemes, the solid tumor is lung cancer.

[0198] In some implementations, the solid tumor is squamous non-small cell lung cancer (NSCLC).

[0199] In some implementations, the solid tumor is non-squamous NSCLC.

[0200] In some implementation schemes, the solid tumor is lung adenocarcinoma.

[0201] In some implementations, the solid tumor is renal cell carcinoma (RCC) (e.g., clear cell renal carcinoma or papillary renal carcinoma) or its metastatic lesions.

[0202] In some implementation schemes, the solid tumor is mesothelioma.

[0203] In some implementation schemes, the solid tumor is nasopharyngeal carcinoma (NPC).

[0204] In some implementation schemes, the solid tumor is colorectal cancer.

[0205] In some implementations, the solid tumor is prostate cancer or castration-resistant prostate cancer.

[0206] In some implementations, the solid tumor is stomach cancer.

[0207] In some implementation schemes, the solid tumor is ovarian cancer.

[0208] In some implementation schemes, the solid tumor is gastric cancer.

[0209] In some implementation schemes, the solid tumor is liver cancer.

[0210] In some implementation schemes, the solid tumor is adenocarcinoma.

[0211] In some implementation schemes, the solid tumor is thyroid cancer.

[0212] In some implementations, the solid tumor is squamous cell carcinoma of the head and neck.

[0213] In some implementations, the solid tumor is cancer of the esophagus or gastrointestinal tract.

[0214] In some implementations, the solid tumor is breast cancer.

[0215] In some implementation schemes, the solid tumor is fallopian tube cancer.

[0216] In some implementation schemes, the solid tumor is brain cancer.

[0217] In some implementation schemes, the solid tumor is urethral cancer.

[0218] In some implementations, the solid tumor is a cancer of the genitourinary system.

[0219] In some implementations, the solid tumor is referred to as endometriosis.

[0220] In some implementation schemes, solid tumors are defined as cervical cancer.

[0221] In some implementations, solid tumors are metastatic lesions of cancer.

[0222] In some implementations, solid tumors lack detectable CD38 expression.

[0223] When CD38 expression in solid tumor tissue or on cells isolated from a solid tumor is not statistically significant compared to a control (e.g., expression detected by anti-CD38 antibody using well-known methods compared to expression detected by isotype control antibody), a solid tumor lacks detectable CD38 expression.

[0224] The anti-CD38 antibody used in the method of the present invention may also be de novo selected, for example, from a phage display library, wherein the phage is engineered to express human immunoglobulin or a portion thereof, such as 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, phage display libraries that express antibody heavy chain and light chain variable regions as fusion proteins with phage pIX coat proteins, as in Shi et al. (2010). J. Mol. Biol As described in 397:385-96 and International Patent Publication WO09 / 085462, phage display methods for isolating human antibodies are screened from an antibody library that binds to the extracellular domain of human CD38. The resulting positive clones are further characterized, Fab is isolated from the clone lysate, and subsequently cloned into full-length antibodies. Such phage display methods for isolating human antibodies are established in the art. See, for example, U.S. Patents 5,223,409, 5,403,484, 5,571,698, 5,427,908, 5,580,717, 5,969,108, 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, and 6,593,081.

[0225] In some implementations, the anti-CD38 antibody is an IgG1, IgG2, IgG3, or IgG4 isotype.

[0226] The Fc portion of an antibody can mediate antibody effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). These functions can be mediated by one or more Fc effector domains binding to Fc receptors on immune cells with phagocytic or lytic activity, or by one or more Fc effector domains binding to components of the complement system. Typically, Fc-mediated effects involving cell or complement components result in the suppression and / or depletion of target cells (e.g., cells expressing CD38). Human IgG isotypes IgG1, IgG2, IgG3, and IgG4 exhibit differential abilities in effector function. ADCC can be mediated by IgG1 and IgG3, ADCP by IgG1, IgG2, IgG3, and IgG4, and CDC by IgG1 and IgG3.

[0227] Antibodies substantially identical to those comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 can be used in the methods of the present invention. As used herein, the term “substantially identical” means that the amino acid sequences of the two antibodies VH or VL being compared are identical or have “non-significant differences.” A non-significant difference means that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids are substituted in the antibody heavy or light chain, and these substitutions do not adversely affect the properties of the antibody. Percentage identity can be determined, for example, by pairwise alignment using the default settings of the AlignX module in Vector NTI v.9.0.0 (Invitrogen, Carlsbad, CA). The protein sequences of the present invention can be used as query sequences for retrieval against public or patent databases to, for example, identify relevant sequences. Exemplary programs used for such retrievals are the XBLAST or BLASTP programs (http: / / www.ncbi.nlm / nih.gov) or GenomeQuest using the default settings. ™(GenomeQuest, Westborough, MA) software package. Exemplary substitutions that can be made to antibodies that specifically bind 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, such as stability or affinity, or to improve antibody effector function. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions can be made to the heavy or light chain of the anti-CD38 antibody. In addition, any native residue in VH or VL can also be substituted with alanine, as previously described for alanine scan mutagenesis (MacLennan et al., Acta Physiol Scand Suppl 643:55-67, 1998; Sasaki et al., Adv Biophys 35:1-24, 1998). The required amino acid substitutions can be determined by those skilled in the art when such substitutions are required. Amino acid substitution can be performed, for example, by PCR mutagenesis (US Patent 4,683,195). A variant library can be generated using well-known methods, such as using random (NNK) or non-random codons (e.g., DVK codons encoding 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp)), and variants with desired properties can be screened from the library. The binding of the generated variants to CD38 and their ability to induce ADCC, ADCP, or apoptosis in vitro, or to modulate CD38 enzyme activity, can be detected using the methods described herein.

[0228] In some implementations, antibodies that specifically bind to CD38 can bind to a range of affinities (K... D ( ) Binding to human CD38. In one embodiment of the invention, and in some embodiments of each of the following numbered embodiments, an antibody that specifically binds to CD38 binds to CD38 with high affinity, for example, as determined by surface plasmon resonance or the Kinexa method, K D Equal to or less than approximately 10 -7 M, such as but not limited to 1-9.9 (or any range or value thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, or 9) × 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 practiced by those skilled in the art. An exemplary affinity is equal to or less than 1 × 10⁻⁶. -8M. Another exemplary affinity is equal to or less than 1 × 10⁻⁶. -9 M.

[0229] In some implementations, the antibody that specifically binds to CD38 is a bispecific antibody. The VL and / or VH regions of existing anti-CD38 antibodies, or the VL and VH regions identified de novo as described herein, can be engineered into a bispecific full-length antibody. Such bispecific antibodies can be prepared by modulating the CH3 interaction between the heavy chains of monospecific antibodies to form bispecific antibodies using techniques such as those described in the following patents: U.S. Patent 7,695,936; International Patent Publication WO04 / 111233; U.S. Patent Publication US2010 / 0015133, U.S. Patent Publication US2007 / 0287170, International Patent Publication WO2008 / 119353, U.S. Patent Publication US2009 / 0182127, U.S. Patent Publication US2010 / 0286374, U.S. Patent Publication US2011 / 0123532, International Patent Publication WO2011 / 131746, International Patent Publication WO2011 / 143545, or U.S. Patent Publication US2012 / 0149876. Additional bispecific structures that can bind the VL and / or VH regions of the antibodies of the present invention are, for example, dual variable domain immunoglobulins (International Patent Publication WO2009 / 134776), or structures comprising multiple dimerizing domains to connect two antibody arms with different specificities, such as leucine zippers or collagen dimerizing domains (International Patent Publication WO2012 / 022811, US Patent 5,932,448, US Patent 6,833,441).

[0230] For example, bispecific antibodies can be generated in vitro in a cell-free environment via the following process: According to the method described in International Patent Publication WO2011 / 131746, an asymmetric mutation is introduced into the CH3 region of two monospecific homodimers, and a bispecific heterodimer is formed from the two parental monospecific homodimers under reducing conditions that allow disulfide isomerization. In this method, a first monospecific bivalent antibody (e.g., anti-CD38 antibody) and a second monospecific bivalent antibody are engineered to have certain substitutions at the CH3 domain that promote heterodimer stability; these antibodies are incubated together under reducing conditions sufficient to cause disulfide isomerization of cysteine ​​residues in the hinge region; thereby generating a bispecific antibody via Fab arm exchange. Ideally, the incubation conditions can be restored to non-reducing conditions. Exemplary reducing agents that can be used are 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. For example, the following conditions can be used: incubation for at least 90 minutes at a pH of 5-8, such as pH 7.0 or pH 7.4, in the presence of at least 25 mM 2-MEA or at least 0.5 mM dithiothreitol, at a temperature of at least 20°C.

[0231] Exemplary CH3 mutations that can be used in the first and second heavy chains of bispecific antibodies are K409R and / or F405L.

[0232] The method of this invention can be used to treat animal patients belonging to any classification. Examples of such animals include mammals, such as humans, rodents, dogs, cats, and livestock.

[0233] Application / Pharmaceutical Composition An antibody that specifically binds to CD38 can be provided in the method of the present invention in a suitable pharmaceutical composition comprising the antibody that specifically binds to CD38 and a pharmaceutically acceptable carrier. The carrier may be a diluent, adjuvant, excipient, or medium to which the antibody that specifically binds to CD38 is administered. Such mediators may be liquids, such as water and oils, including those derived from petroleum, animals, plants, or synthetic sources, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. For example, 0.4% saline and 0.3% glycine may be used. These solutions are sterile and generally free of particulate matter. They can be sterilized using well-known conventional sterilization techniques, such as filtration. The composition may contain, as needed, pharmaceutically acceptable excipients to approximate physiological conditions, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and colorants. The concentration of the CD38-specific antibody in such drug formulations can vary widely, from less than about 0.5% by weight, typically to at least about 1% by weight, up to 15%, or 20%, 25%, 30%, 35%, 40%, 45%, or 50% by weight, and will be selected based primarily on the desired dose, fluid volume, viscosity, etc., depending on the specific route of administration chosen. Suitable mediators and formulations including other human proteins (e.g., human serum albumin) are described, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, Troy, DB editor, Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691–1092 (see especially pp. 958–989).

[0234] The administration of the CD38-specific antibody in the method of the present invention can be by any suitable route, such as parenteral administration, for example intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary, mucosal (oral, nasal, vaginal, rectal) administration, or other methods known to those skilled in the art. The CD38-specific antibody can be administered intratumorally to lymph node drainage sites using known methods for local delivery into the tumor.

[0235] Antibodies that specifically bind to CD38 can be administered to patients via any suitable route, such as intravenous (iv) infusion or bolus injection, either parenteral, intramuscular, subcutaneous, or intraperitoneal. Intravenous infusion can be given over, for example, within 15 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes, 180 minutes, or 240 minutes, or within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours.

[0236] The dose administered to the patient is sufficient to relieve or at least partially suppress the disease being treated (“therapeutic effective dose”), and may sometimes be from 0.005 mg / kg to about 100 mg / kg, for example from about 0.05 mg / kg to about 30 mg / kg, or from about 5 mg / kg 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 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, etc. / kg, 8mg / kg, 9mg / kg or 10mg / kg, but may be even higher, such as about 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22mg / kg, 23mg / kg, 24mg / kg, 25mg / kg, 30mg / kg, 40mg / kg, 50mg / kg, 60mg / kg, 70mg / kg, 80mg / kg, 90mg / kg or 100mg / kg.

[0237] A fixed unit dose can also be given, such as 50 mg, 100 mg, 200 mg, 500 mg, or 1000 mg, or the dose can be based on the patient's surface area, such as 500 mg / m². 2 400mg / m 2 300mg / m 2 250mg / m 2 200mg / m 2 Or 100mg / m 2 Doses between 1 and 8 are usually administered (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), but doses of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more may be given.

[0238] The CD38-specific antibody of the method of the present invention can be repeated after one, two, three, four, five, six, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months, or longer periods. The treatment process can also be repeated, as with chronic administration. Repeated administration can be at the same dose or different doses. For example, the CD38-specific antibody of the method of the present invention can be administered intravenously at a weekly interval of 8 mg / kg or 16 mg / kg for 8 weeks, followed by 16 weeks at a weekly interval of 8 mg / kg or 16 mg / kg, and then at a weekly interval of 8 mg / kg or 16 mg / kg.

[0239] Antibodies that specifically bind to CD38 can be administered as maintenance therapy in the methods of the present invention, such as for example, once a week for 6 months or longer.

[0240] For example, the CD38-specific antibody in the method of the present invention may be present on at least one day of days 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, or 40 after the start of treatment, or alternatively, on days 1, 2, 3, 4, 5, 6, 7, 8, 9, 1 For at least one week of weeks 0, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, a single dose or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof, shall be provided as a daily dose of about 0.1-100 mg / kg, such as 0.5 mg / kg / day, 0.9 mg / kg / day, 1.0 mg / kg / day, 1.1 mg / kg / day, 1.5 mg / kg / day, 2 mg / kg / day, 3 mg / kg / day, etc. g / kg / day, 4mg / kg / day, 5mg / kg / day, 6mg / kg / day, 7mg / kg / day, 8mg / kg / day, 9mg / kg / day, 10mg / kg / day, 11mg / kg / day, 12mg / kg / day, 13mg / kg / day, 14mg / kg / day, 15mg / kg / day, 16mg / kg / day, 17mg / kg / day, 18mg / kg / day, 19mg / kg / day, 20mg / kg / day, 21mg / kg / day, 22mg / kg / day, 23mg / kg / day, 24mg / kg / day, 25mg / kg / day, 26mg / kg / day, 27mg / kg / day, 28mg / kg / day, 29mg / kg / day, 30mg / kg / day, 40mg / kg / day, 45mg / kg / day, 50mg / kg / day, 60mg / kg / day, 70mg / kg / day, 80mg / kg / day, 90mg / kg / day, or 100mg / kg / day.

[0241] The CD38-specific antibody used in the method of this invention can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of events during cancer progression, and / or reduce the risk of recurrence after cancer remission. This may be particularly useful for patients whose tumors are difficult to locate but who are known to have tumors due to other biological factors.

[0242] The CD38-specific antibody used in the method of this invention can be lyophilized for easy storage and reconstituted in a suitable carrier before use. This technique has been proven effective for conventional protein formulations and can be employed using well-known lyophilization and reconstitution techniques.

[0243] The antibody that specifically binds to CD38 in the method of the present invention can be administered in combination with a second therapeutic agent.

[0244] In the method of this invention, the antibody that specifically binds to CD38 can be administered in combination with any one or more chemotherapeutic agents or other anticancer therapeutic agents known to those skilled in the art. Chemotherapy agents are chemical compounds that can be used to treat cancer and include growth inhibitors or other cytotoxic agents, including alkylating agents, antimetabolites, antimicrotubule inhibitors, topoisomerase inhibitors, receptor tyrosine kinase inhibitors, angiogenesis inhibitors, etc. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide. ®Alkyl sulfonates, such as busulfan, endosulfan, and azirsulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylene imines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamine; nitrogen mustards, such as chlornaphazine, cholophosphamide, estramustine, ifosfamide, dichloroethylmethylamine, and mechlorethamine oxide. Hydrochloride, melphalan, novobichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorhexidine, formosine, lomustine, nimustine, ranimnustine;Antibiotics, such as aclacinomysins, actinomycins, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, daunorubicin, detorubicin, 6-diaza-5-oxo-L-leucine, doxorubicin, epirubicin, isopyridine, idarubicin, marcellomycin, mitomycins, and mycophenolic acid. Drugs containing 5-amino acids, such as nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozotocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites, such as methotrexate and 5-FU; and folic acid analogues, such as denoposide, methotrexate, and pteroxate. Trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and fluorouridine; and androgens, such as calusterone and dromostanolone. Propionate, epitiostanol, mepitiostane, testolactone; anti-adrenergic drugs, such as aminoglutethimide, mitotan, trilostane; folic acid supplements, such as frolinic acid; aceglatone.Aldophosphamide glycoside; aminolevulinic acid; acridine; bestrabucil; bisantrene; idatraxa; defofamide; demecolcine; elfornithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoantrone; mopidanmol; nitrarine; pentostatin; phenamet; pirarubicin; podophyllinic acid acid); 2-ethylhydrazide; procarbazine; PSK; ® Razoxane; Sizofiran; Spirogermanium; Tenuazonicacid; Triaziquone; 2,2′,2″-Trichlorotriethylamine; Urethan; Vinpocetine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactalol; Pipobroman; Gacytosine; Arabinoside (“Ara-C”); Cyclophosphamide; Thiotepa; Members of the taxane family of novel taxanes, such as Taxol (TAXOL) ® Dorsetar (TAXOTER) ® ( ) and its analogues; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues, such as cisplatin and carboplatin; vincristine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; norvincristine; novantrone; teniposide; danomycin; aminopterin; capecitabine; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; receptor tyrosine kinase and / or angiogenesis inhibitors, including NEXAVAR ®(Sorafenib), SUTENT ® (Sunitinib), VOTRIENT ™ (Pazopanib), PALLADIA ™ (toceranib), ZACTIMA ™ (Vandertani), RECENTIN ® (Sildenafil), Regorafenib (BAY 73-4506), Axitinib (AG013736), Lettatinib (CEP-701), TARCEVA ® (Erlotinib), IRESSA ™ (Gefitinib), Gilotrif ® (Afatinib), TYKERB ® (lapatinib), neratinib, and pharmaceutically acceptable salts, acids, or derivatives of any of the above substances. This definition also includes antihormonal drugs used to regulate or inhibit the effects of hormones on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trivoxifen, keoxifene, LY 117018, onanasone, and FARESTON. ® (Toremifene); and antiandrogen drugs such as flutamide, nilumet, bicalutamide, leuprorelin, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the above substances. Other conventional cytotoxic chemicals, such as those described in Wiemann et al., 1985, Medical Oncology Those disclosed in Chapter 10 of (Calabresi et al., ed.), McMillan Publishing, are also applicable to the methods of this invention.

[0245] Exemplary formulations that can be used in conjunction with antibodies that specifically bind to CD38 in the methods of this invention include tyrosine kinase inhibitors and targeted anticancer therapies, such as IRESSA. ™ (Gefitinib) and Tarceva ® Erlotinib and other antagonists of HER2, HER3, HER4, or VEGF. Exemplary HER2 antagonists include CP-724-714 and HERCEPTIN. ™ (trastuzumab), OMNITARG ™ (Pertuzumab), TAK-165, TYKERB ®(Lapatinib) (EGFR and HER2 inhibitor) and GW-282974. Exemplary HER3 antagonists include anti-Her3 antibodies (see, for example, U.S. Patent Publication 2004 / 0197332). Exemplary HER4 antagonists include anti-HER4 siRNA (see, for example, Maatta et al., Mol Biol Cell 17: 67-79, 2006). Exemplary VEGF antagonists are (Avastin) ™ (Bevacizumab)

[0246] Exemplary agents that can be used in conjunction with antibodies that specifically bind to CD38 in the methods of the present invention include standard care drugs for solid tumors or immune checkpoint inhibitors.

[0247] The second therapeutic agent in the method of the present invention can be an immune checkpoint inhibitor.

[0248] In some implementations, the immune 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.

[0249] In some implementations, the immune checkpoint inhibitor is an antagonistic anti-PD-1 antibody, an antagonistic anti-PD-L1 antibody, an antagonistic anti-PD-L2 antibody, an antagonistic anti-LAG3 antibody, or an antagonistic anti-TIM3 antibody.

[0250] In some implementations, the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0251] In some implementations, the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0252] In some implementations, the immune checkpoint inhibitor is an anti-PD-L2 antibody.

[0253] In some implementations, the immune checkpoint inhibitor is an anti-LAG3 antibody.

[0254] In some implementations, the immune checkpoint inhibitor is an anti-TIM3 antibody.

[0255] In some implementations, the immune checkpoint inhibitor is an anti-CTLA-4 antibody.

[0256] Any antagonistic anti-PD-1 antibody can be used in the method of the present invention. An exemplary anti-PD-1 antibody that can be used is OPVIDO. ® (nivolumab) and KEYTRUDA ® (pembrolizumab). OPVIDO ®(nivolumab) is described, for example, in U.S. Patent 8,008,449 (antibody 5C4), and comprises VH of SEQ ID NO: 24 and VL of SEQ ID NO: 25. KEYTRUDA ® Pembrolizumab is described, for example, in U.S. Patent 8,354,509, and comprises VH of SEQ ID NO: 22 and VL of SEQ ID NO: 23. The amino acid sequences of nivolumab and pembrolizumab are also available through CAS registration. Other PD-1 antibodies that may be used are described in U.S. Patent 7,332,582, U.S. Patent Publication 2014 / 0044738, International Patent Publication WO2014 / 17966, and U.S. Patent Publication 2014 / 0356363.

[0257] An "antagonist" is a molecule that, when bound to a cellular protein, inhibits at least one response or activity induced by the protein's natural ligand. A molecule is considered an antagonist when at least one response or activity is inhibited by at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the inhibition of at least one response or activity in the absence of an antagonist (e.g., a negative control), or when the inhibition is statistically significant compared to inhibition in the absence of an antagonist. Antagonists can be antibodies, soluble ligands, small molecules, DNA, or RNA (such as siRNA). For example, the typical response or activity induced by PD-1 binding to its receptors PD-L1 or PD-L2 can reduce the antigen-specific CD4+ of T cells. + or CD8 + Cell proliferation or reduced interferon-γ (IFN-γ) production by T cells can suppress immune responses against, for example, tumors. Typical responses or activities induced by TIM-3 binding to its receptors (such as galactolectin-9) can reduce antigen-specific CD4+. + or CD8 + Cell proliferation, reducing IFN-γ production by T cells, or reducing CD4 + or CD8 + CD137 is expressed on the surface of cells, thereby suppressing immune responses against, for example, tumors. Therefore, antagonistic PD-1 antibodies that specifically bind to PD-1, antagonistic PD-L2, and antagonistic antibodies that specifically bind to TIM-3 induce immune responses by inhibiting inhibitory pathways.

[0258] SEQ ID NO: 22 QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS SEQ ID NO: 23 EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK SEQ ID NO: 24 QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS SEQ ID NO: 25 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK Anti-PD-L1 antibodies that enhance the immune response (e.g., antagonistic anti-PD-L1 antibodies) can be used in the methods of the present invention. Exemplary anti-PD-L1 antibodies that can be used are durvalumab, atezolizumab, and avelumab, as well as those described in, for example, U.S. Patent Publication 2009 / 0055944, U.S. Patent 8,552,154, U.S. Patent 8,217,149, and U.S. Patent 8,779,108.

[0259] Durvalumab comprises VH of SEQ ID NO: 26 and VL of SEQ ID NO: 27.

[0260] Atezolizumab comprises VH of SEQ ID NO: 28 and VL of SEQ ID NO: 29.

[0261] Avelumab comprises VH of SEQ ID NO: 30 and VL of SEQ ID NO: 31.

[0262] SEQ ID NO: 26 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREG GWFGELAFDYWGQGTLVTVSS SEQ ID NO: 27 EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK SEQ ID NO: 28 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS SEQ ID NO: 29 DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK SEQ ID NO: 30 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS SEQ ID NO: 31 QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-PD-1 antibody sufficient to treat the solid tumor for a duration thereof, the CD38-specific antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-PD-1 antibody comprising VH of SEQ ID NO: 24 and VL of SEQ ID NO: 25.

[0263] The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-PD-1 antibody for a duration sufficient to treat the solid tumor, the CD38-specific antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-PD-1 antibody comprising VH of SEQ ID NO: 22 and VL of SEQ ID NO: 23.

[0264] The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-PD-L1 antibody for a duration sufficient to treat the solid tumor, the CD38-specific antibody comprising VH of SEQ ID NO:4 and VL of SEQ ID NO:5, and the anti-PD-L1 antibody comprising VH of SEQ ID NO:26 and VL of SEQ ID NO:27.

[0265] The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-PD-L1 antibody for a duration sufficient to treat the solid tumor, the CD38-specific antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-PD-L1 antibody comprising VH of SEQ ID NO: 28 and VL of SEQ ID NO: 29.

[0266] The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-PD-L1 antibody for a duration sufficient to treat the solid tumor, the CD38-specific antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-PD-L1 antibody comprising VH of SEQ ID NO: 30 and VL of SEQ ID NO: 31.

[0267] The present invention also provides a method for enhancing an immune response in a patient, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-PD-1 antibody for a duration sufficient to enhance the immune response, the CD38-specific antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-PD-1 antibody comprising VH of SEQ ID NO: 24 and VL of SEQ ID NO: 25.

[0268] The present invention also provides a method for enhancing an immune response in a patient, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-PD-1 antibody for a duration sufficient to enhance the immune response, the CD38-specific antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-PD-1 antibody comprising VH of SEQ ID NO: 22 and VL of SEQ ID NO: 23.

[0269] The present invention also provides a method for enhancing an immune response in a patient, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-PD-L1 antibody for a duration sufficient to enhance the immune response, the CD38-specific antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-PD-L1 antibody comprising VH of SEQ ID NO: 26 and VL of SEQ ID NO: 27.

[0270] The present invention also provides a method for enhancing an immune response in a patient, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-PD-L1 antibody for a duration sufficient to enhance the immune response, the CD38-specific antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-PD-L1 antibody comprising VH of SEQ ID NO: 28 and VL of SEQ ID NO: 29.

[0271] The present invention also provides a method for enhancing an immune response in a patient, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-PD-L1 antibody for a duration sufficient to enhance the immune response, the CD38-specific antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-PD-L1 antibody comprising VH of SEQ ID NO: 30 and VL of SEQ ID NO: 31.

[0272] The present invention also provides a method for treating a patient with colorectal cancer, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an antagonistic anti-PD-1 antibody for a duration sufficient to treat the colorectal cancer.

[0273] The present invention also provides a method for treating a patient with colorectal cancer, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an antagonistic anti-PD-L1 antibody for a duration sufficient to treat the colorectal cancer.

[0274] The present invention also provides a method for treating a patient with colorectal cancer, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an antagonistic anti-PD-L2 antibody for a duration sufficient to treat the colorectal cancer.

[0275] The present invention also provides a method for treating a patient with lung cancer, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an antagonistic anti-PD-1 antibody for a duration sufficient to treat the lung cancer.

[0276] The present invention also provides a method for treating a patient with lung cancer, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an antagonistic anti-PD-L1 antibody for a duration sufficient to treat the lung cancer.

[0277] The present invention also provides a method for treating a patient with lung cancer, the method comprising administering to the patient in need a therapeutically effective amount of a CD38-specific antibody and an antagonistic anti-PD-L2 antibody for a duration sufficient to treat the lung cancer.

[0278] The present invention also provides a method for treating a patient with prostate cancer, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an antagonistic anti-PD-1 antibody for a duration sufficient to treat the prostate cancer.

[0279] The present invention also provides a method for treating a patient with prostate cancer, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an antagonistic anti-PD-L1 antibody for a duration sufficient to treat the prostate cancer.

[0280] The present invention also provides a method for treating a patient with prostate cancer, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an antagonistic anti-PD-L2 antibody for a duration sufficient to treat the prostate cancer.

[0281] Anti-LAG-3 antibodies that enhance the immune response can be used in the methods of the present invention. Exemplary anti-LAG-3 antibodies that can be used are those described, for example, in International Publication WO2010 / 019570.

[0282] Anti-CTLA-4 antibodies that enhance the immune response can be used in the methods of the present invention. An exemplary anti-CTLA-4 antibody that can be used is ipilimumab.

[0283] Anti-PD-1, anti-PD-L1, anti-PD-L2, anti-LAG3, anti-TIM3, and anti-CTLA-4 antibodies that can be used in the methods of this invention can also be generated de novo using the methods described herein.

[0284] In some implementations, an anti-PD1 antibody comprising VH of SEQ ID NO: 32 and VL of SEQ ID NO: 33 may be used.

[0285] In some implementations, an anti-PD1 antibody comprising VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35 may be used.

[0286] In some implementations, an anti-TIM-3 antibody comprising VH of SEQ ID NO: 36 and VL of SEQ ID NO: 37 may be used.

[0287] In some implementations, an anti-TIM-3 antibody comprising VH of SEQ ID NO: 38 and VL of SEQ ID NO: 39 may be used.

[0288] SEQ ID NO: 32 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFDTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRPGLAAAYDTGSLDYWGQGTLVTVSS SEQ ID NO: 33 EIVLTQSPATLSLSPGERATLSCRASQSVRSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRNYWPLTFGQGTKVEIK SEQ ID NO: 34 EVQLVESGGGLVQPGGSLRLSCAASGFAFSRYDMSWVRQAPGKGLESVAYISGGGANTYYLDNVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYLSYFDVWGQGTLVTVSS SEQ ID NO: 35 EIVMTQSPATLSVSPGERATLSCRASQSLSDYLHWYQQKPGQAPRLLIKSASQSISGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQNGHSFPYTFGQGTKLEIK SEQ ID NO: 36 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSPYAPLDYWGQGTLVTVSS SEQ ID NO: 37 EIVLTQSPATLSLSPGERATLSCRASQSVNDYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQGGHAPITFGQGTKVEIK SEQ ID NO: 38 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWMQWVRQMPGKGLEWMGAIYPGDGDIRYTQNFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARWEKSTTVVQRNYFDYWGQGTTVTVSS SEQ ID NO: 39 DIQMTQSPSSLSASVGDRVTITCKASENVGTFVSWYQQKPGKAPKLLIYGASNRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGQSYSYPTFGQGTKLEIK The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-PD-1 antibody for a duration sufficient to treat the solid tumor, the CD38-specific antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-PD-1 antibody comprising VH of SEQ ID NO: 32 and VL of SEQ ID NO: 33.

[0289] The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-PD-1 antibody for a duration sufficient to treat the solid tumor, the CD38-specific antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-PD-1 antibody comprising VH of SEQ ID NO: 34 and VL of SEQ ID NO: 35.

[0290] The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering, in combination to a patient in need, a therapeutically effective amount of a CD38-specific antibody and an anti-TIM-3 antibody sufficient to treat the solid tumor for a duration, wherein the CD38-specific antibody comprises VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-TIM-3 antibody comprises VH of SEQ ID NO: 36 and VL of SEQ ID NO: 37.

[0291] The present invention also provides a method for treating a patient with a solid tumor, the method comprising administering to a patient in need a therapeutically effective amount of a CD38-specific antibody and an anti-TIM-3 antibody sufficient to treat the solid tumor for a duration thereof, the CD38-specific antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, and the anti-TIM-3 antibody comprising VH of SEQ ID NO: 38 and VL of SEQ ID NO: 39.

[0292] In the method of this invention, the combination of a CD38-specific antibody and a second therapeutic agent can be administered within any convenient timeframe. For example, the CD38-specific antibody and the second therapeutic agent can be administered to the patient on the same day, or even in the same intravenous infusion. However, the CD38-specific antibody and the second therapeutic agent can also be administered every other day, every other week, or every other month. In some methods, the CD38-specific antibody and the second therapeutic agent can be administered at sufficiently close intervals such that they are simultaneously present at detectable levels in the treated patient (e.g., in serum). In some methods, a treatment course consisting of multiple doses of the CD38-specific antibody is followed by, or preceded by, a treatment course consisting of multiple doses of the second therapeutic agent over a period of time. A recovery period of 1 day, 2 days, or several days or weeks can be used between the administration of the CD38-specific antibody and the second therapeutic agent.

[0293] Antibodies that specifically bind to CD38, or combinations of antibodies that specifically bind to CD38 and a second therapeutic agent, may be administered in conjunction with any form of radiotherapy (including external beam radiation, intensity-modulated radiotherapy (IMRT), focused radiation) and any form of radiosurgery (including Gamma Knife, CyberKnife, linear accelerators, and interstitial radiation (e.g., implantation of radioactive particles, GliaSite balloon)) and / or in conjunction with surgery.

[0294] Available focused radiation methods include stereotactic radiosurgery, fractionated stereotactic radiosurgery, and intensity-modulated radiotherapy (IMRT). Notably, stereotactic radiosurgery involves precisely delivering radiation to tumor tissue, such as a brain tumor, while avoiding surrounding non-tumor normal tissue. The radiation dose administered using stereotactic radiosurgery can vary, typically from 1 Gy to approximately 30 Gy, and can include intermediate ranges, including doses of, for example, 1 to 5, 10, 15, 20, 25, and up to 30 Gy. Because of the non-invasive fixation device, stereotactic radiosurgery does not require delivery in a single treatment session. Treatment plans can be reliably repeated daily, allowing for the delivery of multiple fractionated radiation doses. When used to treat tumors over time, radiosurgery is called “fractionated stereotactic radiosurgery” or FSR. In contrast, stereotactic radiosurgery refers to a single treatment session. Fractionated stereotactic radiosurgery can result in a high treatment-to-response ratio, i.e., a high rate of tumor cell killing and low impact on normal tissue. Tumors and normal tissues respond differently to a single high radiation dose and multiple smaller radiation doses. A single large dose of radiation can kill more normal tissue than several smaller doses. Therefore, multiple smaller doses can kill more tumor cells while avoiding normal tissue. The radiation dose delivered using fractionated stereotactic radiotherapy can vary, ranging from 1 Gy to approximately 50 Gy, and can include intermediate ranges, including, for example, fractions of 1 to 5, 10, 15, 20, 25, 30, 40, and up to 50 Gy. Intensity-modulated radiotherapy (IMRT) can also be used. IMRT is an advanced mode of high-precision three-dimensional conformal radiotherapy (3DCRT) that uses a computer-controlled linear accelerator to deliver precise radiation doses to malignant tumors or specific areas within tumors. In 3DCRT, the profile of each beam is conformed to the target shape in the field of view (BEV) using a multi-leaf collimator (MLC), thereby producing multiple beams. IMRT allows for more precise adaptation of the radiation dose to the three-dimensional (3-D) shape of the tumor by adjusting the intensity of the beams in multiple zones. Therefore, IMRT allows a higher radiation dose to be concentrated in the area within the tumor while minimizing the dose to surrounding normal critical structures. IMRT improves the ability to adapt the treatment area to the shape of the tumor, such as when the tumor surrounds vulnerable structures, such as the spinal cord or major organs or blood vessels.

[0295] Subcutaneous administration of a pharmaceutical composition comprising an antibody that specifically binds to CD38 and hyaluronidase Antibodies that specifically bind to CD38 can be administered as pharmaceutical compositions comprising an antibody that specifically binds to CD38 and subcutaneous hyaluronidase.

[0296] The concentration of antibodies that specifically bind to CD38 in a subcutaneously administered pharmaceutical composition can be approximately 20 mg / ml.

[0297] Subcutaneously administered drug compositions may contain an antibody that specifically binds to CD38 in the range of about 1,200 mg to 1,800 mg.

[0298] The drug composition administered subcutaneously may contain approximately 1,200 mg of an antibody that specifically binds to CD38.

[0299] The drug composition administered subcutaneously may contain approximately 1,600 mg of an antibody that specifically binds to CD38.

[0300] The drug composition administered subcutaneously may contain approximately 1,800 mg of an antibody that specifically binds to CD38.

[0301] Subcutaneously administered pharmaceutical compositions may contain hyaluronidase in the range of about 30,000 U to 45,000 U.

[0302] The subcutaneously administered drug composition may contain about 1,200 mg of an antibody that specifically binds to CD38 and about 30,000 U of hyaluronidase.

[0303] The subcutaneously administered drug composition may contain about 1,800 mg of an antibody that specifically binds to CD38 and about 45,000 U of hyaluronidase.

[0304] The subcutaneously administered drug composition may contain about 1,600 mg of an antibody that specifically binds to CD38 and about 30,000 U of hyaluronidase.

[0305] The subcutaneously administered drug composition may contain about 1,600 mg of an antibody that specifically binds to CD38 and about 45,000 U of hyaluronidase.

[0306] The drug composition for subcutaneous administration may contain hyaluronidase rHuPH20 having the amino acid sequence of SEQ ID NO: 40.

[0307] rHuPH20 is a recombinant hyaluronidase (HYLENEX). ® The recombinant is described in international patent publication WO2004 / 078140.

[0308] Hyaluronidase is an enzyme that degrades hyaluronic acid (EC 3.2.1.35) and reduces the viscosity of hyaluronic acid in the extracellular matrix, thereby increasing tissue permeability.

[0309] SEQ ID NO: 40 MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKA KKDITFYMPVDNLGMAVIDWEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWN ESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLC QEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFYNASPSTLSATMFIVSILFLIISSVASL The drug composition containing an antibody that specifically binds to CD38 and hyaluronidase can be repeated after one, two, three, four, five, six, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months, or longer periods. The treatment course can also be repeated as with chronic administration. Repeated administration can be at the same dose or different doses. For example, the drug composition containing an antibody that specifically binds to CD38 and hyaluronidase can be administered once a week for eight weeks, then once every two weeks for 16 weeks, followed by once every four weeks. The drug composition to be administered may contain 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 in the drug composition is about 20 mg / ml. The drug composition to be administered may contain about 1,800 mg of an antibody that specifically binds to CD38 and about 45,000 U of hyaluronidase. The pharmaceutical composition to be administered may contain 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 contain about 1,600 mg of an antibody that specifically binds to CD38 and about 45,000 U of hyaluronidase.

[0310] A pharmaceutical composition containing an antibody that specifically binds to CD38 and hyaluronidase can be administered subcutaneously to the abdominal area.

[0311] A pharmaceutical composition containing an antibody that specifically binds to CD38 and hyaluronidase can be administered in a total volume of about 80 ml, 90 ml, 100 ml, 110 ml or 120 ml.

[0312] For administration, a 20 mg / mL CD38-specific antibody in 25 mM sodium acetate, 60 mM sodium chloride, 140 mM D-mannitol, and 0.04% polysorbate 20 (pH 5.5) can be mixed with 1.0 mg / mL rHuPH20 (75-150 kU / mL) in 10 mM L-histidine, 130 mM NaCl, 10 mM L-methionine, and 0.02% polysorbate 80 (pH 6.5), and the mixture can then be administered to the treated individual.

[0313] Although the invention has been described in general terms, embodiments of the invention will be further disclosed in the following examples, which should not be construed as limiting the scope of the claims.

[0314] Other embodiments of the present invention Other embodiments of the invention are set forth below based on disclosures elsewhere herein. The features of the embodiments of the invention set forth above in relation to the invention disclosed herein also apply to each of all these other numbered embodiments.

[0315] 1. An antibody that specifically binds to CD38 for the treatment of patients with solid tumors.

[0316] 2. An antibody that specifically binds to CD38 for the treatment of patients with regulatory T cell (Treg) mediated diseases.

[0317] 3. An antibody that specifically binds to CD38 for the treatment of patients with myeloid-derived suppressor cell (MDSC) mediated diseases, comprising administering a therapeutically effective amount of the antibody that specifically binds to CD38 to the patient.

[0318] 4. An antibody that specifically binds to CD38 for inhibiting the activity of regulatory T cells (Tregs), comprising contacting the regulatory T cells with the antibody that specifically binds to CD38.

[0319] 5. An antibody that specifically binds to CD38 for inhibiting the activity of bone marrow-derived suppressor cells (MDSCs), comprising contacting MDSCs with the antibody that specifically binds to CD38.

[0320] 6. An antibody that specifically binds to CD38 for the treatment of patients with solid tumors, including reducing the number of regulatory T cells in the patient by administering the antibody that specifically binds to CD38.

[0321] 7. An antibody that specifically binds to CD38 for the treatment of patients with solid tumors, including reducing the number of myeloid-derived suppressor cells (MDSCs) in the patient by administering the antibody that specifically binds to CD38.

[0322] 8. An antibody that specifically binds to CD38, used according to any one of implementation schemes 1 to 7, wherein the antibody elicits an immune response in the patient.

[0323] 9. An antibody that specifically binds to CD38, used according to any one of embodiments 1 to 8, wherein the immune response is an effector T cell (Teff) response.

[0324] 10. An antibody that specifically binds to CD38, used according to any one of embodiments 1 to 9, wherein the Teff response is mediated by CD4. + T cells or CD8 + T cell mediated.

[0325] 11. An antibody that specifically binds to CD38, as used according to embodiment 9 or 10, wherein the Teff response is mediated by CD8. + T cell mediated.

[0326] 12. An antibody that specifically binds to CD38, used according to any one of embodiments 1 to 11, wherein the Teff response is CD8. + Increased number of T cells, CD8 + Increased T cell proliferation, increased T cell clonal expansion, CD8 + Increased formation of memory cells, increased production of antigen-dependent antibodies, or increased production of cytokines, chemokines, or interleukins.

[0327] 13. An antibody that specifically binds to CD38, used according to any one of embodiments 1 to 13, wherein the antibody inhibits the function of immunosuppressive cells.

[0328] 14. An antibody that specifically binds to CD38, used according to any one of embodiments 1 to 14, wherein the immunosuppressive cells are regulatory T cells (Tregs) or bone marrow-derived suppressor cells (MDSCs).

[0329] 15. An antibody that specifically binds to CD38 according to embodiment 15, wherein Treg is CD3 + CD4 + CD25 +CD127 dim T cells.

[0330] 16. An antibody that specifically binds to CD38, used according to implementation scheme 14 or 15, wherein the Treg expresses CD38.

[0331] 17. An antibody that specifically binds to CD38, used according to any one of embodiments 13 to 16, wherein Treg function is inhibited by Treg killing.

[0332] 18. An antibody that specifically binds to CD38 used according to any of embodiments 17, wherein Treg killing is mediated by antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cell-dependent cytotoxicity (CDC), or apoptosis induced by an antibody that specifically binds to CD38.

[0333] 19. An antibody that specifically binds to CD38, used according to any one of embodiments 17 to 18, wherein Treg killing is mediated by ADCC.

[0334] 20. The method according to claims 17 to 19, wherein about 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 Tregs are killed.

[0335] 21. An antibody that specifically binds to CD38, as used according to embodiment 14, wherein the MDSC is CD11b. + HLA-DR - CD14 - CD33 + CD15 + cell.

[0336] 22. The antibody specifically binding to CD38 used according to implementation scheme 21, wherein CD11b + HLA-DR - CD14 - CD33 + CD15 + Cells express CD38.

[0337] 23. An antibody that specifically binds to CD38, used according to any one of embodiments 21 to 22, wherein MDSC function is inhibited by MDSC killing.

[0338] 24. The CD38-specific antibody used according to implementation scheme 23, wherein MDSC killing is mediated by antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), or apoptosis induced by the CD38-specific antibody.

[0339] 25. An antibody that specifically binds to CD38, used according to any one of embodiments 23 to 24, wherein MDSC killing is mediated by ADCC.

[0340] 26. An antibody that specifically binds to CD38, used according to any one of embodiments 23 to 25, wherein approximately 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 MDSCs are killed.

[0341] 27. An antibody that specifically binds to CD38, used according to any one of embodiments 14 to 26, wherein Teff or immunosuppressive cells are present in the bone marrow or peripheral blood.

[0342] 28. An antibody that specifically binds to CD38, used according to any one of embodiments 14 to 27, 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, ovarian cancer, stomach cancer, liver cancer, pancreatic cancer, thyroid cancer, squamous cell carcinoma of the head and neck, esophageal cancer or gastrointestinal cancer, breast cancer, fallopian tube cancer, brain cancer, urethral cancer, genitourinary cancer, endometriosis, cervical cancer or metastatic lesions of cancer.

[0343] 29. An antibody that specifically binds to CD38, used according to any one of implementation schemes 1 to 28, wherein the solid tumor lacks CD38 expression.

[0344] 30. An antibody that specifically binds to CD38, used according to any one of embodiments 1 to 29, wherein the antibody competitively binds to CD38 with an antibody comprising a heavy chain variable region (VH) of SEQ ID NO: 4 and a light chain variable region (VL) of SEQ ID NO: 5.

[0345] 31. An antibody that specifically binds to CD38 according to any one of embodiments 1 to 30, wherein the antibody binds at least to the SKRNIQFSCKNIYR region (SEQ ID NO: 2) and the EKVQTLEAWVIHGG region (SEQ ID NO: 3) of human CD38 (SEQ ID NO: 1).

[0346] 32. An antibody that specifically binds to CD38, used according to any one of embodiments 1 to 31, wherein the antibody comprises heavy chain complementarity-determining region (HCDR) 1 (HCDR1), 2 (HCDR2), and 3 (HCDR3) sequences of SEQ ID NO: 6, 7, and 8, respectively.

[0347] 33. An antibody that specifically binds to CD38, used according to any one of embodiments 1 to 32, wherein the antibody comprises light chain complementarity-determining regions (LCDR) 1 (LCDR1), 2 (LCDR2), and 3 (LCDR3) sequences of SEQ ID NO: 9, 10, and 11, respectively.

[0348] 34. An antibody that specifically binds to CD38, used according to any one of embodiments 1 to 33, wherein the antibody comprises the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 6, 7, 8, 9, 10 and 11, respectively.

[0349] 35. An antibody that specifically binds to CD38, used according to any one of embodiments 1 to 34, wherein the antibody comprises VH having 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 4, and VL having 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 5.

[0350] 36. An antibody that specifically binds to CD38, used according to any one of embodiments 1 to 31, wherein the antibody comprises VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5.

[0351] 37. An antibody specifically binding to CD38 used according to any one of embodiments 1 to 29, wherein the antibody comprises the sequences HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3: a. VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15; b. VH for SEQ ID NO: 16 and VL for 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.

[0352] 38. An antibody specifically binding to CD38 used according to embodiment 37, wherein the anti-CD38 antibody comprises: a. VH of SEQ ID NO: 14 and VL of SEQ ID NO: 15; b. VH for SEQ ID NO: 16 and VL for 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.

[0353] 39. An antibody that specifically binds to CD38, used according to any one of embodiments 1 to 38, wherein the antibody is administered in combination with a second therapeutic agent.

[0354] 40. An antibody that specifically binds to CD38, used according to implementation plan 39, wherein the second therapeutic agent is a chemotherapy agent, a targeted anticancer therapy, a standard care drug for the treatment of solid tumors, or an immune checkpoint inhibitor.

[0355] 41. The CD38-specific antibody used according to implementation scheme 40, wherein the immune 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.

[0356] 42. The CD38-specific antibody used according to implementation scheme 39, wherein the second therapeutic agent is administered simultaneously, sequentially, or separately.

[0357] 43. An antibody that specifically binds to CD38, used according to any one of implementation schemes 39 to 42, wherein the patient is treated with radiotherapy.

[0358] 44. An antibody comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 that specifically binds to CD38, for the treatment of patients with solid tumors.

[0359] 45. An antibody specifically binding to CD38 comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, for treating a patient with a solid tumor, wherein the antibody is administered in combination with an anti-PD-1 antibody comprising VH of SEQ ID NO: 22 and VL of SEQ ID NO: 23.

[0360] 46. ​​An antibody specifically binding to CD38 comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, for treating a patient with a solid tumor, wherein the antibody is administered in combination with an anti-PD-1 antibody comprising VH of SEQ ID NO: 24 and VL of SEQ ID NO: 25.

[0361] 47. An antibody that specifically binds to CD38, comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, for enhancing the immune response in a patient.

[0362] 48. An antibody that specifically binds to CD38, comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, for enhancing an immune response in a patient, wherein the antibody is administered in combination with an anti-PD-1 antibody comprising VH of SEQ ID NO: 22 and VL of SEQ ID NO: 23.

[0363] 49. An antibody specifically binding to CD38 comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, for enhancing an immune response in a patient, wherein the antibody is administered in combination with an anti-PD-1 antibody comprising VH of SEQ ID NO: 24 and VL of SEQ ID NO: 25.

[0364] 50. An antibody specifically binding to CD38 comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, for treating a patient with a solid tumor, wherein the antibody is administered in combination with an anti-PD-L1 antibody comprising VH of SEQ ID NO: 26 and VL of SEQ ID NO: 27.

[0365] 51. An antibody specifically binding to CD38 comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5 for treating a patient with a solid tumor, wherein the antibody is administered in combination with an anti-PD-L1 antibody comprising VH of SEQ ID NO: 28 and VL of SEQ ID NO: 29.

[0366] 52. An antibody specifically binding to CD38 comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, for treating a patient with a solid tumor, wherein the antibody is administered in combination with an anti-PD-L1 antibody comprising VH of SEQ ID NO: 30 and VL of SEQ ID NO: 31.

[0367] 53. An antibody specifically binding to CD38 comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, for enhancing an immune response in a patient, wherein the antibody is administered in combination with an anti-PD-L1 antibody comprising VH of SEQ ID NO: 26 and VL of SEQ ID NO: 27.

[0368] 54. An antibody specifically binding to CD38 comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, for enhancing an immune response in a patient, wherein the antibody is administered in combination with an anti-PD-L1 antibody comprising VH of SEQ ID NO: 28 and VL of SEQ ID NO: 29.

[0369] 55. An antibody specifically binding to CD38 comprising VH of SEQ ID NO: 4 and VL of SEQ ID NO: 5, for enhancing an immune response in a patient, wherein the antibody is administered in combination with an anti-PD-L1 antibody comprising VH of SEQ ID NO: 30 and VL of SEQ ID NO: 31.

[0370] Example 1: General Materials and Methods Sample collection and processing Peripheral blood and bone marrow aspirates were collected into heparinized tubes at baseline immediately prior to the first infusion and at specific time points during treatment. When most samples arrived at the central laboratory 24–48 hours after collection, they were evaluated using real-time flow cytometry. Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood separated by density gradient centrifugation and cryopreserved prior to analysis. Frozen PBMC samples were used to simultaneously perform T-cell activation, clonal analysis, and CD38 assays on samples before and after treatment. +Treg inhibition assay analysis.

[0371] These samples were analyzed by flow cytometry at the BARC Global Central Laboratory using a validated immunophenotyping assay to assess NK cells, T cells, B cells, and myeloma cells (CD138). + ) and CD38 expression. In short, blood and bone marrow samples were stained with the following multi-fluorescent dye antibody combination: Cell lineages: PerCPCy5.5α-CD19 (clone 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 The T cell groups were: APCα-CD25 (2A3; BD), PEα-CD127 (HIL-7R-M21; BD), APC-H7α-HLA-DR (G46-6; BD), and PerCPα-CD4 (L200; BD); and the naive / memory T cell group were: APC-H7α-CD4 (RPA-T4; BD), PerCP-Cy5.5α-CD8 (RPA-T4 BD), PEα-CD62L (SK11; BD), and APCα-CD45RA (HI100; BD). CD38 expression was assessed using mAb 003, an antibody labeled with Alexa 647 containing the VH and VL sequences of SEQ ID NO: 14 and SEQ ID NO: 15, as described in U.S. Patent 7,829,693. Blood samples were prepared using various lysis and washing methods. For bone marrow aspirate samples, membrane or intracellular staining was performed using various antibodies. Peripheral blood cells were lysed using Becton Dickinson FACS lysis buffer, and intracellular staining of bone marrow aspirate samples was performed using Fix and Perm cell permeabilizers from Invitrogen. Stained samples were acquired from a FACS Canto II flow cytometer, and data were analyzed using FacsDiva software. Absolute counts of immune cell populations in blood samples and the percentage of lymphocytes in bone marrow samples were determined at all test time points.

[0372] T-cell receptor (TCR) sequencing T cell diversity was analyzed using genomic DNA from PBMC samples via deep sequencing of TCR rearrangements to assess CD8. + T-cell clonal expansion. Using Adaptive Biotechnologies' commercial Immunoseq.™ The assay performed TCR sequencing and was analyzed using a pre-qualified multiplex polymerase chain reaction (PCR) assay (TR2015CRO-V-019), which consisted of forward and reverse primers for the variable (V) gene (forward primer) and the linker (J) gene (reverse primer) of the direct-targeting family. Each V and J gene primer served as a primer pair to amplify the somatic recombinant TCR, and each primer contained a specific universal DNA sequence. After the initial PCR amplification, each amplicon was amplified a second time using forward and reverse primers containing the universal sequence and adapter sequence required by Illumina for DNA sequencing.

[0373] T-cell responses to viral antigens and allogeneic antigens Patient PBMCs were seeded onto 96-well plates (2 × 10⁻⁶). 5 Cells / well) and combined with human cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza virus (2µg / ml, CEF peptide library; PANATecs) ® The cells were stimulated for 5 days with a mixture of 23 major histocompatibility complex (MHC) class I restricted viral peptides or an equivalent number of allogeneic PBMCs irradiated with 25 Gy from healthy donors. Unstimulated PBMCs and PBMCs stimulated with anti-CD3 / CD28 coated beads served as negative and positive controls, respectively. On day 5, interferon-γ (IFN-γ) in cell-free supernatant was measured by sandwich enzyme-linked immunosorbent assay (ELISA; Human IFN gamma ELISA Ready-SET-Go; eBioscience) and used as a surrogate marker for T cell activation.

[0374] Suppression of regulatory T cells (Tregs) of effector cell function: Carboxyfluorescein succinimide (CFSE) dilution assay PBMCs from healthy donors were labeled 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), and sorted using FACS Aria (BD). Sorted effector cells were labeled with carboxyfluorescein succinimide (CFSE; eBioscience) and differentiated based on the presence or absence of CD38. + Treg or CD38 −Treg cells (Treg to effector cell ratio 1:1) were stimulated with an anti-CD3 / CD28-coated strain in RPMI plus 10% fetal bovine serum. Flow cytometry analysis was performed 72 hours later, and percentage dilutions of CFSE were used as a surrogate for T cell proliferation.

[0375] Myeloid-derived suppressor cells (MDSC) phenotyping and DARZALEX ™ (Daralimumab)-mediated ADCC PBMCs from three healthy donors were co-cultured with myeloma cell lines (RPMI8226, U266, H929) for six days and then processed as described in Gorgund et al., Blood 121:2975-87, 2013, to detect granulocyte MDSCs (G-MDSCs) (CD11b). + CD14 - HLA - DR - CD15 + CD33 + The generation of G-MDSCs was assessed. G-MDSCs are absent in normal healthy PBMCs; however, after co-culturing with all three myeloma cell lines, G-MDSCs were present at 5% to 25% of the total PBMC cell population (data not shown). Gating strategies used for flow cytometry assessment of G-MDSCs included CD11b. + As the first gate, CD14 is the second. - and HLA - DR - Set a door, then CD15 + and CD33 + Gating was performed. G-MDSCs were sorted and CD38 expression levels and their response to DARZALEX were assessed. ™ Sensitivity for daralimumab-mediated ADCC. To evaluate DARZALEX... ™ The effect of daratumumab on ADCC / CDC in MDCC was assessed by adding serum containing complement or isotype control to the ADCC assay.

[0376] Analysis of naive T cells and memory T cells During each DARZALEX infusion ™ (Daratumumab) Heparinized peripheral blood samples were previously obtained from patients. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation and stored in cryopreservation medium (RPMI supplemented with 10% human serum and 10% dimethyl sulfoxide) in liquid nitrogen. For FACS analysis, the PBMCs were thawed and 2 × 10⁶ cells were cultured. 6Each cell / group was resuspended in phosphate-buffered saline (PBS) containing 0.05% azide and 0.1% HAS.

[0377] Data Analysis All data analysis and the generation of correlation diagrams were performed using R software (R: A Language and Environment for Statistical Computing, R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria, 2011, ISBN3-900051-07-0 http: / / www.R-project.org / ). The data analysis included all treated subjects with evaluable responses. Throughout, responders were defined as those with optimal responses according to IRC's sCR, VGPR, and PR, while non-responders were defined as those with optimal responses according to IRC's MR, SD, and PD.

[0378] Different statistical comparisons included: (i) baseline levels between responders and non-responders, (ii) baseline to treatment period between responders and non-responders, (iii) percentage change between responders and non-responders, and (iv) ratio change between baseline and treatment period. Each comparison was first tested for normalization using the Shapiro-Wilk test (Royston (1995) Remark ASR94: A remark on Algorithm AS 181: The W test for normality. Applied Statistics, 44, 547–551). It was found that almost all data were not normally distributed. Difference level tests included the nonparametric Wilcox rank-sum test (Hollander and Wolfe (1973), Nonparametric Statistical Methods. New York: John Wiley & Sons, pp. 27–33 (single sample), 68–75 (two sample)) and the Box-Cox transformed t-test (Weisberg, S., (2014) Applied Linear Regression, Fourth Edition, Wiley Wiley, Chapter 7). For the Box-Cox transformation, the decimal value (1e-0.7) was added to a value equal to zero. In all cases, both tests were consistent. Unless otherwise specified, the p-values ​​for the Wilcox rank-sum test are shown in the tables throughout the instruction manual. When testing for differences in baseline between responders and non-responders during treatment, two paired tests were performed for each subject; in all other cases, two unpaired tests were performed.

[0379] Because samples for analyzing various lymphocyte populations were taken at different time points for different dosing regimens, population modeling was performed. Model fitting was performed according to the order of visits. Population modeling for total NK cells and activated NK cells included fitting a broken stick model (Lutz et al., “Statistical model to estimate athreshold 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). Linear mixed-effects models with random intercepts and slopes were suitable for patient population data of B cells, T cell subsets, and leukocytes, monocytes, neutrophils, and lymphocytes (Bates et al., (2014). “lme4: Linear mixed-effects models using Eigen and S4.” ArXiv e-print; submitted to Journal of Statistical Software , http:_ / / _arxiv_org / abs / _1406.5823 ） This linear mixture modeling was performed on a relative day following the start of treatment (ADY). The linear mixture model fitting was done on log-transformed response variables. When response variable values ​​were equal to zero, all response variable values ​​were incremented by 0.1 to allow for modeling at the logarithmic level.

[0380] Example 2: Study of the 54767414MMY2002 design (SIRIUS) The target population for Study 54767414MMY2002 (SIRIUS) consisted of patients with advanced multiple myeloma who had received at least three prior lines of therapy (including a proteasome inhibitor (PI) and an immunomodulatory drug (IMiD)) or were double-refractory to both PI and IMiD. The primary endpoint / final analysis for response assessment was performed using the 2011 IMWG guidelines (Clinical Study Report: An Open-label, Multicenter, Phase 2 Trial Investigating the Efficacy and Safety of DARZALEXTM (daratumumab) in Subjects With Multiple Myeloma Who Have Received at Least 3 Prior Lines of Therapy (Including a Proteasome Inhibitor and IMiD) or are Double-Refractory to a Proteasome Inhibitor and an IMiD) (EDMS-ERI-92399922; de Weers et al., (2011)). J Immunol 186(3):1840-1848) Based on evaluations from the Independent Review Committee (IRC) and computer algorithms.

[0381] These assessments include: overall response rate (ORR), duration of response, time to response and best response, clinical benefit rate, time to progression (TTP), progression-free survival (PFS), and overall survival (OS).

[0382] In this study, a total of 124 participants received DARZALEX. ™ (Daralimumab) treatment (de Weers et al., (2011)) J Immunol 186(3):1840-1848). Eighteen patients received 8 mg / kg, and 106 patients received 16 mg / kg. The dosing regimens were as follows: Group A: DARZALEX ™ (Daratumumab) 16 mg / kg: Cycle 1 and 2: Days 1, 8, 15 and 22 (weekly); Cycles 3 to 6: Day 1 and Day 15 (every other week); Cycle 7+: Day 1 (every 4 weeks). Each cycle is 4 weeks.

[0383] Group B: DARZALEX ™(Daratumumab) 8mg / kg: Cycle 1+: Day 1 (every 4 weeks).

[0384] The primary objective of this study was to determine the DARZALEX levels as measured by ORR (CR+PR). ™ The efficacy of two treatment regimens of daratumumab in patients with multiple myeloma who have received at least three prior line therapies (including PI and IMiD) or whose disease is doubly refractory to PI and IMiD (Clinical Study Report: An Open-label, Multicenter, Phase 2 Trial Investigating the Efficacy and Safety of DARZALEX). ™ (daratumumab) in Subjects With Multiple Myeloma Who Have Received at Least 3 Prior Lines ofTherapy (Including a Proteasome Inhibitor and IMiD) or Double Refractoryto a Proteasome Inhibitor and an IMiD. EDMS-ERI-92399922).

[0385] Secondary objectives of this study included evaluating DARZALEX. ™ The safety and tolerability of daratumumab, measures of additional efficacy (e.g., clinical benefit, TTP, PFS, and OS), and assessments of pharmacokinetics, immunogenicity, and pharmacodynamics, as well as the exploration of its effects on DARZALEX, are being investigated. ™ Predictive biomarkers for daralimumab response. Additional research-related information is available from the clinical study protocol (Clinical Study Report: An Open-label, Multicenter, Phase 2 Trial Investigating the Efficacy and Safety of DARZALEX). ™(daratumumab) inSubjects With Multiple Myeloma Who Have Received at Least 3 Prior Lines ofTherapy (Including a Proteasome Inhibitor and IMiD) or Double Refractory to a Proteasome Inhibitor and an IMiD. EDMS-ERI-92399922).

[0386] In Phase 1 of Part 1, one patient (6%) responded in the 8 mg / kg group and five patients (31%) responded in the 16 mg / kg group. Therefore, only the 16 mg / kg group was expanded in Phase 2 of Part 1 and Part 2.

[0387] In the 16 mg / kg group, 31 patients achieved a partial response (PR) or better based on IRC assessment; the objective response rate (ORR) was 29% (95% CI: 21%, 39%). Three patients (3%) achieved a stable complete response (sCR), and 13 patients (12%) achieved a volume-to-volume (VGPR) or better.

[0388] Example 3: DARZALEX in patients participating in the 54767414MMY2002 study (SIRIUS) ™ Effects of daratumumab on T cell expansion and activity CD38 is expressed by various immune cells and hematopoietic cells. Extensive immunoassays using flow cytometry were performed to examine DARZALEX. ™ The effects of daratumumab on immune cell subsets and the association between baseline levels of these cells and clinical response. At baseline and subsequent DARZALEX... ™ During daratumumab treatment, flow cytometry was used to assess various cell populations in peripheral blood and bone marrow aspirates, including T cells (CD3+) in peripheral blood and bone marrow aspirates. + Cells, CD4 + Cells, CD8 + Cells and regulatory T cells (Tregs), B cells (CD19) + NK cells, monocytes (CD14) + (1) white blood cells and neutrophils to monitor changes in these cell populations in responders and non-responders.

[0389] Lymphocytes, white blood cells, monocytes and neutrophils The study investigated peripheral blood counts of white blood cells, lymphocytes, monocytes, and neutrophils in responders and non-responders. DARZALEX was administered at doses of 8 mg / kg and 16 mg / kg. ™ An increase in total lymphocytes was observed in all responders treated with daratumumab. Figure 1 Linear mixed-effects modeling shows an increase of 0.8 × 10⁻⁶ per 100 days on a logarithmic scale. 6 Cells / µL (CI=0.06, 0.11). A slight increase in monocytes and leukocytes was observed, with a significant increase of 0.03 × 10⁻⁶ cells per 100 days on a logarithmic scale. 6 Cells / µL (CI=0.01, 0.04) and 0.03 × 10 6 Cells / µL (CI=0.01, 0.05). Although some patients developed neutropenia, the median neutrophil count remained consistent with baseline and did not change significantly.

[0390] The baseline levels of these cell populations were compared between the response groups. Using the Wilcoxon signed-rank test, it was found that the baseline levels of any of these cell types were the same across the different response groups (Table 1).

[0391] Table 1 : NK cells Over time, total NK cells (CD16) + CD56 + ) and activated NK cells (CD16) + CD56 dim (with DARZALEX) ™ (Daratumumab) treatment increased and decreased (data not shown).

[0392] B cells In DARZALEX ™ During daratumumab treatment, B cells (CD45+) in peripheral blood or bone marrow aspirates were measured in both responders and non-responders. + CD3 - CD19 + Absolute counts over time. B cells increase slightly in whole blood but remain constant in bone marrow aspirate. A linear mixture model of B cells in peripheral blood reveals the overall DARZALEX. ™ (Daratumumab) During treatment, the dosage should be increased at least 0.1 × 10⁻⁶ every 100 days on a logarithmic scale. 6 Cells / µl [CI=0.04, 0.16]. B cells (CD45+) in bone marrow aspirates from responders or non-responders during daratumumab treatment.+ CD3 - CD19 + The percentage of B cells ( / lymphocytes) remained unchanged (p = 0.1 and 0.4, respectively). Furthermore, there was no evidence that B cell counts were different at baseline between responders and non-responders (p = 0.5).

[0393] T cells Note that lymphocytes are being used with DARZALEX ™ (Daralimumab) treatment increases ( Figure 1 Although B cells showed only a minimal increase (see above). To further investigate, various T cell populations (CD3+) in peripheral blood and bone marrow were studied. + CD4 + CD8 + T cells, regulatory T cells).

[0394] DARZALEX ™ Following daratumumab treatment, CD3 levels in peripheral blood + T cells, CD4 + T cells and CD8 + Increased T cells (absolute count / µl and percentage of lymphocytes). Figure 2 The peripheral blood CD3 levels of each patient were shown. + T cells (CD45) + CD3 + The percentage change in the absolute count of ( ) relative to baseline over time. The black line in the figure shows the absolute median count × 10 for all patients. 6 Cells / µL. The figure only includes visits with more than two observations. Figure 3 The peripheral blood CD4 levels of each patient were shown. + T cells (CD45) + CD3 + CD4 + The percentage change in the absolute count of patients relative to baseline over time. The black line in the graph represents the median for all patients. The graph only includes visits with more than two observations. Figure 4 The peripheral blood CD8 levels of each patient were shown. + T cells (CD45) + CD3 + CD8 + The percentage change in absolute counts relative to baseline over time. The black line in the figure represents the median for all patients. The figure only includes visits with more than two observations. Linear mixture modeling of absolute counts in peripheral blood reveals DARZALEX ™ Mean total T cells (CD455) after daratumumab treatment + CD3+ It increases by 0.13 × 10⁻¹⁰ every 100 days on a logarithmic scale. 6 Cells / µl (CI=0.1, 0.15). CD8 was found. + T cells increased significantly, increasing by 0.16 × 10⁻⁶ per 100 days on a logarithmic scale. 6 Cells / µl (CI=0.13, 0.19). CD4 was found. + Cell counts increased moderately, increasing by 0.11 × 10⁻⁶ per 100 days on a logarithmic scale. 6 Cells / µl (CI=0.09, 0.13).

[0395] For each T cell subset, responders showed a higher maximum percentage change in absolute count relative to baseline (CD3+) compared to non-responders. + 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 Wilcoxon signed-rank test, used to compare the percentage change in the absolute count of each T cell subset in peripheral blood relative to baseline between responders and non-responders.

[0396] Table 2 : Similarly, in the bone marrow, DARZALEX was found. ™ Total T cells (CD45) in responders and non-responders during daratumumab treatment + CD3 + (as a percentage of lymphocytes) and CD8 + T cells (CD45) + CD3 + CD8 + The percentage of lymphocytes (CD3) increased significantly. + For respondents, p = 3.8147e-06; for non-responders, p = 9.8225e-05; CD8 + (Respondents p=3.8147e-06, non-responders p=0.0003). In any clinical response group, CD4 in the bone marrow... + The number of T cells remained unchanged. Table 3 shows the Wilcoxon signed-rank test results for various T cells as a percentage of lymphocytes in the bone marrow. Figure 5 DARZALEX was shown ™ (Daratumumab) CD45 levels increase over time during treatment. + CD3 +Percentage of cells (%) (The figure includes responders and non-responders). Figure 6 DARZALEX was shown ™ (Daratumumab) CD45 levels increase over time during treatment. + CD3 + CD8 + Percentage of cells (%) (The figure includes responders and non-responders).

[0397] Table 3 : Although T cell increases were observed in peripheral blood and bone marrow in both responders and non-responders, the percentage change relative to baseline was greatest in responders. To differentiate between responders and non-responders, DARZALEX... ™ Did they have different levels of CD3 before daratumumab treatment? + T cells, CD4 + T cells and CD8 + T cells were compared with baseline measurements for each subgroup in peripheral blood.

[0398] Wilcoxon signed-rank test showed no statistically significant difference at baseline between responders and non-responders in absolute T cell counts in peripheral blood (Table 4) or percentage of T cells in total lymphocytes in bone marrow (Table 5).

[0399] Table 4 : Table 5 : T regulatory cells Treg cells were identified as CD3 in the sample. + CD4 + CD25 + CD127 dim Cell population. Evaluation using DARZALEX. ™ CD8+ levels in peripheral blood and bone marrow of patients treated with daratumumab + The ratio of T cells to Tregs over time. This ratio increases in peripheral blood and bone marrow. Figure 7A The peripheral blood CD8 levels of all patients at each time point are shown. + / Treg and CD8 + / CD4 + Median cell ratio. Figure 7B The data show CD8 levels in the bone marrow of all patients at each time point. + / Treg and CD8 + / CD4 + Median T cell ratio. CD8+ levels in peripheral blood (Table 6) and bone marrow (Table 7) over time of treatment were determined using the Wilcoxon signed-rank test. + Treg and CD8 + / CD4 + The ratio of absolute counts changed significantly.

[0400] In a combined data analysis of the SIRIUS and GEN501 studies (Example 6), CD8+ in peripheral blood + / CD4 + With CD8 + The median percentage of / Treg cells at week 8 (for CD8) + / CD4 + p = 5.1 × 10 -5 For CD8 + / Treg, p=1.8×10 -7 ) and week 16 (for CD8) + / CD4 + p=0.00017, for CD8 + / Treg, p=4.1×10 -7 () increases. Similarly, in the bone marrow, CD8... + / CD4 + and CD8 + Median percentage of / Treg cells compared to baseline (for CD8) + / CD4 + p=0.00016; for CD8 + / Treg, p=2.8×10 -7 The levels increased during treatment (week 12 ± cycle 1). No significant difference was observed between responders and non-responders.

[0401] Table 6 : Table 7 : Example 4: Study Design (GEN501) Study GEN501 (NCT00572488) to evaluate DARZALEX ™ (Daralimumab) was used as a monotherapy for patients with doubly refractory MM. Sample isolation, processing, and statistical analysis were performed as described in Examples 1 and 2. This study has been described by Lokhorst et al., N Eng J Med 373:1207-19, 2005.

[0402] In short, the study of GEN501 is DARZALEX ™ The first human clinical study of daratumumab in patients with multiple myeloma (MM). This study is a two-part, phase 1, dose-escalation safety study. Part 1 is an unblinded, dose-escalation study; Part 2 is an unblinded, single-arm study based on the dose level determined in Part 1.

[0403] In Part 1, 10 DARZALEX samples were evaluated. ™ (Daratumumab) Dosage levels: 0.005 mg / kg, 0.05 mg / kg, 0.10 mg / kg, 0.50 mg / kg, 1 mg / kg, 2 mg / kg, 4 mg / kg, 8 mg / kg, 16 mg / kg, and 24 mg / kg. One (+3) patients were assigned to each of the two lowest dose groups, and the remaining eight dose groups were assigned to three (+3) patients using standard methods. Part 2 was a non-blinded, single study comprising two dose levels, 8 mg / kg and 16 mg / kg. Part 1 included 32 patients, and Part 2 included 72 patients.

[0404] Example 5: DARZALEX ™ (Daratumumab) treatment induces clonal T-cell activity in patients. Given that CD8 was found in both peripheral blood and bone marrow in the MY2002 study + T cell expansion using Immunoseq ™ High-throughput next-generation sequencing of T-cell receptors (TCRs) was performed using assays to determine amplified CD8+. + Whether T cells actually possess clonality to indicate an adaptive immune response. A total of 17 patient samples from participants in the GEN501 study were evaluated (n=6 responders, i.e., ≥PR; n=11 non-responders, i.e., MR, SD, PD).

[0405] TCR sequencing revealed DARZALEX ™ (Daratumumab) treatment significantly increases clonal variation among patients. Figure 8A DARZALEX was shown ™ The correlation between T-cell clonality before and after daratumumab treatment (p=0.0056). Figure 8B The fold change in clonal variability is shown for individual patients. Respondents are marked with an asterisk. This data indicates the effectiveness of DARZALEX. ™ The T-cell expansion indicated by daratumumab treatment may actually be clonal.

[0406] Compared to non-responders, responders showed greater overall amplification in the TCR library (as measured by abundance variation; CIA). Figure 8C The CIA% of individual patients is shown. Group A: Responders; Group B: Non-responders. A statistically significant difference was observed between responders and non-responders (p=0.037). Figure 8D The sum of absolute changes in abundance (CIA) for each expanded T cell clone is shown in responders and non-responders. Figure 8E The maximum CIA percentage for each individual patient is shown. Group A: Responders; Group B: Non-responders. A statistically significant difference was observed between responders and non-responders (p=0.048). Figure 8F The maximum CIA of a single T cell clone is shown in responders (Group A) and non-responders (Group B).

[0407] The CIA was obtained by using Fisher's exact test to identify significant differences in clonal abundance between two samples (DeWitt et al., J. Virol. 2015), and the absolute change in abundance of each amplified clone was summed.

[0408] Example 6: DARZALEX ™ Immunomodulatory effects of daratumumab in patients participating in the GEN501 study To assess various T cell and B cell populations in responders and non-responders participating in GEN501.

[0409] lymphocytes Similar to the SIRIUS (MMY2002) study, in DARZALEX ™ During daratumumab treatment, lymphocytes increased in both peripheral blood and bone marrow. This increase was attributed to CD4+. + and CD8 + An increase in the number of cells.

[0410] CD8 + Central memory cells In a subset of 17 patients participating in the GEN 501 study, the effects of using DARZALEX were investigated. ™ (Daratumumab) treatment patients' CD8 counts over time + T-cell phenotype. Using a standard protocol, CD8+ cells from the patient were analyzed. + Cell recognition is initial (CD45RO- / CD62L) + ) (T N ) or central memory (T CM (CD45RO) + / CD62L +high )cell.

[0411] Figure 9A CD8 is shown + Initial cell percentage (CD8) + (cells%), and Figure 9B CD8 is shown + Central memory cells%. DARZALEX ™ (Daratumumab) treatment significantly reduced initial CD8 counts. + Number of T cells (week 8) p =1.82×10 -4 And add CD8 + Number of memory T cells (week 8) p =4.88×10 -2 This indicates the transformation of naïve cytotoxic T cells into memory T cells that can be activated against specific antigens. White squares indicate patients achieving at least a minimal response (≥MR), and black squares indicate patients with stable or progressive disease. In patients who respond to treatment, CD8+... + The significant reduction in naïve T cells was evident (data not shown). Figure 9C DARZALEX was shown ™ (Daratumumab) treatment increased the percentage of HLA-I class-restricted T cells, which in part drove virus-specific and allogeneic reactive T cell responses. Figure 9D The expanded effector memory T cells were shown to express low levels of CD38. It is important to note that these T cells exhibited normal or even increased functional activity against viral peptides and allogeneic antigens (see Example 8). From these functional results, we conclude that in DARZALEX… ™ During daratumumab treatment, antigen-experienced T cells showed improved amplification or activity against viruses and allogeneic antigens. These data suggest that, unlike regulatory cell subsets, effector T cells do not require CD38 expression to function and proliferate normally.

[0412] CD38-positive regulatory T cells The observed potent proliferation and increased activity of cytotoxic T cells, along with recent literature, indicate that several immunosuppressive cell subsets express DARZALEX. ™ The effects of daratumumab on the CD38-mediated effects of regulatory cell populations, including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and regulatory B cells (Bregs), were investigated.

[0413] Regulatory T cells (Tregs) (CD3) were isolated using a standard protocol. + CD4 + CD25 + CD127dim The frequency of Tregs was analyzed using flow cytometry.

[0414] Prior to Treg activation, the peripheral Treg subset (10% ± 10%) expressed high levels of CD38. Figure 10A The above figure shows CD3 at the baseline. + CD4 + Frequency of Treg cells in the cell population (P4 cell population). Figure 10A The figure below shows a subset of Tregs expressing high levels of CD38 (P5 cell population). These CD38... + Treg vs DARZALEX ™ (Daratumumab) is highly sensitive to treatment and is available at DARZALEX. ™ (Daratumumab) showed a significant and almost immediate decrease after the first dose (n=17 patients; compared with baseline, at week 1, P = 8.88×10 -16 DARZALEX ™ The frequency of Tregs after (daratumumab) treatment is shown in Figure 10B The image above shows the P4 cell population. Figure 10B The following diagram shows CD38 high Treg (P5 cells) were infused with DARZALEX during the first infusion. ™ The Treg groups most significantly depleted after (daralimumab) are affected. These CD38 groups... + Treg throughout DARZALEX ™ (Daralimumab) treatment was maintained at exhaustion (compared to baseline, at weeks 1, 4, and 8 respectively). p = 8.88×10 -16 1.11×10 -15 and 1.50×10 -11 ). Figure 10C The results show CD38 levels at baseline, week 1, week 4, week 8, relapse, and month 6 after the end of treatment (EOT). high Treg occupies a total of CD3 + Percentage of cells. CD38 high Treg was restored to baseline at this point in time. CD38 + Changes in Tregs were similar between patients who responded to treatment and those who did not; however, CD8... + T cell:Treg ratio at week 8 against DARZALEX ™ (Daralimumab) showed a significantly higher rate of response in patients ( P = 0.00955; Figure 10D ).

[0415] To evaluate the use of DARZALEX ™ (Daratumumab) treatment for CD38 + Potential biological relevance of Treg consumption, assessing CD38 + Treg and CD38 − Treg on autologous CD3 + The inhibitory capacity of T cells. In a series of experiments using samples from multiple healthy donors, CD38... + Treg compared to CD38 − Treg cells (53.2% cell proliferation observed) or negative control cells (74.9% cell proliferation observed) more strongly inhibited T cell proliferation (9.9% cell proliferation observed). Figure 10E ).

[0416] Because MDSCs are not easily detected in frozen PBMC samples, CD38 + Granulocyte MDSC (CD11b) + CD14 - HLA-DR - CD15 + CD33 + (Separated from patients at baseline and those who have received one DARZALEX) ™ (Daratumumab) infusions are generated in vitro in PBMCs of patients. Figure 11 The flow cytometry histograms of the identified MDSCs are shown. Figure 11 (Top histogram, showing the cell population outlined in the box). Approximately half of the MDSCs express CD38 (…). Figure 11 (Middle image; circled P7 cell population). CD38 high MDSC is using DARZALEX ™ (Daralimumab) treatment almost exhausted the patients ( Figure 11 (Bottom image; circled P7 cell population).

[0417] In non-responders and patients with at least a minimal response to treatment, CD38 high Lineage-specific MDSC using DARZALEX ™ (Daratumumab) is depleted over time during treatment. Figure 12 CD38 is shown high The percentage of MDSCs decreased to near 0% in patients at weeks 1, 4, or 8 post-treatment. CD38 high Lineage-nonspecific MDSCs returned to baseline after treatment.

[0418] It has the largest CD38 in lineage-nonspecific MDSCs. +Patients in the group showed resistance to DARZALEX ™ (Daratumumab) treatment achieves the best and most durable response. Figure 13 It shows the highest CD38 high MDSC percentage (e.g.) Figure 11 Patients 2, 4, 15, 16, and 17, who were classified as PR or MR patients, had at least 8 months of progression-free survival (PFS).

[0419] CD38 high Lineage-specific MDSC also affects DARZALEX ™ (Daratumumab) in vitro induced ADCC sensitivity. CD38 from two donors was used. high ADCC was measured in MDSCs and Daudi cells, which served as control target cells, with an effector cell:target cell ratio of 50:1. Figure 14 The experimental results from a single donor are shown. (DARZALEX) ™ (Daratumumab) induces lysis of MDSC cells.

[0420] CD38 + Breg was measured in patients (n = 16) treated with DARZALEXTM (daratumumab) and was similar to CD38. + Treg, which was consumed after the first dose of DARZALEXTM (daratumumab) (compared to baseline, at week 1), p =0.0018; paired Wilcoxon rank test), and remained low while the patient was undergoing treatment ( Figure 15A FACS-sorted Breg cells produce IL-10 upon stimulation. Figure 15B ).

[0421] In summary, these observations suggest that immunosuppressive CD38 + The consumption of MDSC, Breg, and Treg on DARZALEX ™ (Daratumumab) is an important contributing mechanism to the changes in T cell population and clonalness induced by daratumumab.

[0422] Example 7: CD38 + MDSC cells are present in cancer patients The percentage of MDSCs in the peripheral blood of patients with NSCLC or prostate cancer was studied using flow cytometry. - CD14 - HLADR low / - ) and its CD38 expression.

[0423] In the analyzed samples of PBMCs from NSCLC and prostate cancer patients, the percentage of MDSCs ranged from approximately 10% to 37% and from approximately 10% to 27%, respectively. In PBMCs from NSCLC patients, Lin... - CD14 + HLADR - / low CD38 expression was identified in MDSCs, and in PBMCs from prostate cancer patients, CD38 expression was identified in 70%–100% of MDSCs.

[0424] Example 8: DARZALEX ™ (Daratumumab) enhances antiviral T-cell responses To further evaluate DARZALEX ™ The effects of daratumumab on T cell activation and function, as observed in DARZALEX ™ In patients (n = 7) treated with daratumumab, peripheral T cell IFN-γ production in response to viral and allogeneic antigens was measured, yielding a range of clinical outcomes. Patients with a PR or better response were treated with DARZALEX. ™ Following daratumumab treatment, a significant increase in IFN-γ secretion in response to the virus and allogeneic antigens was observed at at least one time point during treatment compared to baseline, indicating that T cell function was not impaired due to low CD38 expression (see Example 6). Figure 9C Similar to the TCR clonal data, in the DARZALEX... ™ This increase was more pronounced in patients who responded to daratumumab than in those who did not. Figure 16A The antiviral response of a representative patient with VGPR is shown. Figure 16B The antiviral response of a representative patient with complete remission (CR) is shown. Figure 16C The antiviral response of a representative patient with PD is shown. Figure 16D The antiviral response of a representative patient with MR is shown. In the figure, error bars represent the standard error of the mean of two cultures. An asterisk indicates a statistically significant change between the comparisons shown. The best response according to the independent review committee is shown. Consistent with these results, in patients with VGPR ( Figure 16E ) or CR ( Figure 16F Viral reactive T cells in patients with DARZALEX ™ (Daratumumab) treatment showed an increase in proliferative capacity.

[0425] Example 9: CD38-expressing immune cell subtypes against DARZALEX ™Sensitization mechanism of daratumumab Data from both the GEN501 and SIRIUS studies indicate that some CD38-expressing immune cells are depleted (NK cells, regulatory T cells (Tregs), regulatory B cells (Bregs), and myeloid-derived suppressor cells (MDSCs)), while those expressed by DARZALEX... ™ (Daratumumab) therapy increases the number of other immune cells expressing CD38 (cytotoxic T cells and helper T cells).

[0426] To address the sensitivity mechanism, CD38 expression levels were assessed in various subsets of immune cells from healthy donors and multiple myeloma patients participating in the GEN501 or SIRIUS studies. Figure 17A A histogram of CD38 expression in immune cells from healthy donors is shown, and Figure 17B The histogram shows the expression of CD38 in immune cells from multiple myeloma patients. In healthy donors, CD38 expression was highest in NK cells, followed by monocytes, B cells, and T cells. In multiple myeloma patients, CD38 expression was highest in plasma cells, followed by subsets of B cells, NK cells, monocytes, and T cells. Figure 17C The comparison of mean fluorescence intensity (MFI) of CD38 in NK cells, Tregs, Bregs, B cells, and T cells from patients with relapsed and refractory myeloma shows that NK cells express the highest level of CD38 after plasma cells, followed by regulatory T cells (Tregs) and regulatory B cells (Bregs).

[0427] Besides CD38 expression, other cell surface proteins such as complement inhibitory proteins (CIP; CD46, CD55, CD59) can interact with DARZALEX. ™ Sensitivity or resistance to daratumumab is related to its presence. In vitro assessment of CIP in immune cell subsets revealed that NK cells expressed very low levels of CD59 and CD55, while other T cell and B cell populations expressed much higher levels. This may also contribute to the different levels of DARZALEX expression in immune cell subtypes. ™ The sensitivity of (daratumumab) is variable (data not shown).

[0428] discuss This study describes DARZALEX ™ Previously unknown immunomodulatory effects of daratumumab, through reducing CD38 + Immunosuppressive cell populations, along with the induction of helper T cells and cytotoxic T cells, the production of IFN-γ in response to viral peptides, and increased TCR clonality, indicate an improved adaptive immune response.

[0429] This study shows that MDSC and Breg express CD38 and are susceptible to DARZALEX. ™ (Daralimumab) Treatment Effects. These cells are known to exist in the tumor microenvironment and contribute to tumor growth, immune evasion, angiogenesis, metastasis, and inhibition of cytokine production. In addition to these CD38 cells... + In addition to the suppressor cell subset, a new subset of regulatory T cells (CD4+) was also identified. + CD25 + CD127 dim These cells also express high levels of CD38 and exhibit superior autologous T cell suppression ability. These cells respond to DARZALEX... ™ (Daralimumab) was also very effective and significantly reduced in treated patients. DARZALEX ™ (Daralimumab)-mediated effects on these CD38 + Elimination of immunomodulatory cells can reduce local immunosuppression within the myeloma microenvironment and allow positive immune effector cells to proliferate, thus contributing to the antitumor response.

[0430] In fact, a wide population of T cells (including CD4+) has been observed in both peripheral blood and bone marrow. + and CD8 + A significant increase in both (i.e., tumors). Specific CD8 + Subgroup by DARZALEX ™ (Daralimumab) therapy alterations included a significant reduction in naïve T cells and the accompanying effector memory CD8. + The significant increase in T cells indicates a shift in effector T cells towards an antigen-experienced phenotype, which retains immune memory and is responsive to tumor antigens. CD8 + :CD4 + and CD8 + The rate of Tregs also increased significantly with treatment, indicating a shift from positive to negative immunomodulators.

[0431] To evaluate the amplified CD4 + and CD8 + Whether the T cells were essentially clones was examined in a subgroup of patients using DARZALEX. ™ (Daralimumab) treatment significantly increased T-cell clonality, even in patients with optimal SD response or those who had progressed. Therefore, the increased T-cell clonality cannot be simply attributed to a reduction in tumor burden. However, in patients with good clinical response, the bias in T-cell clonality was greater and correlated with CD8+. +The increase in T cells is associated with the observed increase in DARZALEX cells, which suggests that the increase is related to the increase in T cells. ™ T-cell expansion induced by daratumumab treatment is antigen-driven. This is significant in this patient population, which has undergone heavy pretreatment (median of 5 prior lines of therapy) and is not expected to produce a robust anti-tumor immune response. In addition to increased TCR clonality, DARZALEX... ™ Patients who responded to daratumumab showed increased T-cell responses to pre-existing viral antigens and allogeneic antigens, indicating that the immune system was rescued from an immunosuppressive state.

[0432] Use DARZALEX ™ (Daratumumab) treatment causes a decrease in immunosuppressive MDSCs and regulatory T cells and B cells. These decreases are accompanied by CD4 reduction. + Helper T cells and CD8 + Expansion of cytotoxic T cells. Using DARZALEX. ™ (Daralimumab) treatment also increased T cell clonal and functional antiviral responses, as measured by IFN-γ production. These observations suggest that T cells continue to function normally despite low CD38 expression, and indicate that the increased T cell response may be due to the depletion of regulatory cells. Furthermore, these changes in T cell expansion, activity, and clonalness were observed in DARZALEX... ™ The effect was more pronounced in patients who responded to daralimumab than in those who did not. (From DARZALEX) ™ Relapse after daratumumab therapy was associated with the reversal of many of these changes. This suggests that DARZALEX ™ (Daratumumab) may contribute to clinical response and efficacy through another previously uncharacterized mechanism of action via immune modulation.

[0433] Recently, antibodies that promote anti-tumor immune responses rather than directly target cancer have shown efficacy in a range of settings. In patients with advanced solid tumors and hematologic malignancies such as Hodgkin's lymphoma, antibodies that inhibit CTLA-4 and PD-1 promote T cell proliferation and enhance T cell activation, leading to prolonged survival and delayed disease relapse. By enhancing anti-cancer immunity, these immunomodulatory antibodies not only induce clinical responses but also prevent disease relapse.

[0434] Example 10: Use of single agent DARZALEX in Part 2 clinical study of 54767414MMY2002 (SIRIUS) ™ Serum proteomics analysis of multiple myeloma patients treated with daratumumab Biomarker Sample Collection and Processing Peripheral blood samples were collected in standard serum separation tubes (2.5 mL to 5 mL), and the serum aliquots were frozen and shipped to SomaLogic, Inc (Boulder, CO) for multianalyte serum proteomic analysis.

[0435] Serum proteomic analysis was performed at SomaLogic using the pre-validated SOMAscan assay, which measures 1129 protein analytes using SOMAmer affinity-based molecular measurements. SOMAmer reagents are protein affinity reagents based on single-stranded DNA. The assay uses a small input sample (150 µL plasma) and converts the protein signal into a SOMAmer signal quantified by a custom-designed DNA microarray.

[0436] Each SOMAmer contains 4 functional parts: 1. Unique protein recognition sequence 2. Biotin used for capture 3. Optically cut joints 4. Fluorescent molecules used for detection Unique protein recognition sequences utilize DNA and bind chemically modified nucleotides that mimic amino acid side chains, expanding the diversity of standard aptamers and enhancing the specificity and affinity of protein-nucleic acid interactions (Gold et al., PLoS One 5:e15004, 2010). Aptamers are selected by SELEX. SOMAmer reagents use proteins in their native conformation for selection because such SOMAmer reagents require intact tertiary protein structures for binding. SOMAmer reagents do not detect unfolded or denatured, potentially inactive proteins.

[0437] The master mixture of SOMAmer reagents was grouped by sample type and dilution. The reagents were pre-bound to streptavidin beads prior to sample incubation. Proteins in the sample were bound to their homologous SOMAmers during equilibration, washed, incubated with NHS-Biotin, washed again, and then the beads were exposed to UV light to cleave photocleavable linkers. The eluent contained SOMAmer reagents bound to their biotinylated proteins. Strepavidin captured unbound SOMAmer reagents and was subsequently washed away. In the final eluent, SOMAmer molecules were released from their homologous proteins under denaturing conditions. The final eluent was hybridized to a custom-designed Agilent DNA microarray, and fluorophores from the SOMAmer molecules were quantified in relative fluorescence units (RFUs). RFUs were proportional to the amount of protein in the sample.

[0438] The samples from the MMY2002 study were tested in two batches. The first batch of 180 samples comprised paired serum samples from 90 treatment subjects on day 1 of cycle 1 (C1D1, baseline) and day 1 of cycle 3 (C3D1). These 180 samples were analyzed together on three independent SomaScan plates. The second batch consisted of 50 C1D1 samples, including 35 replicates from batch 1.

[0439] Data Analysis Input dataset and definition The data analysis included treated subjects with evaluable responses. Throughout the report, responders were defined as subjects with overall best responses of sCR, VGPR, and PR (according to IRC, for MMY2002), as well as stable disease (SD) subjects with minimal response (MR) or SD, while non-responders were defined as subjects with overall best responses of progressive disease (PD) (according to IRC, for MMY2002).

[0440] Somalogic data preprocessing Batch comparison Batch 1 and batch 2 of the MMY2002 samples were tested on two different versions of the SOMAscan platform. The differences between the two versions were small and included three SOMAmer sequences that varied between versions (CTSE: 3594-6_1 -> 3594-6_5, FCN1: 3613-62_1 -> 3613-62_5, BMPER: 3654-27_1 -> 3654-27_4). These differences were removed from the analysis.

[0441] Following SomaLogic's standard inter-plate calibration workflow, plate measurements from three batches 1 are calibrated by defining a plate-width calibration scaling factor for each SOMAmer and by calculating the ratio of a master mixture-specific global reference value to the median of 7-inch plate control calibration measurements. The plate-specific scaling factor for each SOMAmer reagent is applied equally to each sample on the plate.

[0442] Given the different SOMAscan platform versions for batches 1 and 2, inter-batch system variability corrections were performed by utilizing repeated measurements from 35 samples across batches and employing a modified implementation of SomaLogic's standard inter-board calibration workflow. For each SOMAmer, for each of the 35 repeated samples, the ratio of the calibrated measurement from batch 1 to the uncalibrated measurement from batch 2 was calculated. ri,jThe median of these 35 ratios was used to define the modified SOMAmer-specific calibration scaling factor for batch 2 samples. Then, these calibration scaling factors are implemented in the same manner as the standard SOMAscan procedure.

[0443] Once the corrected calibration scaling factor was calculated, the distribution of all scaling factors was plotted for each batch of the analysis to assess the presence of outliers. Due to poor reproducibility, nine SOMAmers with extremely large or small calibration values ​​(>0.25 and <3) were removed from the analysis.

[0444] After completing batch alignment and SOMAmer filtering for MMY2002, log2 transformation was applied to all protein concentration values ​​of MMY2002 to make the data more consistent with a normal distribution and improve the performance of parametric statistical tests.

[0445] Confounding variable correction Principal component analysis (PCA) on the central and extended datasets was used to estimate the portion of dataset variance explained by metavariables (such as demographics, response categories, and sample time points) and identify potential confounding factors. Simple linear models were fitted to identify the highest-ranking PCs significantly associated with each variable of interest. The significance of these associations was determined using the Wald test, and the fraction of PC variability explained by this model was estimated using the R² of the fit. For the MMY2002 data, locus ID was found to be associated with PC1 and explained the largest portion of dataset variability (≥7.37%, p-value = 3.71 × 10⁻⁹). To reduce the influence of the sampling locus-related effect in the data, ComBat28 was used to correct for the locus ID effect.

[0446] Duplicate sample merging Data from 35 samples repeated between MMY2002 batch 1 and batch 2 were combined by calculating the average value for each protein.

[0447] Protein concentration difference analysis Respondents to non-respondents The following two complementary methods were used to statistically compare the protein concentration distributions in responders and non-responders of DARZALEXTM (daratumumab) at baseline and during treatment: (i) Wilcoxon rank-sum test for each individual SOMAmer (Hollander and Wolfe, Ninparametric Statistical Methods. New York: John Wiley & Sons. 1973. 27-33 (single sample), 68-75 (two sample); and (ii) Limma analysis simultaneously for all SOMAmers (Ritchie, ME et al., Nucleic Acids Res. 2015; 20:43(7):e47). The Benjamini-Hochberg (BH) method for multiple hypothesis correction was used (Benjamini and Hochberg, (1995)). JR Statist. 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) Adjust all p-values. When the adjusted p-value < 0.05, the null hypothesis of no difference in expression was rejected.

[0448] During treatment, at baseline The following three alternative statistical methods were used to compare protein levels at baseline during treatment: (i) two-way repeated measures ANOVA; (ii) Wilcoxon signed-rank test; and (iii) Friedman test (Johnson et al., (2007)). Biostatistics8(1):118-127). All p-values ​​were adjusted to control for FDR using the BH method for multiple hypothesis correction (Benjamini and Hochberg, JR Statist. 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) was also used to determine whether significant time point interactions with response categories occurred for each SOMAmer. A modified Wilcoxon rank-sum test was applied as a post-hoc comparison test to specifically determine whether responders and non-responders exhibited different treatment effects by calculating the difference between protein concentration values ​​during treatment and at baseline for each treated individual and performing the Wilcoxon rank-sum test. The significance value was adjusted using the BH method, and the null hypothesis was rejected when the adjusted p-value was <0.05.

[0449] Classifier training A response prediction classifier was constructed using baseline protein level MMY2002 data. A nested, hierarchical, 10x cross-validation approach was repeated 30 times using four different machine learning tools: Support Vector Machine (SVM), Random Forest (RF), Naive Bayes (NB), and j48 Decision Tree. For each learner, the training process began by creating 10 balanced folds of the dataset (outer loop). One of these folds was designated as the test set, and the remaining nine were passed to the inner loop as training sets. In the inner loop, the training sets were again divided into 10 balanced folds, forming inner training and inner test sets. A learner was trained for each of these inner training sets, and the method was repeated 30 times for each set within the outer loop. Features were selected and model parameters optimized using the accuracy of each inner loop learner in predicting the inner test set. Once 30 inner loops had been completed for each training set, the performance of the outer loop was evaluated for each corresponding test set (using optimized parameters and features). The entire outer loop process was then repeated 30 times to generate 30 response predictions for each sample within the dataset. The AUC, sensitivity, and specificity statistics obtained from this iterative approach are approximations of how the final model trained on the complete original dataset will perform in new test cases.

[0450] Results of the MMY2002 study Various comparisons were made, including treatment-induced response-dependent changes in protein expression. One of the proteins that showed decreased expression over time in responders was PD-L1, while PD-L1 protein expression increased over time in non-responders. PD-L1 binding to T cells leads to decreased T cell function and increased Treg development. Figure 18 The protein expression profiles of PD-L1 in responders, non-responders, and patients with stable disease are shown in cycles 1 and 3.

[0451] PD-L1 binding to its receptor PD-1 suppresses antitumor responses and drives T-cell anergy and exhaustion. While not wishing to be bound by any particular theory, downregulation of PD-L1 following CD38 therapy may also lead to an enhanced effect on improved antitumor immune responses in solid tumors.

Claims

1. A pharmaceutical composition for use in inhibiting the activity of immunosuppressive cells in a patient with cancer, said pharmaceutical composition comprising an antibody that specifically binds to CD38, said antibody having an IgG1 isotype, and said antibody comprising: a) The amino acid sequence of the heavy chain variable region (VH) of SEQ ID NO: 4, and b) The amino acid sequence of the light chain variable region (VL) of SEQ ID NO:

5.

2. The pharmaceutical composition according to claim 1, wherein the immunosuppressive cells comprise regulatory T cells (Tregs).

3. The pharmaceutical composition according to claim 1, wherein the immunosuppressive cells comprise bone marrow-derived suppressor cells (MDSCs).

4. The pharmaceutical composition according to claim 1, wherein the immunosuppressive cells comprise regulatory B cells (Breg).

5. The pharmaceutical composition according to any one of claims 1-4, wherein the antibody that specifically binds to CD38 is daratumumab.

6. A pharmaceutical composition for treating a patient with a solid tumor, wherein the solid tumor lacks detectable CD38 expression, the pharmaceutical composition comprising an antibody that specifically binds to CD38, wherein the antibody comprises the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 4 and the light chain variable region (VL) amino acid sequence of SEQ ID NO: 5, and wherein the antibody has an IgG1 isotype.

7. A pharmaceutical composition for inhibiting the activity of immunosuppressive cells, the pharmaceutical composition comprising an antibody that specifically binds to CD38, wherein the antibody comprises the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 4 and the light chain variable region (VL) amino acid sequence of SEQ ID NO: 5, and wherein the antibody has an IgG1 isotype.

8. The pharmaceutical composition according to claim 7, wherein the immunosuppressive cell is a Treg.

9. The pharmaceutical composition according to claim 7, wherein the immunosuppressive cells are MDSCs.

10. The pharmaceutical composition according to claim 7, wherein the immunosuppressive cell is Breg.

11. A pharmaceutical composition for enhancing a patient’s immune response, the pharmaceutical composition comprising an antibody that specifically binds to CD38, wherein the antibody comprises the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 4 and the light chain variable region (VL) amino acid sequence of SEQ ID NO: 5, and wherein the antibody has an IgG1 isotype.

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